ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine （N6-mA）, a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the...ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine （N6-mA）, a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells （MSCs） was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBHl-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation.展开更多
AFF1 and AFF4 belong to the AFF （AF4/FMR2） family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b （P-TEFb） and ELL1/2, in su...AFF1 and AFF4 belong to the AFF （AF4/FMR2） family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b （P-TEFb） and ELL1/2, in super elongation complexes （SECs）. Both AFF1 and AFF4 regulate gene transcription through elongation and chromatln remodeling. However, their function in the osteogenic differentiation of mesenchymal stem cells （MSCs） is unknown. In this study, we show that small interfering RNA （siRNA）-mediated depletion of AFF1 in human MSCs leads to increased alkaline phosphatase （ALP） activity, enhanced mineralization and upregulated expression of osteogenic-related genes. On the contrary, depletion of AFF4 significantly inhibits the osteogenic potential of MSCs. In addition, we confirm that overexpression of AFF1 and AFF4 differentially affects osteogenic differentiation in vitro and MSC-mediated bone formation in vivo. Mechanistically, we find that AFFI regulates the expression of DKK1 via binding to its promoter region. Depletion of DKK1 in HA-AFFl-overexpressing MSCs abrogates the impairment of osteogenic differentiation. Moreover, we detect that AFF4 is enriched in the promoter region of ID1. AFF4 knockdown blunts the BRE luciferase activity, SP7 expression and ALP activity induced by BMP2 treatment. In conclusion, our data indicate that AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.AFF1 and AFF4 belong to the AFF （AF4/FMR2） family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b （P-TEFb） and ELL1/2, in super elongation complexes （SECs）. Both AFFI and AFF4 regulate gene transcription through elongation and chromatln remodeling. However, their function in the osteogenic differentiation of mesenchymal stem cells （MSCs） is unknown. In this study, we show that small interfering RNA （siRNA）-mediated depletion of AFF1 in human MSCs leads to increased alkaline phosphatase （ALP） activity, enhanced mineralization and upregulated expression of osteogenic-related genes. On the contrary, depletion of AFF4 significantly inhibits the osteogenic potential of MSCs. In addition, we confirm that overexpression of AFF1 and AFF4 differentially affects osteogenic differentiation in vitro and MSC-mediated bone formation in vivo. Mechanistically, we find that AFFI regulates the expression of DKK1 via binding to its promoter region. Depletion of DKK1 in HA-AFFl-overexpressing MSCs abrogates the impairment of osteogenic differentiation. Moreover, we detect that AFF4 is enriched in the promoter region of ID1. AFF4 knockdown blunts the BRE luciferase activity, SP7 expression and ALP activity induced by BMP2 treatment. In conclusion, our data indicate that AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.展开更多
BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution; however, hippocampal ...BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution; however, hippocampal astrocyte conditioned medium (HCAM) can induce in vitro differentiation from hADASC to neuron-like cells is still unclear. OBJECTIVE: To observe whether HCAM can induce in vitro differentiation from hADASC to neuron-like cells. DESIGN: Randomized control study. SETTING: Department of Neurology, Taixing People's Hospital; Central Laboratory, North China Coal Medical College. MATERIALS: Donor of adipose tissue was donated by female volunteers suffering from caesarean section in the department of obstetrics & gynecology in our hospital and aged 20-35 years. Adipose tissue was collected from subcutaneous tissue of abdomen during the operation. In addition, 8 male newborn Wistar rats within 24 hours with average body mass of 20 g were provided by Animal Institute of Chinese Academy of Medical Sciences. Rabbit-anti-human Nestin polyclonal antibody, rabbit-anti-human glial fibriliary acidic protein (GFAP) polyclonal antibody, rabbit-anti-human neuro-specific enolase polyclonal antibody and mouse-anti-human microtubal associated protein 2 (MAP-2) polyclonal antibody were provided by Wuhan Boster Company. METHODS: The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005. hADASC was cultured with HCAM and its growth and morphological changes were observed under inverted phase contrast microscope. Immunocytochemistry, immunofluorescence and Western blotting were used to evaluate the expressions of Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. MAIN