大鼠骨髓间充质干细胞体外诱导肝星状细胞系凋亡
发布时间：2018-08-29 13:15
【摘要】： 目的研究大鼠骨髓间充质干细胞(MSCs)体外诱导大鼠肝星状细胞(HSCs)凋亡及其机制。 方法MSCs的分离：从SD雄性大鼠的股骨骨髓中获得,经分离,培养,传代至第4代使用；大鼠肝星状细胞系(HSC-T6)及纤维原细胞系复苏后传代使用。用6孔塑料培养板,在半透膜(transwell insert)上层接种MSCs(2×105cells／well),在下层接种HSC-T6细胞(2X105cells/well),建立上下双层细胞共培养体系,常规培养。实验分组：①空白对照组：HSCs单独培养；②阴性对照组：纤维原细胞(Fiberoblasts)与HSCs共培养；③MSCs组：MSCs与HSCs共培养。以上体系培养观察24h,48h和72h,于倒置相差显微镜下动态观察HSCs细胞形态；免疫组化法检测HSCs a-SMA表达；WST-8法检测HSCs增殖抑制率；流式细胞仪Annexin-V-FITC/PI双染法和DNA凝胶电泳(DNA Ladder)检测HSCs细胞凋亡；RT-PCR检测HSCs Caspase-3, Bax基因mRNA表达；Western blot检测HSCs Caspase-3, Bax蛋白表达。 结果(1)MSCs与HSCs共培养24h,48h和72h, HSCs表现明显增殖抑制(P0.01),且呈现时间依赖性,MSCs组与空白对照组、阴性对照组比较均有显著性差异(P0.01；P0.01)。(2)流式细胞仪Annexin-V-FITC/PI双染法检测MSCs与HSCs共培养后HSCs的凋亡率：共培养24h后HSCs凋亡率呈现时间依赖性,与空白对照组和阴性对照组比较有统计学差异(P0.01；P0.01)。(3)DNA凝胶电泳(DNA Ladder测定)MSCs组中出现了明显的DNA Ladder,空白对照组和阴性对照组中无DNA Ladder。(4)RT-PCR检测共培养后各组HSCs的Caspase-3, Bax mRNA表达：共培养24h后,MSCs组Caspase-3, Bax mRNA表达呈现时间依赖性,与空白对照组、阴性对照组比较均有统计学差异(P0.01；P0.01)。(5)Western blot检测共培养后各组HSCs的Caspase-3, Bax蛋白质表达：MSCs组24h后Caspase-3, Bax表达呈现时间依赖性,与空白对照组,阴性对照组比较均有统计学差异(P0.01；P0.01)。 结论(1)MSCs可在体外抑制HSCs增殖和诱导其凋亡。(2)MSCs可能通过旁分泌途径诱导HSCs凋亡。(3)MSCs诱导HSCs凋亡发生是通过上调Caspase-3, Bax表达发挥作用。
[Abstract]:Objective to investigate the apoptosis of rat hepatic stellate cell (HSCs) induced by rat bone marrow mesenchymal stem cell (MSCs) in vitro and its mechanism. Methods MSCs was isolated from the femur bone marrow of SD male rats. It was isolated, cultured and subcultured to the 4th passage. The rat hepatic stellate cell line (HSC-T6) and fibroblast cell line were resuscitated and then subcultured. MSCs (2 脳 105cells/well) was inoculated on the semi-permeable membrane (transwell insert) and HSC-T6 cell (2X105cells/well) was inoculated in the lower layer with a 6-well plastic culture plate. The co-culture system of upper and lower double layer cells was established and cultured routinely. The control group was divided into two groups: the control group was isolated and the control group was isolated from the control group: the fibroblast (Fiberoblasts) and HSCs co-cultured in the control group, and the control group was co-cultured with HSCs. The morphology of HSCs cells was observed by inverted phase contrast microscope for 48h and 72h respectively, and the expression of HSCs a-SMA was detected by immunohistochemistry and the inhibitory rate of HSCs proliferation was detected by WST-8 method. Flow cytometry Annexin-V-FITC/PI double staining and DNA gel electrophoresis (DNA Ladder) were used to detect the apoptosis of HSCs cells. RT-PCR was used to detect the mRNA expression of HSCs Caspase-3, Bax gene. Western blot was used to detect the expression of HSCs Caspase-3, Bax protein. Results (1) MSCs and HSCs co-cultured for 48h and 72h showed obvious proliferation inhibition (P0.01), and there were significant differences between the control group and the control group (P0.01). P0.01). (2) flow cytometry Annexin-V-FITC/PI double staining method was used to detect the apoptosis rate of HSCs after co-culture of MSCs and HSCs. The apoptosis rate of HSCs was time-dependent after 24 hours of co-culture, which was significantly different from that of blank control group and negative control group (P0.01). P0.01). (3) DNA gel electrophoresis (DNA Ladder assay) in MSCs group, there were no DNA Ladder. (4 in DNA Ladder, blank control group and no DNA Ladder. (4 in negative control group. Caspase-3, Bax mRNA expression of HSCs in each group after co-culture was detected. Caspase-3, Bax mRNA expression in MSCs group was time-dependent after 24 hours of co-culture. Compared with the control group and the negative control group, there were significant differences between the control group and the negative control group (P0.01P0.01). (5) Western blot was used to detect the Caspase-3, Bax protein expression of HSCs in each group in a time-dependent manner after 24 hours, compared with the blank control group. There was statistical difference in negative control group (P 0.01, P 0.01). Conclusion (1) MSCs can inhibit the proliferation and induce apoptosis of HSCs in vitro. (2) MSCs may induce HSCs apoptosis through paracrine pathway. (3) MSCs induces HSCs apoptosis through up-regulation of Caspase-3, Bax expression.