BACKGROUND:The lack of a stable,easy-to-operate animal model for severe trauma has hindered the research progress.The aim of this study is to develop a mouse model that replicates the pathophysiological conditions of ...BACKGROUND:The lack of a stable,easy-to-operate animal model for severe trauma has hindered the research progress.The aim of this study is to develop a mouse model that replicates the pathophysiological conditions of severe trauma,providing a reliable research tool.METHODS:Male C57BL/6J mice(aged 8-10 weeks and weighting approximately 20 g)were used to establish the severe trauma model.Under anesthesia,a midshaft femoral fracture was created and packed with sterile cotton.A midline incision was made from the inguinal region to the sternum,exposing the abdominal organs for 30 min.The right femoral artery was cannulated to induce controlled blood loss at 30%,35%,40%,and 50%of the total blood volume.Survival rates were monitored for 24 h post-induction.In the mice that experienced 30%blood loss,the mean arterial pressure,body temperature,blood gas parameters,peripheral blood inflammatory markers,and major organ pathological changes were assessed.RESULTS:Mice with femoral fractures,soft tissue injuries,abdominal organ exposure,and 30%blood loss exhibited stable survival rates.Increased blood loss significantly reduced survival rates.Mean arterial pressure decreased initially,recovering within 0-15 min and returning to baseline by 50 min.Similarly,the body temperature decreased initially and gradually recovered to baseline within 50 min.Levels of peripheral blood inflammatory markers remained elevated for 12 h post-injury.Distant organs,including intestines,lungs,liver,spleen and kidneys,displayed varying degrees of injury.CONCLUSION:The established mouse model replicates the pathophysiological responses to severe trauma,indicating stability and reproducibility,which could be an useful tool for further trauma research.展开更多
Calvarial nerves,along with vasculature,influence skull formation during development and following injury,but it remains unclear how calvarial nerves are spatially distributed during postnatal growth and aging.Studyin...Calvarial nerves,along with vasculature,influence skull formation during development and following injury,but it remains unclear how calvarial nerves are spatially distributed during postnatal growth and aging.Studying the spatial distribution of nerves in the skull remains a challenge due to a lack of methods to quantify 3D structures in intact bone.To visualize calvarial 3D neurovascular architecture,we imaged nerves and endothelial cells with lightsheet microscopy.展开更多
Macrophages have a diverse set of functions based upon their activation states. The activation states, including resting（M0） and polarizing（M1 and M2） states, of macrophages derived from the mouse bone marrow, spl...Macrophages have a diverse set of functions based upon their activation states. The activation states, including resting（M0） and polarizing（M1 and M2） states, of macrophages derived from the mouse bone marrow, spleen, and peritoneal cavity（BMs, SPMs, and PCMs, respectively） were compared. We evaluated the macrophage yield per mouse and compared the surface markers major histocompatibility complex（MHC） Ⅱ and CD86 by flow cytometry. The relative mRNA levels of tumor necrosis factor-α（TNF-α）, interleukin（IL）-1β, mannose receptor（MR）, and Ym1 in the M0, M1, and M2 states were also compared using real-time polymerase chain reaction（PCR） analysis. Bone marrow yielded the most macrophages with the best homogeneity, but they were polarized toward the M2 phenotype. All three types of macrophages had the capacity to polarize into the M1 and M2 states, but SPMs had a stronger capacity to polarize into M1. The three types of macrophages showed no differences in their capacity to polarize into the M2 state. Therefore, the three types of macrophages have distinct characteristics regardless of their resting or polarizing states. Although bone marrow can get large amounts of homogeneous macrophages, the macrophages cannot replace tissue-derived macrophages.展开更多
Objective:To evaluate the in vitro activities of the ethyl acetate(EA) fraction of Houttuynia cordata(H.cordata) Thunb.(Saururaceae) and three of its constituent flavonoids(quercetin.quercitrin and rutin) against muri...Objective:To evaluate the in vitro activities of the ethyl acetate(EA) fraction of Houttuynia cordata(H.cordata) Thunb.(Saururaceae) and three of its constituent flavonoids(quercetin.quercitrin and rutin) against murine coronavirus and dengue virus(DENV).Methods:The antiviral activities of various concentrations of the EA fraction of H.cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus(MHV) and DENV type 2(DENV-2).Cinanserin hydrochloride was also tested against MHV.The EA fraction of H.cordata was tested for acute oral toxicity in C57BL/6 mice.Results:The EA fraction of H.cordata inhibited viral infectivity up to 6 d.Cinanserin hydrochloride was able to inhibit MHV for only 2 d.The 50%inhibitory concentrations(IC_(50)) of the EA fraction of H.cordata added before the viral adsorption stage were 0.98 μg/mL for MHV and 7.50 μg/mL for DENV-2with absence of cytotoxicity.The mice fed with the EA fraction up to 2 000 mg/kg did not induce any signs of acute toxicity,with normal histological features of major organs.Certain flavonoids exhibited comparatively weaker antiviral activity,notably quercetin which could inhibit both MHV and DENV-2.This was followed by quercitrin which could inhibit DENV-2but not MHV,whereas rutin did not exert any inhibitory effect on either virus.When quercetin was combined with quercitrin,enhancement of anti-DENV-2 activity and reduced cytotoxicity were observed.However,the synergistic efficacy of the flavonoid combination was still less than that of the EA fraction.Conclusions:The compounds in H.cordata contribute to the superior antiviral efficacy of the EA fraction which lacked cytotoxicity in vitro and acute toxicity in vim.H.cordata has much potential for the development of antiviral agents against coronavirus and dengue infections.展开更多