OUTCOME MEASURES: Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. RESULTS: On the 3rd day of culture, partial hADASC started deformation from slender shuttle-shape cells to neuron-like cells. It suggested that cells stretched out apophysis, which were mainly double-pole or multiple-pole cells. Five days later, immunohistochemical detection suggested that expression of Nestin (10.5±0.037) was found out in cells; meanwhile, expressions of GFAP (38.4±0.052) and neuro-specific enolase (NSE) (15.7±0.023) were also found out in cells; however, expression of MAP-2 was not observed. Western blot indicated that, 5 days after effect of HCAM, Nestin was found out in hADASC; meanwhile, expressions of GFAP and neuro-specific enolase were also found out; however, expression of MAP-2 was not observed. CONCLUSION: HCAM can induce the differentiation from hADASC to neuron-like cells in vitro.展开更多
Human adipose-derived stem cells(hASCs)are a promising cell type for bone tissue regeneration.Circular RNAs(circRNAs)have been shown to play a critical role in regulating various cell differentiation and involve in me...Human adipose-derived stem cells(hASCs)are a promising cell type for bone tissue regeneration.Circular RNAs(circRNAs)have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis.However,how circRNAs regulate hASCs in osteogenesis is still unclear.Herein,we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs.Knockdown of circ_0003204 by si RNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs.We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p.We predicted and confirmed that miR-370-3p had targets in the 3′-UTR of HDAC4 m RNA.The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis.Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model,while overexpression of circ_0003204 inhibited bone defect repair.Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.展开更多
For providing some experimental basis in establishing malignant phenotypic reversed indexes of gastric carcinoma cells, human gastric adenocar-cinoma cell line MGc80-3 was induced by dBcAMP in vitro to appraise the ef...For providing some experimental basis in establishing malignant phenotypic reversed indexes of gastric carcinoma cells, human gastric adenocar-cinoma cell line MGc80-3 was induced by dBcAMP in vitro to appraise the effect of gastric carcinoma cell differentiation by chemical inducers.Under light microscope, MGc80-3 cells, after treated with 1 mM dBcAMP, tended to be flat and disperse, and their volume gradually enlarged, with their uncleus relatively smaller and their shape rather regular. Morphological changes, like norma differentiated epithelial cells, were observed. The cells attached firmly, grew slowly, their growth curve showed inhibitory rate amounted to 52.87%, and cellular division exponent displayed their peak value 1.5 times less than that of MGc80-3 cells. It was clear that dBcAMP could effectively inhibit the multiplication activity of MGc80-3 cells. After dBcAMP treatment, remarkable changes of cell surface charges was indicated by cell electrophoresis, the ratio dropped to 3.043 from 3.988, and their re-tardant ratio reached up to 31.2%. cAMP content in cells after this treatment, detected by cAMP and cGMP radioimmunoassay, was enhanced by 2.42 times, and cAMP/cGMP ratio, by 1.73 times. Thus, cAMP level within MGc80-3 cells was raised obviously by dBcAMP. Heterotransplantation experiments showed that tuntorigenic rate of MGc80-5 cells (transplanted subcutaneously to BALB/c mice) amounted to 100%, and that of the cells after this treatment was only 5.6%. Their tumorigenic ability was extremely reduced.These results confirmed that dBcAMP was able to change malignant phenotypic characteristics of MGc80-3 cells and produce a reversed alteration: Thus, it has a remarkable inductive effect in differentiating gastric carcinoma cells. All these characteristics were also considered as the reference indexes in appraising reversed effect for the homologous cancer cells.展开更多
Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural dif...Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.展开更多
Objective To explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMCs) induced by growth differentiation ...Objective To explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMCs) induced by growth differentiation factor-5 (GDF-5)展开更多
Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in v...Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in vitro culture.展开更多
