Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and ce...Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and cell growth. Methods: MCT1 antisense gene recombinant vector was introduced into human lung cancer cell line A549 by electroporation. The transfected A549 cells resistant to G418 were selected. Positive clones were examined by using PCR. The changes of intracellular pH and lactate were examined with spec-trophotometric method. Cell growth was studied with cell growth curve. Results: Intracellular pH and lactate were remarkably decreased in the cells transfected pLXSN-MCT1 in comparison with A549 cells without transfection (P<0. 001). The growth of A549 cells transfected pLXSN-MCTl was also inhibited remarkably. Conclusion: MCT1 gene may play an important role in pHi regulation, lactate transportation and cell growth in tumor cells.展开更多
Objective:The aim of this study was to study the changes of SGC-7901 cells transfecting small hairpin RNA(shRNA) targeted Bcl-2 and celecoxib in vitro.Methods:To use the effective siRNA targeted to Bcl-2 by Lipofectam...Objective:The aim of this study was to study the changes of SGC-7901 cells transfecting small hairpin RNA(shRNA) targeted Bcl-2 and celecoxib in vitro.Methods:To use the effective siRNA targeted to Bcl-2 by Lipofectamine 2000,the rate of cell growth inhibition of all groups was detected by multiply-table tournament(MTT) method,apoptosis rate was examined by flow cytometry and the expression of Bcl-2 was assayed by Elisa.Results:After siRNA was transfected to SGC-7901 cells,the rate of cell growth inhibition was increased,the joint group was higher by flow cytometry and the expression of Bcl-2 was lower by Elisa.Conclusion:The growth of SGC-7901 cells which was transfected siRNA has been inhibited,and the sensitivity to celecoxib has been increased.展开更多
The recent commercialization of gene products has sparked significant interest in gene therapy,necessitating efficient and precise gene delivery via various vectors.Currently,viral vectors and lipid-based nanocarriers...The recent commercialization of gene products has sparked significant interest in gene therapy,necessitating efficient and precise gene delivery via various vectors.Currently,viral vectors and lipid-based nanocarriers are the predominant choices and have been extensively investigated and reviewed.Beyond these vectors,polymeric nanocarriers also hold the promise in therapeutic gene delivery owing to their versatile functionalities,such as improving the stability,cellar uptake and endosomal escape of nucleic acid drugs,along with precise delivery to targeted tissues.This review presents a brief overview of the status quo of the emerging polymeric nanocarriers for therapeutic gene delivery,focusing on key cationic polymers,nanocarrier types,and preparation methods.It also highlights targeted diseases,strategies to improve delivery efficiency,and potential future directions in this research area.The review is hoped to inspire the development,optimization,and clinical translation of highly efficient polymeric nanocarriers for therapeutic gene delivery.展开更多
BACKGROUND Mesenchymal stem cells(MSCs)are promising candidates for regenerative therapy due to their self-renewal capability,multilineage differentiation potential,and immunomodulatory effects.The molecular character...BACKGROUND Mesenchymal stem cells(MSCs)are promising candidates for regenerative therapy due to their self-renewal capability,multilineage differentiation potential,and immunomodulatory effects.The molecular characteristics of MSCs are influenced by their location.Recently,epidural fat(EF)and EF-derived MSCs(EF-MSCs)have garnered attention due to their potential benefits to the spinal microenvironment and their high expression of neural SC markers.However,their clinical applications are limited due to cell senescence and limited accessibility of EF.Although many studies have attempted to establish an immortalized,stable SC line,the characteristics of immortalized EF-MSCs remain to be clarified.AIM To establish and analyze stable immortalized EF-MSCs.METHODS The phenotypes of EF-MSCs were analyzed using optical microscopy.Cell immortalization was performed using lentiviral vectors.The biomolecular characteristics of the cells were analyzed by immunoblotting,quantitative PCR,and proteomics.RESULTS The immortalized EF-MSCs demonstrated a significantly extended lifespan compared to the control group,with well-preserved adipogenic potential and SC surface marker expression.Introduction of human telomerase reverse transcriptase genes markedly increased the lifespan of EF-MSCs.Proteomics analysis revealed substantial increase in the expression of DNA replication pathway components in immortalized EF-MSCs.CONCLUSION Immortalized EF-MSCs exhibited significantly enhanced proliferative capacity,retained adipogenic potential,and upregulated the expression of DNA replication pathway components.展开更多