AIM： To evaluate the expression of fibrinogenlike protein 2 （fgl2） and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS： Balb/cJ or A/J mice were infected int...AIM： To evaluate the expression of fibrinogenlike protein 2 （fgl2） and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS： Balb/cJ or A/J mice were infected intraperitoneally （ip） with 100 PFU of murine hepatitis virus type 3 （MHV-3）, liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic （AOC） hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuciear cells （PBMC） were isolated from 30 patients （unpaired） with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase （ALT） and total bilirubin （TBil） in serum were measured to assess the severity of liver injury.RESULTS： Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. House fgl2 （mfgl2） protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 （hfgl2） was detected in 21 of 23 patients （91.30%） with severe AOC hepatitis B, while only 1 of 13 patients （7.69%） with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients （93.33%） with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B. CONCLUSION： The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.展开更多
AIM： To explore the relation between B-cell-specific Moloney murine leukemia virus insertion site 1 （Bmi-1） expression and the clinicopathological features of gastric carcinoma （GC）.METHODS： Immunohistochemistry...AIM： To explore the relation between B-cell-specific Moloney murine leukemia virus insertion site 1 （Bmi-1） expression and the clinicopathological features of gastric carcinoma （GC）.METHODS： Immunohistochemistry was used to detect the expression of Bmi-1 and ki-67. Doublelabeling staining was used to display the distribution of Bcl-2^＋/ki-67 cells in 162 cases of GC and its matched normal mucosa and precancerous lesion.RESULTS： The positive rate of Bmi-1 expression in GC（52.5%） was significantly higher than that in normal gastric mucosa （21.6%, X^2 = 33.088, P 〈 0.05）. The Bmi-1 expression in GC was closely related with the Lauren＇s and Borrmann＇s classification and clinicalstage （X^2 = 4.400, 6.122 and 11.190, respectively, P〈 0.05）. The expression of ki-67 was related to the Borrmann＇s classification （X^2 = 13.380, P 〈 0.05）.Bcl-2 expression was correlated with the Lauren＇s classification （Z2 = 4.725, P 〈 0.05）, and the Bmi-1 expression both in GC （rk = 0.157, P 〈 0.05） and inintestinal metaplasia （rk = 0.270, P 〈 0.05）.CONCLUSION： Abnormal Bmi-1 expression in GCmay be involved in cell proliferation, apoptosis andcancerization. This marker can objectively indicate theclinicopathological characteristics of GC.展开更多
AIM:To investigate the potential anti-Helicobacter pylori(H.pylori ) and anti-inflammation in vivo effects of two lactobacillus strains from human stomach.METHODS:Forty H.pylori infected Balb/c mice were randomly divi...AIM:To investigate the potential anti-Helicobacter pylori(H.pylori ) and anti-inflammation in vivo effects of two lactobacillus strains from human stomach.METHODS:Forty H.pylori infected Balb/c mice were randomly divided into 4 groups:proton pump inhibitor and antibiotics triple treated group,Lactobacillus fermenti(L.fermenti ) treated group,Lactobacillus acidophilus treated group and normal saline control group.Ten uninfected mice were also included as blank control group.The infection of H.pylori was detected by rapid urease tests,Giemsa staining and bacterial culture.The colonization of H.pylori was assessed in bacterial density score and gastric inflammation was assessed in histological score.The colonization of L.fermenti was performed by fluorescent probe.RESULTS:Histopathologic evaluation showed significant release of mucosal inflammation in gastric antrum and gastric body in lactobacillus treated groups and triple treated group.H.pylori eradication rate in both lactobacillus treated groups and triple treated group were higher than normal saline control group.Lactobacillus treated groups and triple treated group showed significant decrease of H.pylori bacterial density.CONCLUSION:Both lactobacillus strains have a significant anti-H.pylori activity;L.fermenti displays more efficient antagonistic activity in vivo against H.pylori infection.展开更多
Aim： To study the effect and mechanism of gonadotrophin-releasing hormone （GnRH） on murine Leydig cell steroidogenesis. Methods： Purified murine Leydig cells were treated with GnRH-Ⅰ and -Ⅱ agonists, and testost...Aim： To study the effect and mechanism of gonadotrophin-releasing hormone （GnRH） on murine Leydig cell steroidogenesis. Methods： Purified murine Leydig cells were treated with GnRH-Ⅰ and -Ⅱ agonists, and testosterone production and steroidogenic enzyme expressions were determined. Results： GnRH-Ⅰ and -Ⅱ agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner （P 〈 0.05）. The mRNA expressions of steroidogenic acute regulatory （STAR） protein, P450scc, 3β-hydroxysteroid dehydrogenase （HSD）, but not 17β-hydroxylase or 17β-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase （P 〈 0.05）. However, only 3β-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase （P 〈 0.05）. Conclusion： GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3β-HSD enzyme expression.展开更多
Idiopathic pulmonary fibrosis(IPF),characterized by aggravated alveolar destruc-tion and fibrotic matrix deposition,tendentiously experiences the stage called acute exacerbation IPF(AE-IPF)and progresses to multiple o...Idiopathic pulmonary fibrosis(IPF),characterized by aggravated alveolar destruc-tion and fibrotic matrix deposition,tendentiously experiences the stage called acute exacerbation IPF(AE-IPF)and progresses to multiple organ damage,especially liver injury.Recent studies have found a variety of immune microenvironment disorders associated with elevated IPF risk and secondary organ injury,whereas current animal models induced with bleomycin(BLM)could not completely reflect the pathologi-cal manifestations of AE-IPF patients in clinic,and the exact underlying mechanisms are not yet fully explored.In the current study,we established an AE-IPF model by tracheal administration of a single dose of BLM and then repeated administrations of lipopolysaccharide in mice.This mouse model successfully recapitulated the clinical features of AE-IPF,including excessive intrapulmonary inflammation and fibrosis and extrapulmonary manifestations,as indicated by significant upregulation of Il6,Tnfa,Il1b,Tgfb,fibronectin,and Col1a1 in both lungs and liver and elevated serum aspartate transaminase and alanine transaminase levels.These effects might be attributed to the regulation of Th17 cells.By sharing this novel murine model,we expect to pro-vide an appropriate experimental platform to investigate the pathogenesis of AE-IPF coupled with liver injury and contribute to the discovery and development of targeted interventions.展开更多
AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue te...AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue tetrazolium bromide(M一)assay.Effects of RSV on choroidal endothelial cell(CEC)migration were evaluated with a modified Boyden chamber assay,while tube formation was evaluated in a 2-D gel assay.CNV was induced by laser photocoagulation in mice.The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography(FA),confocal analysis of isolectin B4 labeled choroidal flat mounts,and histologic examination of CNV membranes.Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2(VEGFR2).RESULTS:No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 pmol/L(P>0.05).RSV inhibited vascular endothelial growth factor(VEGF)-induced CEC migration(P<0.05)and tube formation(P<0.05)invitro.Furthermore,intravitrealinjectionof RSV significantly inhibited laser induced CNV formation in mice.The FA leakage,CNV volume and CNV area analysis revealed that there were 41%,45%,and 58%reduction in RSV-treated eyes(1.691±0.1032,178163±78623μm^3 and 6508±619.0μm^2,respectively)compared with those in control(2.724±0.08447,379676±98382μm3and16576±2646μm^2,respectively;P<0.05).Phospho-VEGFR2expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control(P<0.05).CONCLUSION:Intravitreal injection of RSV exerts an inhibitory effect on CNV,which may through suppressing endothelial cell migration,tube formation and VEGFR2 phosphorylation.展开更多
Increasing evidence has revealed that maternal cytomegalovirus （CMV） infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related...Increasing evidence has revealed that maternal cytomegalovirus （CMV） infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 （TLR2） and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL- 10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV （MCMV） infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide （LPS）-treated and uninfected mice were used as controls. At E13.5, E 14.5 and E 18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 ktg/kg could decrease the IL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more pro- inflammatory cytokine IL-6. High dose of LPS stimulation （50 gg/kg） during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.展开更多
The aim of this study was to evaluate the effects of low concentrations of DEHP and MEHP on steroidogenesis in a murine Leydig tumor cell line （MLTC-1） in vitro. The result of flow cytometry analysis revealed that t...The aim of this study was to evaluate the effects of low concentrations of DEHP and MEHP on steroidogenesis in a murine Leydig tumor cell line （MLTC-1） in vitro. The result of flow cytometry analysis revealed that the proportion of apoptotic cells was significantly increased after the exposure to DEHP. All three genes （P450scc, P450c17, and 38HSD） under study showed an increased expression following exposure to DEHP or MEHP, although some insignificant inhibitory effects appeared in the 10μmol/L treatment group as compared with the controls. It was also found that DEHP or MEHP stimulated INSL3 mRNA and protein especially in the 0.001 μmol/L treatment group. Testosterone secretions were stimulated after the exposure to DEHP or MEHP. Alterations of steroidogenic enzymes and INSL3 in MLTC-1 cells might be involved in the biphasic effects of DEHP/MEHP on androgen production.展开更多
To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed...To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1 （IL-2）/Th2 （IL-4, IL-10, TGF-β1） cytokines in murine vaginal tissues was determined by RT-PCR. The cykotine in local tissues was increased to different extent under normal immune condition. IL-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-β1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10, and TGF-β1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans （C. albicans）, and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.展开更多
BACKGROUND Acetaminophen overdose is the most frequent cause of drug-induced liver failure in developed countries.Substantial progress has been made in understanding the mechanism of hepatocellular injury,but N-acetyl...BACKGROUND Acetaminophen overdose is the most frequent cause of drug-induced liver failure in developed countries.Substantial progress has been made in understanding the mechanism of hepatocellular injury,but N-acetylcysteine remains the only effective treatment despite its short therapeutic window.Thus,other hepatoprotective drugs are needed for the delayed treatment of acetaminopheninduced hepatotoxicity.Our interest focused on glycyrrhizin for its role as an inhibitor of high mobility group box 1(HMGB1)protein,a member of the family of damage-associated molecular pattern,known to play an important pathological role in various diseases.AIM To investigate the efficacy of the N-acetylcysteine/glycyrrhizin combination compared to N-acetylcysteine alone in the prevention of liver toxicity.METHODS Eight-week-old C57BL/6J wild-type female mice were used for all our experiments.Mice fasted for 15 h were treated with acetaminophen(500 mg/kg)or vehicle(phosphate-buffered saline)by intraperitoneal injection and separated into the following groups:Glycyrrhizin(200 mg/kg);N-acetylcysteine(150 mg/kg);and N-acetylcysteine/glycyrrhizin.In all groups,mice were sacrificed 12 h following acetaminophen administration.The assessment of hepatotoxicity was performed by measuring plasma levels of alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase.Hepatotoxicity was also evaluated by histological examination of hematoxylin and eosin-stained tissues sections.Survival rates were compared between various groups using Kaplan-Meier curves.RESULTS Consistent with data published in the literature,we confirmed that intraperitoneal administration of acetaminophen(500 mg/kg)in mice induced severe liver injury as evidenced by increases in alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase but also by liver necrosis score.Glycyrrhizin administration was shown to reduce the release of HMGB1 and significantly decreased the severity of liver injury.Thus,the co-administration of glycyrrhizin and N-acetylcysteine was investigated.Administered concomitantly with acetaminophen,the combination significantly reduced the severity of liver injury.Delayed administration of the combination of drugs,2 h or 6 h after acetaminophen,also induced a significant decrease in hepatocyte necrosis compared to mice treated with N-acetylcysteine alone.In addition,administration of N-acetylcysteine/glycyrrhizin combination was associated with an improved survival rate compared to mice treated with only N-acetylcysteine.CONCLUSION We demonstrate that,compared to N-acetylcysteine alone,co-administration of glycyrrhizin decreases the liver necrosis score and improves survival in a murine model of acetaminophen-induced liver injury.Our study opens a potential new therapeutic pathway in the prevention of acetaminophen hepatotoxicity.展开更多