Human neural stem cells（h NSCs） are a useful tool to assess the developmental effects of various environmental contaminants; however, the application of h NSCs to evaluate water disinfection byproducts（DBPs） is sc...Human neural stem cells（h NSCs） are a useful tool to assess the developmental effects of various environmental contaminants; however, the application of h NSCs to evaluate water disinfection byproducts（DBPs） is scarce. Comprehensive toxicological results are essential to the prioritization of DBPs for further testing and regulation. Therefore, this study examines the effects of DBPs on the proliferation and differentiation of h NSCs. Prior to DBP treatment, characteristic protein markers of h NSCs from passages 3 to 6 were carefully examined and it was determined that h NSCs passaged 3 or 4 times maintained stem cell characteristics and can be used for DBP analysis. Two regulated DBPs, monobromoacetic acid（BAA） and monochloroacetic acid（CAA）, and two emerging DBPs, 2,6-dibromo-1,4-benzoquinone（2,6-DBBQ） and 2,6-dichloro-1,4-benzoquinone（2,6-DCBQ）, were chosen for h NSC treatment. Both 2,6-DBBQ and 2,6-DCBQ induced cell cycle arrest at S-phase at concentrations up to 1 μmol/L. Comparatively, BAA and CAA at 0.5 μmol/L affected neural differentiation. These results suggest DBP-dependent effects on h NSC proliferation and differentiation. The DBP-induced cell cycle arrest and inhibition of normal h NSC differentiation demonstrate the need to assess the developmental neurotoxicity of DBPs.展开更多
The phenomenon of adult neurogenesis is now an accepted occurrence in mammals and also in humans.At least two discrete places house stem cells for generation of neurons in adult brain. These are olfactory system and t...The phenomenon of adult neurogenesis is now an accepted occurrence in mammals and also in humans.At least two discrete places house stem cells for generation of neurons in adult brain. These are olfactory system and the hippocampus. In animals, newly generated neurons have been directly or indirectly demonstrated to generate a significant amount of new neurons to have a functional role. However, the data in humans on the extent of this process is still scanty and such as difficult to comprehend its functional role in humans. This paper explores the available data on as extent of adult hippocampal neurogenesis in humans and makes comparison to animal data.展开更多
BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human gr...BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor （rhG-MCSF） and recombinant human interleukin-4 （rhlL-4） can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJEETIVE： To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN ： Open experiment SEI-FING： Department of Pediatrics, the Medical School Hospital of Qingdao University MATERIALS ： The study was carried out in the Shandong Provincial Key Laboratory （Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University） during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments： type 3111 CO2 incubator （Forma Scientific, USA）, type 550 ELISA Reader （Bio-Rad, USA）. Main reagents： neuroblastoma cell line SK-N-SH （Shanghai Institute of Life Science, Chinese Academy of Sciences）, RPMI-1640 culture fluid and fetal bovine serum （Hyclone）, rhlL-4 （Promega, USA）, rhG-MCSF （Harbin Pharmaceutic Group Bioengineering Co.Ltd）, rat anti-human CDla monoclonal antibody and FITC-labeled rabbit anti-rat IgG （Xiehe Stem cell Gene Engineering Co.Ltd）. METHODS： ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhlL-4. ② Different concentrations of dendritic cells[ dendritic cells： neuroblastoma cells=20：1,50：1,100：1 （2×10^8 L^-1,5×10^8 L^-1,1×10^9 L^-1）], 1×10^9 L^-1 T cells and 1×10^7 L^-1 neuroblastoma cells were added in the experimental group. 1 ×10^9 L^-1 T cells and 1 ×10^7 L^-1 neuroblastoma cells were added in the control group. ③ Main surface marker CDla molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a^＋ cells. ④Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect（%）= （1-A experimentat well-A effector cell /A target cell well）×100%.⑤The expenmental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES： ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a^＋ cellular expression rate. ③Lethal effect of dendntic cells on neuroblastoma cells. RESULTS： ①Morphological characters of dendritic cells in the process of induction and differentiation： On the 15^th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhlL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a^＋ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells： Lethal effect of dendritic cells stimulated T cells in each experimental group （ dendritic cells： neuroblastoma cells=100：1,50：1, 20：1 respectively） on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41 ）%, （30.92±5.27）%,（33.57±5.35）%,（26.23±5.20）%, t=3.51,2.98,4.24, P〈 0.01 ）; But the lethal effect of dendntic cells on neuroblastoma was significantly lower when their ratio was 100：1 and 50：1 in comparison with 20：1 （t=2.01,2.36, P 〈 0.05）, and no significant difference in lethal effect existed between the ratio at 100：1 and 50：1 （t=0.06,P 〉 0.05）. CONCLUSION： Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.展开更多
BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. H...BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.展开更多
Introduction Atherosclerosis is a potentially life-threatening disease of large arteries that is strongly associated with systemic risk factors such as hypercholesterolemia,hypertension,smoking,and diabetes. However,a...Introduction Atherosclerosis is a potentially life-threatening disease of large arteries that is strongly associated with systemic risk factors such as hypercholesterolemia,hypertension,smoking,and diabetes. However,atherosclerosis develops as a展开更多
Background:Habitual physical activity is known to support cardiovascular health.However,when intensive exercise is performed for long durations,it can negatively affect the cardiovascular system.We evaluated the exerc...Background:Habitual physical activity is known to support cardiovascular health.However,when intensive exercise is performed for long durations,it can negatively affect the cardiovascular system.We evaluated the exercise-induced physiological responses of cardiac markers in male marathon runners based on basal plasma trimethylamine-N-oxide(TMAO)levels,a metabolite related to major adverse cardiovascular events.Methods:Blood samples from 28 marathon runners were collected two weeks before the marathon run(baseline),after finishing the race(post-marathon),and two weeks after the race(2 weeks post-marathon).Serum cardiac troponin I(c Tn I),endothelin-1(ET-1),galectin-3,pentraxin-3,human growth differentiation factor 15,and neopterin were determined by the enzyme immunoassay method.Plasma trimethylamine(TMA)and TMAO were measured by the ultra-high-performance liquid chromatography-mass spectrometry method.Results:Running a marathon increased levels of circulating biomarkers.A greater post-marathon elevation of cTn I and ET-1 was associated with baseline plasma TMAO levels(R=0.400,p=0.035 and R=0.476,p=0.012,respectively).Moreover,we observed an increase in post-marathon TMA levels.Conclusion:Greater post-marathon elevation of cTn I and ET-1 was associated with higher baseline plasma TMAO levels.Therefore,TMAO could potentially serve as a new marker in assessing the response of cardiovascular stress biomarkers to marathon running.展开更多
The accumulation of advanced glycation end products(AGEs)plays a crucial role in chronic inflammation and delayed wound healing in individuals with diabetes.In this context,fibronectin has been identified as a crucial...The accumulation of advanced glycation end products(AGEs)plays a crucial role in chronic inflammation and delayed wound healing in individuals with diabetes.In this context,fibronectin has been identified as a crucial protein that promotes the differentiation of human periodontal ligament stem cells(hPDLSCs)into myofibroblasts,which play a vital role in the repair of diabetic skin ulcers.This process is intimately associated with the integrinβ1 receptor and the NF-κB signaling pathway,both crucial for cellular responses to fibronectin.To validate our hypothesis,we expressed rhFN1024,a recombinant protein containing the integrinβ1 affinity-binding domain from human fibronectin segments 12–14.This protein was used to formulate a hydrogel for hPDLSCs.rhFN1024's binding affinity to integrinβ1 was confirmed by molecular docking and the cellular thermal shift assay(CETSA).We developed sh-ITGB1-hPDLSCs with stable ITGB1 knockdown using shRNA-ITGB1 and compared their proliferation,migration and adhesion to wildtype hPDLSCs.Morphological changes were observed via SEM,andα-SMA expression levels were measured in AGEs-damaged hPDLSCs.We created full-thickness wound models in diabetic mice to assess pharmacodynamics.The study showed that rhFN1024 stimulated hPDLSCs differentiation into myofibroblasts by boosting ITGB1 expression.rhFN1024 also reduced AGEs'negative effects on hPDLSCs,as seen through SEM analysis andα-SMA levels.In full-thickness wound models,hPDLSCs and rhFN1024 accelerated re-epithelialization and collagen synthesis.rhFN1024 is proposed to interact with the ITGB1 receptor on hPDLSCs,activating the NF-κB pathway to neutralize AGEs-induced pro-inflammatory cytokines.This study suggests rhFN1024 as a potential biomedical material for tissue repair.展开更多
基金supported by grants from the National Natural Science Foundation of China (No.81271178 and 81470777)
文摘ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine （N6-mA）, a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells （MSCs） was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBHl-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation.