With the deepening of epigenetic research,studies have shown that N6-methyladenosine(m^(6)A)is closely related to the development of rheumatoid arthritis(RA),but the mechanism is still unclear.In the study,we collecte...With the deepening of epigenetic research,studies have shown that N6-methyladenosine(m^(6)A)is closely related to the development of rheumatoid arthritis(RA),but the mechanism is still unclear.In the study,we collected synovial tissues from normal controls and patients with osteoarthritis(OA)or RA.The levels of m^(6)A and inflammation were analyzed by immunofluorescence staining and western blotting.The roles of IGF2BP3 in cell proliferation and inflammatory activation were explored using transfection and RNA immunoprecipitation assays.IGF2BP3^(−/−)mice were generated and used to establish an arthritis mouse model by transferring serum from adult arthritis K/BxN mice.We found m^(6)A levels were markedly increased in RA patients and mouse models,and the expression of IGF2BP3 was upregulated in individuals with RA and related to the levels of inflammatory markers.IGF2BP3 played an important part in RA-fibroblast-like synoviocytes(FLS)by promoting cell proliferation,migration,invasion,inflammatory cytokine release and inhibiting autophagy.In addition,IGF2BP3 inhibited autophagy to reduce ROS production,thereby decreasing the inflammatory activation of macrophages.More importantly,RASGRF1-mediated mTORC1 activation played a crucial role in the ability of IGF2BP3 to promote cell proliferation and inflammatory activation.In an arthritis model of IGF2BP3^(−/−)mice,IGF2BP3 knockout inhibited RA-FLS proliferation and inflammatory infiltration,and further ameliorated RA joint injury.Our study revealed an important role for IGF2BP3 in RA progression.The targeted inhibition of IGF2BP3 reduced cell proliferation and inflammatory activation and limited RA development,providing a potential strategy for RA therapy.展开更多
Fish cell line provide a useful tool for studies in virology and molecular biology.To establish a novel continuous marine fish cell line(EFF)from caudal fin tissue of brown-marbled grouper Epinephelus fuscoguttatus,it...Fish cell line provide a useful tool for studies in virology and molecular biology.To establish a novel continuous marine fish cell line(EFF)from caudal fin tissue of brown-marbled grouper Epinephelus fuscoguttatus,its susceptibility to the crustacean covert mortality nodavirus(CMNV)was evaluated.The primary cell cultures were initiated first by incomplete digestion of fin tissue blocks with dispase and collagenase,and then the explant was cultured in L-15 medium supplemented with 20%fetal bovine serum,10%grouper muscle extract,and 20-ng/mL growth factors of basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF).The EFF cells were continuously passaged beyond 50 times in fibroblast-like morphology,and they grew well in L-15 medium supplemented with a lower concentration fetal bovine serum(10%)at 28℃ after passage 10,without muscle extract and the growth factors.In addition,their grouper origin was confirmed by chromosome analysis and cytochrome oxidase 1(CO 1)gene analysis of these EFF cells.Transfection experiment via lipofectamine 8000 indicated that the EFF cells had a high transfection potential with a transfection efficiency up to 32%.Cross-species viral susceptibility analysis showed that CMNV not only successfully infected the EFF cells as evidenced by obvious cytopathic effects like vacuolation,detachment and death of cells,but also multiplied in the EFF cells as indicated by the results of semi-quantitative RT-PCR in transmission electron microscopy.Therefore,the establishment of immortal EFF cell line provided a useful cell model for future works on the isolation,multiplication,and pathogenic mechanism of cross-species infection of CMNV as well as genetic manipulation.展开更多
Chitosan （CS） is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains we...Chitosan （CS） is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains were scissioned under the γ-ray radiation, and the molecu- lar weight （MW） of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the molecular weight of CS decreased about an order of magnitude. The γ-ray-radiation-scissioned CS can effectively bind with plasmid （pEGFP） through complex coacervation method, forming pEGFP/γ-ray-radiation-scissioned CS complex particles with a size of 200-300 nm. The complex particles have good stability and little cytotoxicity. The in vitro gene transfection efficiencies of the pEGFP/γ-ray-radiation-scissioned CS complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector. This work provides a green and simple method on the preparation of CS-based gene vectors with high efficiency and biosafety.展开更多
OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided in...OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.展开更多
SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP...SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP-1 mutants were made： △SH2-SHIP-1, △Ptase-SHIP-1, △Cter-SHIP-1. These mutant forms were subcloned as well as the wild type （WT） of murine SHIP-1 cDNA were subcloned into pcDNA3 expression vector, then transfected into and overexpressed SHIP-1 and its mutants in a Src-transformed 3Y1 cellline （SR3Y1）. The results showed that overexpression of wild type of SHIP-1 does not affect the MMP2 secretion in both SR3Y1 and 3Y1 cells, but can induce MMP9 secretion, while either WT SHIP-1, the SH2 domain, phosphatase domain, or C terminus deletion mutants could significantly block the MMP2 and MMP9 secretion in SR3Y1 cells and suppress cell invasion ability. The results confirmed SHIP-1 as a negative regulator for cell migration and invasion in transformed cells, and implied that it may function through each of its three domains.展开更多