Acute myeloid leukemia(AML) is an aggressive malignant disease defined by abnormal expansion of myeloid blasts. Despite recent advances in understanding AML pathogenesis and identifying their molecular subtypes based ...Acute myeloid leukemia(AML) is an aggressive malignant disease defined by abnormal expansion of myeloid blasts. Despite recent advances in understanding AML pathogenesis and identifying their molecular subtypes based on somatic mutations, AML is still characterized by poor outcomes, with a 5-year survival rate of only 30%-40%, the majority of the patients dying due to AML relapse. Leukemia stem cells(LSC) are considered to be at the root of chemotherapeutic resistance and AML relapse. Although numerous studies have tried to better characterize LSCs in terms of surface and molecular markers, a specific marker of LSC has not been found, and still the most universally accepted phenotypic signature remains the surface antigens CD34+CD38- that is shared with normal hematopoietic stem cells. Animal models provides the means to investigate the factors responsible for leukemic transformation, the intrinsic differences between secondary post-myeloproliferative neoplasm AML and de novo AML, especially the signaling pathways involved in inflammation and hematopoiesis. However, AML proved to be one of the hematological malignancies that is difficult to engraft even in the most immunodeficient mice strains, and numerous ongoing attempts are focused to develop "humanized mice" that can support the engraftment of LSC. This present review is aiming to in-troduce the field of AML pathogenesis and the concept of LSC, to present the current knowledge on leukemic blasts surface markers and recent attempts to develop best AML animal models.展开更多
AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the sp...AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.展开更多
EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mo...EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation （IP） qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation （CHIP） assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells （MEL） in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase Ⅱ, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE （HS26）. This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.展开更多
Homeostasis of platelet number in human and other mammals is well maintained for prevention of minor bleeding and for other im- munological functions, but the exact molecular mechanism responsible for immune thrombocy...Homeostasis of platelet number in human and other mammals is well maintained for prevention of minor bleeding and for other im- munological functions, but the exact molecular mechanism responsible for immune thrombocytopenic purpura （ITP） has not been fully understood. In an effort to identify genetic factors involved in initiation of platelet production in response to bleeding injury or platelet destruction, we have successfully generated an animal model of human ITP via intraperitoneal injection of anti-platelet antibody into the Balb/c mouse. Platelet counts were dropped dramatically in animals that received antibody injection within 4 h, maintained at the mini- mum level for a period of 44 h, started to rebound after 48 h, and reached to the maximum at 144 h （6 days）. Final homeostasis reached at approximately 408 h （17 days）, following a minor cycle of platelet number fluctuation. Using semi-quantitative RT-PCR, we assessed and compared mRNA level of CD41, c-myb, c-mpl, caspase-3, caspase-9, GATA-1, and Bcl-xl in bone marrow and spleen. Alteration of mRNA expression was correlated with the change of platelet level, and an inverse relationship was found for expression of the genes be- tween bone marrow and spleen. No transcription was detectable for any of the seven genes in bone marrow at the time when platelet number reached the maximum （144 h）. In contrast, mRNA transcripts of the seven genes were found to be at the highest level in spleen tissue. This is the first study of simultaneous detection of multiple platelet related genes in a highly reproducible ITP animal model. Our results provided the supportive evidence that expression of the above seven genes are more related to negative regulation of platelet number in spleen tissue, at least in the model animals.展开更多
Osteocytes reside as three-dimensionally（3D） networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of os...Osteocytes reside as three-dimensionally（3D） networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because：（1） current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and（2） primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to：（1） construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and（2） reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to：（1） distribute and entrap cells within the interstitial spaces between the microbeads and（2） maintain average cell-to-cell distance to be about 19 mm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions（SOST and FGF23） and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of：（1） studying flow-induced shear stress on the mechanotransduction function of primary osteocytes,（2） studying physiological functions of 3D-networked osteocytes with in vitro convenience,and（3） developing clinically relevant human bone disease models.展开更多
基金supported by the National Natural Science Foundation of China(82102315).