基金supported by grants from the National Natural Science Foundation of China(NSFC,81722014,81571001,81500354,and 81621062)Sichuan Province Science and Technology Innovation Team Program(2017TD0016)State Key Laboratory of Oral Diseases(SKLOD201704)
文摘AFF1 and AFF4 belong to the AFF （AF4/FMR2） family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b （P-TEFb） and ELL1/2, in super elongation complexes （SECs）. Both AFF1 and AFF4 regulate gene transcription through elongation and chromatln remodeling. However, their function in the osteogenic differentiation of mesenchymal stem cells （MSCs） is unknown. In this study, we show that small interfering RNA （siRNA）-mediated depletion of AFF1 in human MSCs leads to increased alkaline phosphatase （ALP） activity, enhanced mineralization and upregulated expression of osteogenic-related genes. On the contrary, depletion of AFF4 significantly inhibits the osteogenic potential of MSCs. In addition, we confirm that overexpression of AFF1 and AFF4 differentially affects osteogenic differentiation in vitro and MSC-mediated bone formation in vivo. Mechanistically, we find that AFFI regulates the expression of DKK1 via binding to its promoter region. Depletion of DKK1 in HA-AFFl-overexpressing MSCs abrogates the impairment of osteogenic differentiation. Moreover, we detect that AFF4 is enriched in the promoter region of ID1. AFF4 knockdown blunts the BRE luciferase activity, SP7 expression and ALP activity induced by BMP2 treatment. In conclusion, our data indicate that AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.AFF1 and AFF4 belong to the AFF （AF4/FMR2） family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b （P-TEFb） and ELL1/2, in super elongation complexes （SECs）. Both AFFI and AFF4 regulate gene transcription through elongation and chromatln remodeling. However, their function in the osteogenic differentiation of mesenchymal stem cells （MSCs） is unknown. In this study, we show that small interfering RNA （siRNA）-mediated depletion of AFF1 in human MSCs leads to increased alkaline phosphatase （ALP） activity, enhanced mineralization and upregulated expression of osteogenic-related genes. On the contrary, depletion of AFF4 significantly inhibits the osteogenic potential of MSCs. In addition, we confirm that overexpression of AFF1 and AFF4 differentially affects osteogenic differentiation in vitro and MSC-mediated bone formation in vivo. Mechanistically, we find that AFFI regulates the expression of DKK1 via binding to its promoter region. Depletion of DKK1 in HA-AFFl-overexpressing MSCs abrogates the impairment of osteogenic differentiation. Moreover, we detect that AFF4 is enriched in the promoter region of ID1. AFF4 knockdown blunts the BRE luciferase activity, SP7 expression and ALP activity induced by BMP2 treatment. In conclusion, our data indicate that AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.
文摘BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution; however, hippocampal astrocyte conditioned medium (HCAM) can induce in vitro differentiation from hADASC to neuron-like cells is still unclear. OBJECTIVE: To observe whether HCAM can induce in vitro differentiation from hADASC to neuron-like cells. DESIGN: Randomized control study. SETTING: Department of Neurology, Taixing People's Hospital; Central Laboratory, North China Coal Medical College. MATERIALS: Donor of adipose tissue was donated by female volunteers suffering from caesarean section in the department of obstetrics & gynecology in our hospital and aged 20-35 years. Adipose tissue was collected from subcutaneous tissue of abdomen during the operation. In addition, 8 male newborn Wistar rats within 24 hours with average body mass of 20 g were provided by Animal Institute of Chinese Academy of Medical Sciences. Rabbit-anti-human Nestin polyclonal antibody, rabbit-anti-human glial fibriliary acidic protein (GFAP) polyclonal antibody, rabbit-anti-human neuro-specific enolase polyclonal antibody and mouse-anti-human microtubal associated protein 2 (MAP-2) polyclonal antibody were provided by Wuhan Boster Company. METHODS: The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005. hADASC was cultured with HCAM and its growth and morphological changes were observed under inverted phase contrast microscope. Immunocytochemistry, immunofluorescence and Western blotting were used to evaluate the expressions of Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. MAIN OUTCOME MEASURES: Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. RESULTS: On the 3rd day of culture, partial hADASC started deformation from slender shuttle-shape cells to neuron-like cells. It suggested that cells stretched out apophysis, which were mainly double-pole or multiple-pole cells. Five days later, immunohistochemical detection suggested that expression of Nestin (10.5±0.037) was found out in cells; meanwhile, expressions of GFAP (38.4±0.052) and neuro-specific enolase (NSE) (15.7±0.023) were also found out in cells; however, expression of MAP-2 was not observed. Western blot indicated that, 5 days after effect of HCAM, Nestin was found out in hADASC; meanwhile, expressions of GFAP and neuro-specific enolase were also found out; however, expression of MAP-2 was not observed. CONCLUSION: HCAM can induce the differentiation from hADASC to neuron-like cells in vitro.