[Objective] This study was to improve the virus replication efficiency of full length infectious cDNA clones by making use of the ribozyme＇s self incision property.[Method] By employing three-step PCR,HDV ribozyme（H...[Objective] This study was to improve the virus replication efficiency of full length infectious cDNA clones by making use of the ribozyme＇s self incision property.[Method] By employing three-step PCR,HDV ribozyme（HdvRz）cDNA was isolated,and cloned into the downstream flanking the genome of the porcine reproductive and respiratory syndrome virus,and into which the bovine growth hormone polyadenylation sequence（BGH）was inserted via enzyme digestion and ligation,yielding pAPRRS-HB.The newly constructed pAPRRS-HB was used to transfect MARC-145 cells,in which the N protein and non-structural protein（nsp2）were determined by indirect immunofluorescence assay after 72 h of expression;meanwhile the virus titer of cell supernatant was tested using TCID50 assay.[Result] pAPRRS-HB containing complete infectious PRRSV cDNA has been successfully developed,and it performed about 10-fold higher virus rescue rate than pAPRRS without the engineered ribozyme element.[Conclusion] The results laid a foundation for revealing the structure and function of PRRSV gene.展开更多
[Objective] The aim of this study was to optimize conditions of exogenous gene mediated by liposome to transfect yak mammary epithelial cells in Vitro.[Method] Yak mammary epithelial cells were isolated and cultivated...[Objective] The aim of this study was to optimize conditions of exogenous gene mediated by liposome to transfect yak mammary epithelial cells in Vitro.[Method] Yak mammary epithelial cells were isolated and cultivated in Vitro by the methods of collagenase digestion and tissue adhesion.The expression of cytokeratin in yak mammary epithelial cell was detected by immunocytochemistry technique.With green fluorescence protein as the report gene,yak mammary epithelial cells were transfected with exogenous gene m...展开更多
Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticl...Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticles (SDION) and the feasibility of SDION used as a novel gene carrier for plasmid DNA in vitro. Methods: SDION were prepared by chemical coprecipitation and separated by gel filtration on Sephacryl S-300HR, characterized by TEM, laser scattering system and Vibrating Sample Magnetometer Signal Processor. The green fluorescent protein (pGFP-C2) plasmid DNA was used as target gene. SDION-pGFP-C2 conjugate compounds were produced by means of oxidoreduction reaction. The connection ratio of SDION and pGFP-C2 DNA was analyzed and evaluated by agarose electrophoresis and the concentration of pGFP-C2 in supernatant was measured. Using liposome as control, the transfection efficiency of SDION and liposome was respectively evaluated under fluorescence microscope in vitro. Results: The diameter of SDION ranges from 3 nm to 8 nm, the effective diameter was 59.2 nm and the saturation magnetization was 0.23 emu/g. After SDION were reasonably oxidized, SDION could connect with pGFP-C2 to a high degree. The transfection efficiency of SDION as gene carrier was higher than that of liposome. Conclusion: The successes in connecting SDION with pGFP-C2 plasmid by means of oxidoreduction reaction and in transferring pGFP-C2 gene into human bladder cancer BIU-87 cells in vitro provided the experimental evidence for the feasibility of SDION used as a novel gene carrier.展开更多
AIM： To investigate the effect of hepatitis B virus （HBV） X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS： The HBV X gene eukaryon expression vector pcDNA3-X was ...AIM： To investigate the effect of hepatitis B virus （HBV） X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS： The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in.three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group.RESULTS： HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferationcapacity was obviously lower in HepG2/pcDNA3-X cells （0.08910±0.003164） than in HepG2 （0.14410±0.004927） and HepG2/pcDNA3 cells （0.12150±0.007159） （P〈0.05 and P〈0.01）. Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells （980/2 000） than HepG2 （420/2 000）, HepG2/pcDNA3 cells （520/2 000） （P〈0.05 and P〈0.01）. Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）. Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells （P〈0.05 and P〈0.01）. Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）.CONCLUSION： HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.展开更多
AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS: KAI1 gene was transfected into pancreatic cancer cell line MiaP...AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS: KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane (Matrigel) assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student's two tailed t test.RESULTS: By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h (MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P < 0.05). Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells (MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P < 0.05). In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively (P < 0.05).CONCLUSION: High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting metastasis of pancreatic cancer after direct injection into pancreatic adenocarcinoma. These results show that the suppressed invasion and motor function of pancreatic cancer cells may be a key reason why the KAI1 gene controls pancreatic cancer cell metastasis.展开更多