文摘BACKGROUND:The lack of a stable,easy-to-operate animal model for severe trauma has hindered the research progress.The aim of this study is to develop a mouse model that replicates the pathophysiological conditions of severe trauma,providing a reliable research tool.METHODS:Male C57BL/6J mice(aged 8-10 weeks and weighting approximately 20 g)were used to establish the severe trauma model.Under anesthesia,a midshaft femoral fracture was created and packed with sterile cotton.A midline incision was made from the inguinal region to the sternum,exposing the abdominal organs for 30 min.The right femoral artery was cannulated to induce controlled blood loss at 30%,35%,40%,and 50%of the total blood volume.Survival rates were monitored for 24 h post-induction.In the mice that experienced 30%blood loss,the mean arterial pressure,body temperature,blood gas parameters,peripheral blood inflammatory markers,and major organ pathological changes were assessed.RESULTS:Mice with femoral fractures,soft tissue injuries,abdominal organ exposure,and 30%blood loss exhibited stable survival rates.Increased blood loss significantly reduced survival rates.Mean arterial pressure decreased initially,recovering within 0-15 min and returning to baseline by 50 min.Similarly,the body temperature decreased initially and gradually recovered to baseline within 50 min.Levels of peripheral blood inflammatory markers remained elevated for 12 h post-injury.Distant organs,including intestines,lungs,liver,spleen and kidneys,displayed varying degrees of injury.CONCLUSION:The established mouse model replicates the pathophysiological responses to severe trauma,indicating stability and reproducibility,which could be an useful tool for further trauma research.
基金supported by funding from NIDCR(1R01DE027957)Maryland Stem Cell Research Fund(2022-MSCRFV-5782)the NSF GRFP and NCI(5R01CA237597-05,2R01CA196701-06A1).
文摘Calvarial nerves,along with vasculature,influence skull formation during development and following injury,but it remains unclear how calvarial nerves are spatially distributed during postnatal growth and aging.Studying the spatial distribution of nerves in the skull remains a challenge due to a lack of methods to quantify 3D structures in intact bone.To visualize calvarial 3D neurovascular architecture,we imaged nerves and endothelial cells with lightsheet microscopy.
基金Project supported by the National Natural Science Foundation of China(Nos.81270829,81102247,and 81670681)
文摘Macrophages have a diverse set of functions based upon their activation states. The activation states, including resting（M0） and polarizing（M1 and M2） states, of macrophages derived from the mouse bone marrow, spleen, and peritoneal cavity（BMs, SPMs, and PCMs, respectively） were compared. We evaluated the macrophage yield per mouse and compared the surface markers major histocompatibility complex（MHC） Ⅱ and CD86 by flow cytometry. The relative mRNA levels of tumor necrosis factor-α（TNF-α）, interleukin（IL）-1β, mannose receptor（MR）, and Ym1 in the M0, M1, and M2 states were also compared using real-time polymerase chain reaction（PCR） analysis. Bone marrow yielded the most macrophages with the best homogeneity, but they were polarized toward the M2 phenotype. All three types of macrophages had the capacity to polarize into the M1 and M2 states, but SPMs had a stronger capacity to polarize into M1. The three types of macrophages showed no differences in their capacity to polarize into the M2 state. Therefore, the three types of macrophages have distinct characteristics regardless of their resting or polarizing states. Although bone marrow can get large amounts of homogeneous macrophages, the macrophages cannot replace tissue-derived macrophages.
基金supported by a research grant from the National University of Singapore
文摘Objective:To evaluate the in vitro activities of the ethyl acetate(EA) fraction of Houttuynia cordata(H.cordata) Thunb.(Saururaceae) and three of its constituent flavonoids(quercetin.quercitrin and rutin) against murine coronavirus and dengue virus(DENV).Methods:The antiviral activities of various concentrations of the EA fraction of H.cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus(MHV) and DENV type 2(DENV-2).Cinanserin hydrochloride was also tested against MHV.The EA fraction of H.cordata was tested for acute oral toxicity in C57BL/6 mice.Results:The EA fraction of H.cordata inhibited viral infectivity up to 6 d.Cinanserin hydrochloride was able to inhibit MHV for only 2 d.The 50%inhibitory concentrations(IC_(50)) of the EA fraction of H.cordata added before the viral adsorption stage were 0.98 μg/mL for MHV and 7.50 μg/mL for DENV-2with absence of cytotoxicity.The mice fed with the EA fraction up to 2 000 mg/kg did not induce any signs of acute toxicity,with normal histological features of major organs.Certain flavonoids exhibited comparatively weaker antiviral activity,notably quercetin which could inhibit both MHV and DENV-2.This was followed by quercitrin which could inhibit DENV-2but not MHV,whereas rutin did not exert any inhibitory effect on either virus.When quercetin was combined with quercitrin,enhancement of anti-DENV-2 activity and reduced cytotoxicity were observed.However,the synergistic efficacy of the flavonoid combination was still less than that of the EA fraction.Conclusions:The compounds in H.cordata contribute to the superior antiviral efficacy of the EA fraction which lacked cytotoxicity in vitro and acute toxicity in vim.H.cordata has much potential for the development of antiviral agents against coronavirus and dengue infections.