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2211317
[Abstract]:Objective to investigate the apoptosis of rat hepatic stellate cell (HSCs) induced by rat bone marrow mesenchymal stem cell (MSCs) in vitro and its mechanism. Methods MSCs was isolated from the femur bone marrow of SD male rats. It was isolated, cultured and subcultured to the 4th passage. The rat hepatic stellate cell line (HSC-T6) and fibroblast cell line were resuscitated and then subcultured. MSCs (2 脳 105cells/well) was inoculated on the semi-permeable membrane (transwell insert) and HSC-T6 cell (2X105cells/well) was inoculated in the lower layer with a 6-well plastic culture plate. The co-culture system of upper and lower double layer cells was established and cultured routinely. The control group was divided into two groups: the control group was isolated and the control group was isolated from the control group: the fibroblast (Fiberoblasts) and HSCs co-cultured in the control group, and the control group was co-cultured with HSCs. The morphology of HSCs cells was observed by inverted phase contrast microscope for 48h and 72h respectively, and the expression of HSCs a-SMA was detected by immunohistochemistry and the inhibitory rate of HSCs proliferation was detected by WST-8 method. Flow cytometry Annexin-V-FITC/PI double staining and DNA gel electrophoresis (DNA Ladder) were used to detect the apoptosis of HSCs cells. RT-PCR was used to detect the mRNA expression of HSCs Caspase-3, Bax gene. Western blot was used to detect the expression of HSCs Caspase-3, Bax protein. Results (1) MSCs and HSCs co-cultured for 48h and 72h showed obvious proliferation inhibition (P0.01), and there were significant differences between the control group and the control group (P0.01). P0.01). (2) flow cytometry Annexin-V-FITC/PI double staining method was used to detect the apoptosis rate of HSCs after co-culture of MSCs and HSCs. The apoptosis rate of HSCs was time-dependent after 24 hours of co-culture, which was significantly different from that of blank control group and negative control group (P0.01). P0.01). (3) DNA gel electrophoresis (DNA Ladder assay) in MSCs group, there were no DNA Ladder. (4 in DNA Ladder, blank control group and no DNA Ladder. (4 in negative control group. Caspase-3, Bax mRNA expression of HSCs in each group after co-culture was detected. Caspase-3, Bax mRNA expression in MSCs group was time-dependent after 24 hours of co-culture. Compared with the control group and the negative control group, there were significant differences between the control group and the negative control group (P0.01P0.01). (5) Western blot was used to detect the Caspase-3, Bax protein expression of HSCs in each group in a time-dependent manner after 24 hours, compared with the blank control group. There was statistical difference in negative control group (P 0.01, P 0.01). Conclusion (1) MSCs can inhibit the proliferation and induce apoptosis of HSCs in vitro. (2) MSCs may induce HSCs apoptosis through paracrine pathway. (3) MSCs induces HSCs apoptosis through up-regulation of Caspase-3, Bax expression.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
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