AIM To observe the tumor inhibitory effects bytransfecting IL-6 cDNA into colon cancer cell lineHT-29 with retroviral vector pZIP cDNA.METHODS Human IL-6 gene was reconstructedin retrovirus vector and transfected into...AIM To observe the tumor inhibitory effects bytransfecting IL-6 cDNA into colon cancer cell lineHT-29 with retroviral vector pZIP cDNA.METHODS Human IL-6 gene was reconstructedin retrovirus vector and transfected into incasingcells PA317 by lipofectamine mediated method,the clones of the cells transferred with hlL-6were selected by G418,and targeted HT-29 cellswere infected with the virus granules secretedfrom PA317 and also selected by G418.Test genetranscription and expression level byhybridization,ELISA and MTT assay,etc.Analyze tumor inhibitory effects according to thecell growth curve,plating forming rate andtumorigenicity in nude mice.RESULT Successfully constructed andtransfected recombinant expressing vectorspZIPIL-6 cDNA and got positive transfected celllines.The colon cancer cell line(HT-29 IL-5)transfected with the hlL-5 gene by retroviralvector was established.The log proliferationperiod and the doubling time of this cell line wasbetween 4 to 7 days and 2.5 days according tothe direct cell count,the cell proliferation wasobviously inhibited with MTT assay,the platinginhibitory rate was 50% by plating efficiencytest.When HT-29 IL-6 cells were inoculated intothe nude mice subcutaneously,carcinogenicactivity of the solid tumor was found superior tothe control group and the size of tumor was notsignificantly enlarged.Injection of combinationvirus fluid containing 11.-6 gene intotransplantation tumors could inhibit the growthand development of the tumor.CONCLUSION IL-6 could inhibit the growth andproliferation of colon cancer cells by retroviralvector-mediated transduction.展开更多
AIM： To establish a rapid and convenient animal model with hepatitis B virus （HBV） replication.METHODS： A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, us...AIM： To establish a rapid and convenient animal model with hepatitis B virus （HBV） replication.METHODS： A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen （HBcAg） and hepatitis B surface antigen （HBsAg） in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen （HBeAg） was detected by enzyme- linked immunosorbent assay （ELISA）. Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid （polyIC） or phosphate-buffered saline （PBS）.RESULTS： After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS.CONCLUSION： A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.展开更多
AIM To investigate the hepatitis C virus(HCV)infection in the tissues of carcinoma ofextrahepatic bile duct and study theircorrelation.METHODS HCV NS5 protein and HCV RNA weredetected by labeled streptavidin biotin(LS...AIM To investigate the hepatitis C virus(HCV)infection in the tissues of carcinoma ofextrahepatic bile duct and study theircorrelation.METHODS HCV NS5 protein and HCV RNA weredetected by labeled streptavidin biotin(LSAB)method and in situ reverse transcriptionpolymerase chain reaction(IS-RT-PCR)insections of 51 cases of carcinoma ofextrahepatic bile duct and 34 cases of controlgroup(without malignant biliary disease).RESULTS In 51 cases of carcinoma ofextrahepatic bile duct,HCV NS5 protein wasdetected in 14(27.5%),which was clearlystained in the cytoplasm of cancer cell but not inthe nucleus or cell membrane.HCV RNA wasdetected in 18(35.4%),which was located inthe nucleus of cancer cell in 12 cases and in thecytoplasm in 6 cases.HCV NS5 protein and RNAcoexistence was found in 2 cases.In 34 cases ofcontrol group,HCV RNA was detected in 2(5.9%).HCV NS5 protein and RNA positive cellswere found either scattered or in clusters.CONCLUSION The prevalence of hepatitis C viral infection in the tissues of carcinoma ofextrahepatic bile duct was significantly higherthan in control group(X^2=9.808,P=0.002).The findings suggest a correlation between HCVinfection and carcinoma of extrahepatic bileduct,which is different from the traditionalviewpoint.HCV infection might be involved inthe development of carcinoma of extrahepaticbile duct.展开更多
AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS:...AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS: PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characterristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow-cytometry. RESULTS: PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population-doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P<0.01). The cloning efficiencies of DPC4+-SW620 cells (12%) were markedly lower than those of SW620 cells (69%) and PcDNA3.1-Sw620 cells (67%) (P<0.01). Compared with SW620 cells and PcDNA3.1-Sw620 cells, the Go-G1% of DPC4+-SW620 cells was obviously higher and the S% was markedly lower (P<0.05). Apoptosis rate of DPC4+-SW620 cells was significantly higher than that of SW620 cells and PcDNA3.1-SW620 cells. CONCLUSION: PcDNA3.1-DPC4 plasmid can be successfully re-constructed and stably transfected into human SW620cells, thereby the cells can steadily express Smad4. DPC4 protein may regulate proliferation of colorectal carcinoma cells by inhibiting cell growth and inducing cell apoptosis.展开更多
文摘Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and cell growth. Methods: MCT1 antisense gene recombinant vector was introduced into human lung cancer cell line A549 by electroporation. The transfected A549 cells resistant to G418 were selected. Positive clones were examined by using PCR. The changes of intracellular pH and lactate were examined with spec-trophotometric method. Cell growth was studied with cell growth curve. Results: Intracellular pH and lactate were remarkably decreased in the cells transfected pLXSN-MCT1 in comparison with A549 cells without transfection (P<0. 001). The growth of A549 cells transfected pLXSN-MCTl was also inhibited remarkably. Conclusion: MCT1 gene may play an important role in pHi regulation, lactate transportation and cell growth in tumor cells.