基金Supported by the National Natural Science Foundation of China for Distinguished Young Scholars, No. 30225040 for Dr Ning Q,No. 30123019 for Dr Luo XP
文摘AIM： To evaluate the expression of fibrinogenlike protein 2 （fgl2） and its correlation with disease progression in both mice and patients with severe viral hepatitis. METHODS： Balb/cJ or A/J mice were infected intraperitoneally （ip） with 100 PFU of murine hepatitis virus type 3 （MHV-3）, liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic （AOC） hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuciear cells （PBMC） were isolated from 30 patients （unpaired） with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinase activity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase （ALT） and total bilirubin （TBil） in serum were measured to assess the severity of liver injury.RESULTS： Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice. In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. House fgl2 （mfgl2） protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 （hfgl2） was detected in 21 of 23 patients （91.30%） with severe AOC hepatitis B, while only 1 of 13 patients （7.69%） with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients （93.33%） with severe AOC hepatitis B and 1 of 10 with mild chronic hepatitis B had detectable hfgl2 expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2 expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B. CONCLUSION： The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2 prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2 protein play a pivotal role in initiating severe hepatitis. The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.
基金Supported by A special fund for Key University Laboratories from Department of Education of Liaoning Province, No. 2008S233
文摘AIM： To explore the relation between B-cell-specific Moloney murine leukemia virus insertion site 1 （Bmi-1） expression and the clinicopathological features of gastric carcinoma （GC）.METHODS： Immunohistochemistry was used to detect the expression of Bmi-1 and ki-67. Doublelabeling staining was used to display the distribution of Bcl-2^＋/ki-67 cells in 162 cases of GC and its matched normal mucosa and precancerous lesion.RESULTS： The positive rate of Bmi-1 expression in GC（52.5%） was significantly higher than that in normal gastric mucosa （21.6%, X^2 = 33.088, P 〈 0.05）. The Bmi-1 expression in GC was closely related with the Lauren＇s and Borrmann＇s classification and clinicalstage （X^2 = 4.400, 6.122 and 11.190, respectively, P〈 0.05）. The expression of ki-67 was related to the Borrmann＇s classification （X^2 = 13.380, P 〈 0.05）.Bcl-2 expression was correlated with the Lauren＇s classification （Z2 = 4.725, P 〈 0.05）, and the Bmi-1 expression both in GC （rk = 0.157, P 〈 0.05） and inintestinal metaplasia （rk = 0.270, P 〈 0.05）.CONCLUSION： Abnormal Bmi-1 expression in GCmay be involved in cell proliferation, apoptosis andcancerization. This marker can objectively indicate theclinicopathological characteristics of GC.
文摘AIM:To investigate the potential anti-Helicobacter pylori(H.pylori ) and anti-inflammation in vivo effects of two lactobacillus strains from human stomach.METHODS:Forty H.pylori infected Balb/c mice were randomly divided into 4 groups:proton pump inhibitor and antibiotics triple treated group,Lactobacillus fermenti(L.fermenti ) treated group,Lactobacillus acidophilus treated group and normal saline control group.Ten uninfected mice were also included as blank control group.The infection of H.pylori was detected by rapid urease tests,Giemsa staining and bacterial culture.The colonization of H.pylori was assessed in bacterial density score and gastric inflammation was assessed in histological score.The colonization of L.fermenti was performed by fluorescent probe.RESULTS:Histopathologic evaluation showed significant release of mucosal inflammation in gastric antrum and gastric body in lactobacillus treated groups and triple treated group.H.pylori eradication rate in both lactobacillus treated groups and triple treated group were higher than normal saline control group.Lactobacillus treated groups and triple treated group showed significant decrease of H.pylori bacterial density.CONCLUSION:Both lactobacillus strains have a significant anti-H.pylori activity;L.fermenti displays more efficient antagonistic activity in vivo against H.pylori infection.
文摘Aim： To study the effect and mechanism of gonadotrophin-releasing hormone （GnRH） on murine Leydig cell steroidogenesis. Methods： Purified murine Leydig cells were treated with GnRH-Ⅰ and -Ⅱ agonists, and testosterone production and steroidogenic enzyme expressions were determined. Results： GnRH-Ⅰ and -Ⅱ agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner （P 〈 0.05）. The mRNA expressions of steroidogenic acute regulatory （STAR） protein, P450scc, 3β-hydroxysteroid dehydrogenase （HSD）, but not 17β-hydroxylase or 17β-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase （P 〈 0.05）. However, only 3β-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase （P 〈 0.05）. Conclusion： GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3β-HSD enzyme expression.
基金supported by the Innovation Team and Talents Cultivation Program of the National Administration of Traditional Chinese Medicine(grant no.:ZYYCXTD-C-202006 to XG and Xiaojiaoyang Li)Beijing Municipal Science and Technology Commission(grant no.:7212174 to Xiaojiaoyang Li)+2 种基金National Natural Science Foundation of China(grant no.:82004045 to Xiaojiaoyang Li)Beijing Nova Program of Science and Technology(grant no.:Z191100001119088 to Xiaojiaoyang Li)the Young Elite Scientists Sponsorship Program by CACM(grant no.:2020-QNRC2-01 to Xiaojiaoyang Li).