基金supported by grants from the National Natural Science Foundation of China(82071150,82170934,81870743,8190104 and 82171001)。
文摘Human adipose-derived stem cells(hASCs)are a promising cell type for bone tissue regeneration.Circular RNAs(circRNAs)have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis.However,how circRNAs regulate hASCs in osteogenesis is still unclear.Herein,we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs.Knockdown of circ_0003204 by si RNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs.We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p.We predicted and confirmed that miR-370-3p had targets in the 3′-UTR of HDAC4 m RNA.The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis.Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model,while overexpression of circ_0003204 inhibited bone defect repair.Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.
文摘For providing some experimental basis in establishing malignant phenotypic reversed indexes of gastric carcinoma cells, human gastric adenocar-cinoma cell line MGc80-3 was induced by dBcAMP in vitro to appraise the effect of gastric carcinoma cell differentiation by chemical inducers.Under light microscope, MGc80-3 cells, after treated with 1 mM dBcAMP, tended to be flat and disperse, and their volume gradually enlarged, with their uncleus relatively smaller and their shape rather regular. Morphological changes, like norma differentiated epithelial cells, were observed. The cells attached firmly, grew slowly, their growth curve showed inhibitory rate amounted to 52.87%, and cellular division exponent displayed their peak value 1.5 times less than that of MGc80-3 cells. It was clear that dBcAMP could effectively inhibit the multiplication activity of MGc80-3 cells. After dBcAMP treatment, remarkable changes of cell surface charges was indicated by cell electrophoresis, the ratio dropped to 3.043 from 3.988, and their re-tardant ratio reached up to 31.2%. cAMP content in cells after this treatment, detected by cAMP and cGMP radioimmunoassay, was enhanced by 2.42 times, and cAMP/cGMP ratio, by 1.73 times. Thus, cAMP level within MGc80-3 cells was raised obviously by dBcAMP. Heterotransplantation experiments showed that tuntorigenic rate of MGc80-5 cells (transplanted subcutaneously to BALB/c mice) amounted to 100%, and that of the cells after this treatment was only 5.6%. Their tumorigenic ability was extremely reduced.These results confirmed that dBcAMP was able to change malignant phenotypic characteristics of MGc80-3 cells and produce a reversed alteration: Thus, it has a remarkable inductive effect in differentiating gastric carcinoma cells. All these characteristics were also considered as the reference indexes in appraising reversed effect for the homologous cancer cells.
基金supported by the National Natural Science Foundation of China,No.31340024
文摘Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.
文摘Objective To explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMCs) induced by growth differentiation factor-5 (GDF-5)
文摘Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in vitro culture.
基金supported by funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, Alberta Innovates-Energy and Environmental Solutions, and Alberta Health
文摘Human neural stem cells（h NSCs） are a useful tool to assess the developmental effects of various environmental contaminants; however, the application of h NSCs to evaluate water disinfection byproducts（DBPs） is scarce. Comprehensive toxicological results are essential to the prioritization of DBPs for further testing and regulation. Therefore, this study examines the effects of DBPs on the proliferation and differentiation of h NSCs. Prior to DBP treatment, characteristic protein markers of h NSCs from passages 3 to 6 were carefully examined and it was determined that h NSCs passaged 3 or 4 times maintained stem cell characteristics and can be used for DBP analysis. Two regulated DBPs, monobromoacetic acid（BAA） and monochloroacetic acid（CAA）, and two emerging DBPs, 2,6-dibromo-1,4-benzoquinone（2,6-DBBQ） and 2,6-dichloro-1,4-benzoquinone（2,6-DCBQ）, were chosen for h NSC treatment. Both 2,6-DBBQ and 2,6-DCBQ induced cell cycle arrest at S-phase at concentrations up to 1 μmol/L. Comparatively, BAA and CAA at 0.5 μmol/L affected neural differentiation. These results suggest DBP-dependent effects on h NSC proliferation and differentiation. The DBP-induced cell cycle arrest and inhibition of normal h NSC differentiation demonstrate the need to assess the developmental neurotoxicity of DBPs.