文摘Objective:The aim of this study was to study the changes of SGC-7901 cells transfecting small hairpin RNA(shRNA) targeted Bcl-2 and celecoxib in vitro.Methods:To use the effective siRNA targeted to Bcl-2 by Lipofectamine 2000,the rate of cell growth inhibition of all groups was detected by multiply-table tournament(MTT) method,apoptosis rate was examined by flow cytometry and the expression of Bcl-2 was assayed by Elisa.Results:After siRNA was transfected to SGC-7901 cells,the rate of cell growth inhibition was increased,the joint group was higher by flow cytometry and the expression of Bcl-2 was lower by Elisa.Conclusion:The growth of SGC-7901 cells which was transfected siRNA has been inhibited,and the sensitivity to celecoxib has been increased.
基金supported by National Natural Science Foundation of China(82104082)Natural Science Foundation of Qinghai Province(2024-ZJ-911).
文摘The recent commercialization of gene products has sparked significant interest in gene therapy,necessitating efficient and precise gene delivery via various vectors.Currently,viral vectors and lipid-based nanocarriers are the predominant choices and have been extensively investigated and reviewed.Beyond these vectors,polymeric nanocarriers also hold the promise in therapeutic gene delivery owing to their versatile functionalities,such as improving the stability,cellar uptake and endosomal escape of nucleic acid drugs,along with precise delivery to targeted tissues.This review presents a brief overview of the status quo of the emerging polymeric nanocarriers for therapeutic gene delivery,focusing on key cationic polymers,nanocarrier types,and preparation methods.It also highlights targeted diseases,strategies to improve delivery efficiency,and potential future directions in this research area.The review is hoped to inspire the development,optimization,and clinical translation of highly efficient polymeric nanocarriers for therapeutic gene delivery.
文摘BACKGROUND Mesenchymal stem cells(MSCs)are promising candidates for regenerative therapy due to their self-renewal capability,multilineage differentiation potential,and immunomodulatory effects.The molecular characteristics of MSCs are influenced by their location.Recently,epidural fat(EF)and EF-derived MSCs(EF-MSCs)have garnered attention due to their potential benefits to the spinal microenvironment and their high expression of neural SC markers.However,their clinical applications are limited due to cell senescence and limited accessibility of EF.Although many studies have attempted to establish an immortalized,stable SC line,the characteristics of immortalized EF-MSCs remain to be clarified.AIM To establish and analyze stable immortalized EF-MSCs.METHODS The phenotypes of EF-MSCs were analyzed using optical microscopy.Cell immortalization was performed using lentiviral vectors.The biomolecular characteristics of the cells were analyzed by immunoblotting,quantitative PCR,and proteomics.RESULTS The immortalized EF-MSCs demonstrated a significantly extended lifespan compared to the control group,with well-preserved adipogenic potential and SC surface marker expression.Introduction of human telomerase reverse transcriptase genes markedly increased the lifespan of EF-MSCs.Proteomics analysis revealed substantial increase in the expression of DNA replication pathway components in immortalized EF-MSCs.CONCLUSION Immortalized EF-MSCs exhibited significantly enhanced proliferative capacity,retained adipogenic potential,and upregulated the expression of DNA replication pathway components.
基金supported by the National Natural Science Foundation of China(U22A20374,52373273)National High Level Hospital Clinical Research Funding of China-Japan Friendship Hospital(Grant number:2024-NHLHCRF-JBGSWZ-02).