文摘Idiopathic pulmonary fibrosis(IPF),characterized by aggravated alveolar destruc-tion and fibrotic matrix deposition,tendentiously experiences the stage called acute exacerbation IPF(AE-IPF)and progresses to multiple organ damage,especially liver injury.Recent studies have found a variety of immune microenvironment disorders associated with elevated IPF risk and secondary organ injury,whereas current animal models induced with bleomycin(BLM)could not completely reflect the pathologi-cal manifestations of AE-IPF patients in clinic,and the exact underlying mechanisms are not yet fully explored.In the current study,we established an AE-IPF model by tracheal administration of a single dose of BLM and then repeated administrations of lipopolysaccharide in mice.This mouse model successfully recapitulated the clinical features of AE-IPF,including excessive intrapulmonary inflammation and fibrosis and extrapulmonary manifestations,as indicated by significant upregulation of Il6,Tnfa,Il1b,Tgfb,fibronectin,and Col1a1 in both lungs and liver and elevated serum aspartate transaminase and alanine transaminase levels.These effects might be attributed to the regulation of Th17 cells.By sharing this novel murine model,we expect to pro-vide an appropriate experimental platform to investigate the pathogenesis of AE-IPF coupled with liver injury and contribute to the discovery and development of targeted interventions.
基金Supported by the National Natural Science Foundation of China(No.81703134,No.81770952)Henan Province Nature Science Foundation of China(No.162300410296)Hunan Province Nature Science Foundation of China(No.2018JJ3772)。
文摘AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue tetrazolium bromide(M一)assay.Effects of RSV on choroidal endothelial cell(CEC)migration were evaluated with a modified Boyden chamber assay,while tube formation was evaluated in a 2-D gel assay.CNV was induced by laser photocoagulation in mice.The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography(FA),confocal analysis of isolectin B4 labeled choroidal flat mounts,and histologic examination of CNV membranes.Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2(VEGFR2).RESULTS:No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 pmol/L(P>0.05).RSV inhibited vascular endothelial growth factor(VEGF)-induced CEC migration(P<0.05)and tube formation(P<0.05)invitro.Furthermore,intravitrealinjectionof RSV significantly inhibited laser induced CNV formation in mice.The FA leakage,CNV volume and CNV area analysis revealed that there were 41%,45%,and 58%reduction in RSV-treated eyes(1.691±0.1032,178163±78623μm^3 and 6508±619.0μm^2,respectively)compared with those in control(2.724±0.08447,379676±98382μm3and16576±2646μm^2,respectively;P<0.05).Phospho-VEGFR2expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control(P<0.05).CONCLUSION:Intravitreal injection of RSV exerts an inhibitory effect on CNV,which may through suppressing endothelial cell migration,tube formation and VEGFR2 phosphorylation.
文摘Increasing evidence has revealed that maternal cytomegalovirus （CMV） infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 （TLR2） and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL- 10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV （MCMV） infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide （LPS）-treated and uninfected mice were used as controls. At E13.5, E 14.5 and E 18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 ktg/kg could decrease the IL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more pro- inflammatory cytokine IL-6. High dose of LPS stimulation （50 gg/kg） during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.
基金supported by the National Natural Science Foundation of China(No.81273028)
文摘The aim of this study was to evaluate the effects of low concentrations of DEHP and MEHP on steroidogenesis in a murine Leydig tumor cell line （MLTC-1） in vitro. The result of flow cytometry analysis revealed that the proportion of apoptotic cells was significantly increased after the exposure to DEHP. All three genes （P450scc, P450c17, and 38HSD） under study showed an increased expression following exposure to DEHP or MEHP, although some insignificant inhibitory effects appeared in the 10μmol/L treatment group as compared with the controls. It was also found that DEHP or MEHP stimulated INSL3 mRNA and protein especially in the 0.001 μmol/L treatment group. Testosterone secretions were stimulated after the exposure to DEHP or MEHP. Alterations of steroidogenic enzymes and INSL3 in MLTC-1 cells might be involved in the biphasic effects of DEHP/MEHP on androgen production.
基金the Science Research Foundation of Health Department of Hubei Prov-ince (No. JXIB048)Janssen Research Foundation
文摘To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1 （IL-2）/Th2 （IL-4, IL-10, TGF-β1） cytokines in murine vaginal tissues was determined by RT-PCR. The cykotine in local tissues was increased to different extent under normal immune condition. IL-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-β1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10, and TGF-β1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans （C. albicans）, and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.