基金supported by the Consortium for Advanced Research Training in Africa(CARTA).CARTA is jointly led by the African Population and Health Research Center(APHRC)and the University of the Witwatersrandfunded by the Wellcome Trust(UK)(Grant No.087547/Z/08/Z)+5 种基金the Department for International Development(DfID)under the Development Partnerships in Higher Education(DelPHE),the Carnegie Corporation of New York(Grant No.B 8606)the Ford Foundation(Grant No.11000399)Google.Org(Grant No.191994)Sida(Grant No.54100029)Mac Arthur Foundation(Grant No.10-95915-000-INP)British Council
文摘The phenomenon of adult neurogenesis is now an accepted occurrence in mammals and also in humans.At least two discrete places house stem cells for generation of neurons in adult brain. These are olfactory system and the hippocampus. In animals, newly generated neurons have been directly or indirectly demonstrated to generate a significant amount of new neurons to have a functional role. However, the data in humans on the extent of this process is still scanty and such as difficult to comprehend its functional role in humans. This paper explores the available data on as extent of adult hippocampal neurogenesis in humans and makes comparison to animal data.
文摘BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor （rhG-MCSF） and recombinant human interleukin-4 （rhlL-4） can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJEETIVE： To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN ： Open experiment SEI-FING： Department of Pediatrics, the Medical School Hospital of Qingdao University MATERIALS ： The study was carried out in the Shandong Provincial Key Laboratory （Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University） during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments： type 3111 CO2 incubator （Forma Scientific, USA）, type 550 ELISA Reader （Bio-Rad, USA）. Main reagents： neuroblastoma cell line SK-N-SH （Shanghai Institute of Life Science, Chinese Academy of Sciences）, RPMI-1640 culture fluid and fetal bovine serum （Hyclone）, rhlL-4 （Promega, USA）, rhG-MCSF （Harbin Pharmaceutic Group Bioengineering Co.Ltd）, rat anti-human CDla monoclonal antibody and FITC-labeled rabbit anti-rat IgG （Xiehe Stem cell Gene Engineering Co.Ltd）. METHODS： ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhlL-4. ② Different concentrations of dendritic cells[ dendritic cells： neuroblastoma cells=20：1,50：1,100：1 （2×10^8 L^-1,5×10^8 L^-1,1×10^9 L^-1）], 1×10^9 L^-1 T cells and 1×10^7 L^-1 neuroblastoma cells were added in the experimental group. 1 ×10^9 L^-1 T cells and 1 ×10^7 L^-1 neuroblastoma cells were added in the control group. ③ Main surface marker CDla molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a^＋ cells. ④Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect（%）= （1-A experimentat well-A effector cell /A target cell well）×100%.⑤The expenmental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES： ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a^＋ cellular expression rate. ③Lethal effect of dendntic cells on neuroblastoma cells. RESULTS： ①Morphological characters of dendritic cells in the process of induction and differentiation： On the 15^th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhlL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a^＋ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells： Lethal effect of dendritic cells stimulated T cells in each experimental group （ dendritic cells： neuroblastoma cells=100：1,50：1, 20：1 respectively） on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41 ）%, （30.92±5.27）%,（33.57±5.35）%,（26.23±5.20）%, t=3.51,2.98,4.24, P〈 0.01 ）; But the lethal effect of dendntic cells on neuroblastoma was significantly lower when their ratio was 100：1 and 50：1 in comparison with 20：1 （t=2.01,2.36, P 〈 0.05）, and no significant difference in lethal effect existed between the ratio at 100：1 and 50：1 （t=0.06,P 〉 0.05）. CONCLUSION： Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.