文摘With the deepening of epigenetic research,studies have shown that N6-methyladenosine(m^(6)A)is closely related to the development of rheumatoid arthritis(RA),but the mechanism is still unclear.In the study,we collected synovial tissues from normal controls and patients with osteoarthritis(OA)or RA.The levels of m^(6)A and inflammation were analyzed by immunofluorescence staining and western blotting.The roles of IGF2BP3 in cell proliferation and inflammatory activation were explored using transfection and RNA immunoprecipitation assays.IGF2BP3^(−/−)mice were generated and used to establish an arthritis mouse model by transferring serum from adult arthritis K/BxN mice.We found m^(6)A levels were markedly increased in RA patients and mouse models,and the expression of IGF2BP3 was upregulated in individuals with RA and related to the levels of inflammatory markers.IGF2BP3 played an important part in RA-fibroblast-like synoviocytes(FLS)by promoting cell proliferation,migration,invasion,inflammatory cytokine release and inhibiting autophagy.In addition,IGF2BP3 inhibited autophagy to reduce ROS production,thereby decreasing the inflammatory activation of macrophages.More importantly,RASGRF1-mediated mTORC1 activation played a crucial role in the ability of IGF2BP3 to promote cell proliferation and inflammatory activation.In an arthritis model of IGF2BP3^(−/−)mice,IGF2BP3 knockout inhibited RA-FLS proliferation and inflammatory infiltration,and further ameliorated RA joint injury.Our study revealed an important role for IGF2BP3 in RA progression.The targeted inhibition of IGF2BP3 reduced cell proliferation and inflammatory activation and limited RA development,providing a potential strategy for RA therapy.
基金Supported by the Key Research&Development Program of Shandong Province(No.2023 CXGC 010710)the National Natural Science Foundation of China(No.32273116)the Fundamental Research Funds for the Central Universities(No.202261023)。
文摘Fish cell line provide a useful tool for studies in virology and molecular biology.To establish a novel continuous marine fish cell line(EFF)from caudal fin tissue of brown-marbled grouper Epinephelus fuscoguttatus,its susceptibility to the crustacean covert mortality nodavirus(CMNV)was evaluated.The primary cell cultures were initiated first by incomplete digestion of fin tissue blocks with dispase and collagenase,and then the explant was cultured in L-15 medium supplemented with 20%fetal bovine serum,10%grouper muscle extract,and 20-ng/mL growth factors of basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF).The EFF cells were continuously passaged beyond 50 times in fibroblast-like morphology,and they grew well in L-15 medium supplemented with a lower concentration fetal bovine serum(10%)at 28℃ after passage 10,without muscle extract and the growth factors.In addition,their grouper origin was confirmed by chromosome analysis and cytochrome oxidase 1(CO 1)gene analysis of these EFF cells.Transfection experiment via lipofectamine 8000 indicated that the EFF cells had a high transfection potential with a transfection efficiency up to 32%.Cross-species viral susceptibility analysis showed that CMNV not only successfully infected the EFF cells as evidenced by obvious cytopathic effects like vacuolation,detachment and death of cells,but also multiplied in the EFF cells as indicated by the results of semi-quantitative RT-PCR in transmission electron microscopy.Therefore,the establishment of immortal EFF cell line provided a useful cell model for future works on the isolation,multiplication,and pathogenic mechanism of cross-species infection of CMNV as well as genetic manipulation.
基金This work was supported by the National Natural Science Foundation of China （No.81171829, No.51473152, and No.51573175） and the Fundamental Research hinds for the Central Universities （WK2060200012, WK3450000001）. We also thank Prof. Li-hua Yang and Prof. Ye-zi You at the University of Science and Technology of China （USTC） for their kind help ill providing experimental reagents and instrunlents.
文摘Chitosan （CS） is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains were scissioned under the γ-ray radiation, and the molecu- lar weight （MW） of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the molecular weight of CS decreased about an order of magnitude. The γ-ray-radiation-scissioned CS can effectively bind with plasmid （pEGFP） through complex coacervation method, forming pEGFP/γ-ray-radiation-scissioned CS complex particles with a size of 200-300 nm. The complex particles have good stability and little cytotoxicity. The in vitro gene transfection efficiencies of the pEGFP/γ-ray-radiation-scissioned CS complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector. This work provides a green and simple method on the preparation of CS-based gene vectors with high efficiency and biosafety.
文摘OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.
基金the Sasagawa Medical Fellowship from Japan-Sino Medical Association with funds from the Nippon Foundation.