基金Supported by the Bourse du Conseil Médical de l’hôpital Erasme,Fonds E.et S.Jacobs and Novartis GrantThe CMMI is supported by the European Regional Development Fund and Wallonia
文摘BACKGROUND Acetaminophen overdose is the most frequent cause of drug-induced liver failure in developed countries.Substantial progress has been made in understanding the mechanism of hepatocellular injury,but N-acetylcysteine remains the only effective treatment despite its short therapeutic window.Thus,other hepatoprotective drugs are needed for the delayed treatment of acetaminopheninduced hepatotoxicity.Our interest focused on glycyrrhizin for its role as an inhibitor of high mobility group box 1(HMGB1)protein,a member of the family of damage-associated molecular pattern,known to play an important pathological role in various diseases.AIM To investigate the efficacy of the N-acetylcysteine/glycyrrhizin combination compared to N-acetylcysteine alone in the prevention of liver toxicity.METHODS Eight-week-old C57BL/6J wild-type female mice were used for all our experiments.Mice fasted for 15 h were treated with acetaminophen(500 mg/kg)or vehicle(phosphate-buffered saline)by intraperitoneal injection and separated into the following groups:Glycyrrhizin(200 mg/kg);N-acetylcysteine(150 mg/kg);and N-acetylcysteine/glycyrrhizin.In all groups,mice were sacrificed 12 h following acetaminophen administration.The assessment of hepatotoxicity was performed by measuring plasma levels of alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase.Hepatotoxicity was also evaluated by histological examination of hematoxylin and eosin-stained tissues sections.Survival rates were compared between various groups using Kaplan-Meier curves.RESULTS Consistent with data published in the literature,we confirmed that intraperitoneal administration of acetaminophen(500 mg/kg)in mice induced severe liver injury as evidenced by increases in alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase but also by liver necrosis score.Glycyrrhizin administration was shown to reduce the release of HMGB1 and significantly decreased the severity of liver injury.Thus,the co-administration of glycyrrhizin and N-acetylcysteine was investigated.Administered concomitantly with acetaminophen,the combination significantly reduced the severity of liver injury.Delayed administration of the combination of drugs,2 h or 6 h after acetaminophen,also induced a significant decrease in hepatocyte necrosis compared to mice treated with N-acetylcysteine alone.In addition,administration of N-acetylcysteine/glycyrrhizin combination was associated with an improved survival rate compared to mice treated with only N-acetylcysteine.CONCLUSION We demonstrate that,compared to N-acetylcysteine alone,co-administration of glycyrrhizin decreases the liver necrosis score and improves survival in a murine model of acetaminophen-induced liver injury.Our study opens a potential new therapeutic pathway in the prevention of acetaminophen hepatotoxicity.
基金Supported by The project Competitiveness Operational Programme(COP)A1.1.4.,No.P_37_798,Contract 149/26.10.2016(My SMIS2014+:106774)
文摘Acute myeloid leukemia(AML) is an aggressive malignant disease defined by abnormal expansion of myeloid blasts. Despite recent advances in understanding AML pathogenesis and identifying their molecular subtypes based on somatic mutations, AML is still characterized by poor outcomes, with a 5-year survival rate of only 30%-40%, the majority of the patients dying due to AML relapse. Leukemia stem cells(LSC) are considered to be at the root of chemotherapeutic resistance and AML relapse. Although numerous studies have tried to better characterize LSCs in terms of surface and molecular markers, a specific marker of LSC has not been found, and still the most universally accepted phenotypic signature remains the surface antigens CD34+CD38- that is shared with normal hematopoietic stem cells. Animal models provides the means to investigate the factors responsible for leukemic transformation, the intrinsic differences between secondary post-myeloproliferative neoplasm AML and de novo AML, especially the signaling pathways involved in inflammation and hematopoiesis. However, AML proved to be one of the hematological malignancies that is difficult to engraft even in the most immunodeficient mice strains, and numerous ongoing attempts are focused to develop "humanized mice" that can support the engraftment of LSC. This present review is aiming to in-troduce the field of AML pathogenesis and the concept of LSC, to present the current knowledge on leukemic blasts surface markers and recent attempts to develop best AML animal models.
基金Supported by the Public Health Department Foundation of Hunan province, China, No. Y02-42
文摘AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.
文摘EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation （IP） qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation （CHIP） assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells （MEL） in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase Ⅱ, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE （HS26）. This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.
文摘Homeostasis of platelet number in human and other mammals is well maintained for prevention of minor bleeding and for other im- munological functions, but the exact molecular mechanism responsible for immune thrombocytopenic purpura （ITP） has not been fully understood. In an effort to identify genetic factors involved in initiation of platelet production in response to bleeding injury or platelet destruction, we have successfully generated an animal model of human ITP via intraperitoneal injection of anti-platelet antibody into the Balb/c mouse. Platelet counts were dropped dramatically in animals that received antibody injection within 4 h, maintained at the mini- mum level for a period of 44 h, started to rebound after 48 h, and reached to the maximum at 144 h （6 days）. Final homeostasis reached at approximately 408 h （17 days）, following a minor cycle of platelet number fluctuation. Using semi-quantitative RT-PCR, we assessed and compared mRNA level of CD41, c-myb, c-mpl, caspase-3, caspase-9, GATA-1, and Bcl-xl in bone marrow and spleen. Alteration of mRNA expression was correlated with the change of platelet level, and an inverse relationship was found for expression of the genes be- tween bone marrow and spleen. No transcription was detectable for any of the seven genes in bone marrow at the time when platelet number reached the maximum （144 h）. In contrast, mRNA transcripts of the seven genes were found to be at the highest level in spleen tissue. This is the first study of simultaneous detection of multiple platelet related genes in a highly reproducible ITP animal model. Our results provided the supportive evidence that expression of the above seven genes are more related to negative regulation of platelet number in spleen tissue, at least in the model animals.
基金the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (1R21AR065032 to W.Y.L and J.Z.)the National Science Foundation (DMR 1409779 to W.Y.L and J.Z.)
文摘Osteocytes reside as three-dimensionally（3D） networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because：（1） current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and（2） primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to：（1） construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and（2） reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to：（1） distribute and entrap cells within the interstitial spaces between the microbeads and（2） maintain average cell-to-cell distance to be about 19 mm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions（SOST and FGF23） and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of：（1） studying flow-induced shear stress on the mechanotransduction function of primary osteocytes,（2） studying physiological functions of 3D-networked osteocytes with in vitro convenience,and（3） developing clinically relevant human bone disease models.