基金supported by grants from the Chinese High-Tech Research&Development(863)Program(2013AA020102 and 2012AA020204)Science Fund for Creative Research Groups of the National Natural Science Foundation of China(81121002)+3 种基金Fundamental Research Funds for the Central Universities(2014XZZX008 and 2014FZA7010)Zhejiang CTM Science and Technology Project(2011ZB061)Zhejiang Health Science Foundation(2016KYA148)the National Health and Medical Research Council of Australia and Cancer Council of Western Australia
文摘BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.
基金support from National Heart Lung and Blood Institute Grants P50-HL56985 and R01-HL61794
文摘Introduction Atherosclerosis is a potentially life-threatening disease of large arteries that is strongly associated with systemic risk factors such as hypercholesterolemia,hypertension,smoking,and diabetes. However,atherosclerosis develops as a
基金supported by the Polish Ministry of Science and Higher Education,grant no.2/566516/SPUB/SP/2023。
文摘Background:Habitual physical activity is known to support cardiovascular health.However,when intensive exercise is performed for long durations,it can negatively affect the cardiovascular system.We evaluated the exercise-induced physiological responses of cardiac markers in male marathon runners based on basal plasma trimethylamine-N-oxide(TMAO)levels,a metabolite related to major adverse cardiovascular events.Methods:Blood samples from 28 marathon runners were collected two weeks before the marathon run(baseline),after finishing the race(post-marathon),and two weeks after the race(2 weeks post-marathon).Serum cardiac troponin I(c Tn I),endothelin-1(ET-1),galectin-3,pentraxin-3,human growth differentiation factor 15,and neopterin were determined by the enzyme immunoassay method.Plasma trimethylamine(TMA)and TMAO were measured by the ultra-high-performance liquid chromatography-mass spectrometry method.Results:Running a marathon increased levels of circulating biomarkers.A greater post-marathon elevation of cTn I and ET-1 was associated with baseline plasma TMAO levels(R=0.400,p=0.035 and R=0.476,p=0.012,respectively).Moreover,we observed an increase in post-marathon TMA levels.Conclusion:Greater post-marathon elevation of cTn I and ET-1 was associated with higher baseline plasma TMAO levels.Therefore,TMAO could potentially serve as a new marker in assessing the response of cardiovascular stress biomarkers to marathon running.
基金funded by the National Key Research and Development Program of China(2022YFC2403102)Kea-Area Research and Development Program of Guangdong Province,China(2022B1111080007)Guangzhou Research and Development Plan in Key Fields,China(202103030003).
文摘The accumulation of advanced glycation end products(AGEs)plays a crucial role in chronic inflammation and delayed wound healing in individuals with diabetes.In this context,fibronectin has been identified as a crucial protein that promotes the differentiation of human periodontal ligament stem cells(hPDLSCs)into myofibroblasts,which play a vital role in the repair of diabetic skin ulcers.This process is intimately associated with the integrinβ1 receptor and the NF-κB signaling pathway,both crucial for cellular responses to fibronectin.To validate our hypothesis,we expressed rhFN1024,a recombinant protein containing the integrinβ1 affinity-binding domain from human fibronectin segments 12–14.This protein was used to formulate a hydrogel for hPDLSCs.rhFN1024's binding affinity to integrinβ1 was confirmed by molecular docking and the cellular thermal shift assay(CETSA).We developed sh-ITGB1-hPDLSCs with stable ITGB1 knockdown using shRNA-ITGB1 and compared their proliferation,migration and adhesion to wildtype hPDLSCs.Morphological changes were observed via SEM,andα-SMA expression levels were measured in AGEs-damaged hPDLSCs.We created full-thickness wound models in diabetic mice to assess pharmacodynamics.The study showed that rhFN1024 stimulated hPDLSCs differentiation into myofibroblasts by boosting ITGB1 expression.rhFN1024 also reduced AGEs'negative effects on hPDLSCs,as seen through SEM analysis andα-SMA levels.In full-thickness wound models,hPDLSCs and rhFN1024 accelerated re-epithelialization and collagen synthesis.rhFN1024 is proposed to interact with the ITGB1 receptor on hPDLSCs,activating the NF-κB pathway to neutralize AGEs-induced pro-inflammatory cytokines.This study suggests rhFN1024 as a potential biomedical material for tissue repair.