文摘SHIP-1 is an SH2 domain containing inositol-5-phosphatase that appears to be a negative regulator of hematopoiesis. To the potential effects of SHIP-1 on MMP2 secretion and migration of cancer cells, three murine SHIP-1 mutants were made： △SH2-SHIP-1, △Ptase-SHIP-1, △Cter-SHIP-1. These mutant forms were subcloned as well as the wild type （WT） of murine SHIP-1 cDNA were subcloned into pcDNA3 expression vector, then transfected into and overexpressed SHIP-1 and its mutants in a Src-transformed 3Y1 cellline （SR3Y1）. The results showed that overexpression of wild type of SHIP-1 does not affect the MMP2 secretion in both SR3Y1 and 3Y1 cells, but can induce MMP9 secretion, while either WT SHIP-1, the SH2 domain, phosphatase domain, or C terminus deletion mutants could significantly block the MMP2 and MMP9 secretion in SR3Y1 cells and suppress cell invasion ability. The results confirmed SHIP-1 as a negative regulator for cell migration and invasion in transformed cells, and implied that it may function through each of its three domains.
基金Supported by National Science and Technology R&D Program during 11th 5-year Plan Period(2006BAD06A01)~~
文摘[Objective] This study was to improve the virus replication efficiency of full length infectious cDNA clones by making use of the ribozyme＇s self incision property.[Method] By employing three-step PCR,HDV ribozyme（HdvRz）cDNA was isolated,and cloned into the downstream flanking the genome of the porcine reproductive and respiratory syndrome virus,and into which the bovine growth hormone polyadenylation sequence（BGH）was inserted via enzyme digestion and ligation,yielding pAPRRS-HB.The newly constructed pAPRRS-HB was used to transfect MARC-145 cells,in which the N protein and non-structural protein（nsp2）were determined by indirect immunofluorescence assay after 72 h of expression;meanwhile the virus titer of cell supernatant was tested using TCID50 assay.[Result] pAPRRS-HB containing complete infectious PRRSV cDNA has been successfully developed,and it performed about 10-fold higher virus rescue rate than pAPRRS without the engineered ribozyme element.[Conclusion] The results laid a foundation for revealing the structure and function of PRRSV gene.
基金Supported by National Natural Science Foundation(30771550)~~
文摘[Objective] The aim of this study was to optimize conditions of exogenous gene mediated by liposome to transfect yak mammary epithelial cells in Vitro.[Method] Yak mammary epithelial cells were isolated and cultivated in Vitro by the methods of collagenase digestion and tissue adhesion.The expression of cytokeratin in yak mammary epithelial cell was detected by immunocytochemistry technique.With green fluorescence protein as the report gene,yak mammary epithelial cells were transfected with exogenous gene m...
基金This project was supported by a grant from the National Natural Science Foundation of China (No. 30271300).
文摘Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticles (SDION) and the feasibility of SDION used as a novel gene carrier for plasmid DNA in vitro. Methods: SDION were prepared by chemical coprecipitation and separated by gel filtration on Sephacryl S-300HR, characterized by TEM, laser scattering system and Vibrating Sample Magnetometer Signal Processor. The green fluorescent protein (pGFP-C2) plasmid DNA was used as target gene. SDION-pGFP-C2 conjugate compounds were produced by means of oxidoreduction reaction. The connection ratio of SDION and pGFP-C2 DNA was analyzed and evaluated by agarose electrophoresis and the concentration of pGFP-C2 in supernatant was measured. Using liposome as control, the transfection efficiency of SDION and liposome was respectively evaluated under fluorescence microscope in vitro. Results: The diameter of SDION ranges from 3 nm to 8 nm, the effective diameter was 59.2 nm and the saturation magnetization was 0.23 emu/g. After SDION were reasonably oxidized, SDION could connect with pGFP-C2 to a high degree. The transfection efficiency of SDION as gene carrier was higher than that of liposome. Conclusion: The successes in connecting SDION with pGFP-C2 plasmid by means of oxidoreduction reaction and in transferring pGFP-C2 gene into human bladder cancer BIU-87 cells in vitro provided the experimental evidence for the feasibility of SDION used as a novel gene carrier.
文摘AIM： To investigate the effect of hepatitis B virus （HBV） X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS： The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in.three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group.RESULTS： HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferationcapacity was obviously lower in HepG2/pcDNA3-X cells （0.08910±0.003164） than in HepG2 （0.14410±0.004927） and HepG2/pcDNA3 cells （0.12150±0.007159） （P〈0.05 and P〈0.01）. Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells （980/2 000） than HepG2 （420/2 000）, HepG2/pcDNA3 cells （520/2 000） （P〈0.05 and P〈0.01）. Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）. Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells （P〈0.05 and P〈0.01）. Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）.CONCLUSION： HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.
基金Grant-in-aid No. 39970344 and No. 30470798the National Nature Science Foundation, China in 1999 and 2004
文摘AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS: KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane (Matrigel) assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student's two tailed t test.RESULTS: By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h (MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P < 0.05). Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells (MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P < 0.05). In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively (P < 0.05).CONCLUSION: High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting metastasis of pancreatic cancer after direct injection into pancreatic adenocarcinoma. These results show that the suppressed invasion and motor function of pancreatic cancer cells may be a key reason why the KAI1 gene controls pancreatic cancer cell metastasis.
文摘AIM To observe the tumor inhibitory effects bytransfecting IL-6 cDNA into colon cancer cell lineHT-29 with retroviral vector pZIP cDNA.METHODS Human IL-6 gene was reconstructedin retrovirus vector and transfected into incasingcells PA317 by lipofectamine mediated method,the clones of the cells transferred with hlL-6were selected by G418,and targeted HT-29 cellswere infected with the virus granules secretedfrom PA317 and also selected by G418.Test genetranscription and expression level byhybridization,ELISA and MTT assay,etc.Analyze tumor inhibitory effects according to thecell growth curve,plating forming rate andtumorigenicity in nude mice.RESULT Successfully constructed andtransfected recombinant expressing vectorspZIPIL-6 cDNA and got positive transfected celllines.The colon cancer cell line(HT-29 IL-5)transfected with the hlL-5 gene by retroviralvector was established.The log proliferationperiod and the doubling time of this cell line wasbetween 4 to 7 days and 2.5 days according tothe direct cell count,the cell proliferation wasobviously inhibited with MTT assay,the platinginhibitory rate was 50% by plating efficiencytest.When HT-29 IL-6 cells were inoculated intothe nude mice subcutaneously,carcinogenicactivity of the solid tumor was found superior tothe control group and the size of tumor was notsignificantly enlarged.Injection of combinationvirus fluid containing 11.-6 gene intotransplantation tumors could inhibit the growthand development of the tumor.CONCLUSION IL-6 could inhibit the growth andproliferation of colon cancer cells by retroviralvector-mediated transduction.
基金Supported by the National Science Fund for Distinguished Young Scholars from the National Natural Science Foundation of China,No.30325036a grant from the National Natural Science Foundation of China,No.30571640
文摘AIM： To establish a rapid and convenient animal model with hepatitis B virus （HBV） replication.METHODS： A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen （HBcAg） and hepatitis B surface antigen （HBsAg） in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen （HBeAg） was detected by enzyme- linked immunosorbent assay （ELISA）. Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid （polyIC） or phosphate-buffered saline （PBS）.RESULTS： After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS.CONCLUSION： A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.
文摘AIM To investigate the hepatitis C virus(HCV)infection in the tissues of carcinoma ofextrahepatic bile duct and study theircorrelation.METHODS HCV NS5 protein and HCV RNA weredetected by labeled streptavidin biotin(LSAB)method and in situ reverse transcriptionpolymerase chain reaction(IS-RT-PCR)insections of 51 cases of carcinoma ofextrahepatic bile duct and 34 cases of controlgroup(without malignant biliary disease).RESULTS In 51 cases of carcinoma ofextrahepatic bile duct,HCV NS5 protein wasdetected in 14(27.5%),which was clearlystained in the cytoplasm of cancer cell but not inthe nucleus or cell membrane.HCV RNA wasdetected in 18(35.4%),which was located inthe nucleus of cancer cell in 12 cases and in thecytoplasm in 6 cases.HCV NS5 protein and RNAcoexistence was found in 2 cases.In 34 cases ofcontrol group,HCV RNA was detected in 2(5.9%).HCV NS5 protein and RNA positive cellswere found either scattered or in clusters.CONCLUSION The prevalence of hepatitis C viral infection in the tissues of carcinoma ofextrahepatic bile duct was significantly higherthan in control group(X^2=9.808,P=0.002).The findings suggest a correlation between HCVinfection and carcinoma of extrahepatic bileduct,which is different from the traditionalviewpoint.HCV infection might be involved inthe development of carcinoma of extrahepaticbile duct.
文摘AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS: PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characterristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow-cytometry. RESULTS: PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population-doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P<0.01). The cloning efficiencies of DPC4+-SW620 cells (12%) were markedly lower than those of SW620 cells (69%) and PcDNA3.1-Sw620 cells (67%) (P<0.01). Compared with SW620 cells and PcDNA3.1-Sw620 cells, the Go-G1% of DPC4+-SW620 cells was obviously higher and the S% was markedly lower (P<0.05). Apoptosis rate of DPC4+-SW620 cells was significantly higher than that of SW620 cells and PcDNA3.1-SW620 cells. CONCLUSION: PcDNA3.1-DPC4 plasmid can be successfully re-constructed and stably transfected into human SW620cells, thereby the cells can steadily express Smad4. DPC4 protein may regulate proliferation of colorectal carcinoma cells by inhibiting cell growth and inducing cell apoptosis.
