骨髓间充质干细胞体外调控肝星状细胞死亡受体5的表达

发布时间：2018-06-08 08:00

  本文选题：骨髓间充质干细胞 + 肝星状细胞　； 参考：《广西医科大学》2012年硕士论文

【摘要】：目的：观察大鼠骨髓间充质干细胞(BMSCs)对肝星状细胞(HSCs)死亡受体5和Caspase-3蛋白表达的影响,探讨BMSCs诱导HSCs凋亡及其机制。 方法：贴壁筛选法培养、纯化SD大鼠BMSCs,传代至第3代使用,大鼠肝纤维原细胞系和原代HSCs细胞冻融后传至第4代使用。在半透膜(transwell insert)上分别接种肝纤维原细胞和BMSCs细胞,在6孔培养板上接种大鼠HSCs细胞,建立上下双层共培养体系。实验分4组：①空白对照组：BMSCs和HSCs分别单独培养；②阴性对照组：HSCs与肝纤维原细胞共培养(模拟肝星状细胞体内生长环境)；③共培养组：HSCs与BMSCs共培养；④实验组：用肿瘤坏死因子相关凋亡诱导配体(TRAIL)多克隆抗体先与BMSCs作用6h后,不换液,再与HSCs共培养。在倒置相差显微镜下,于24h,48h,72h各时间段动态观察HSCs和BMSCs细胞形态学改变；MTT法检测BMSCs对HSCs增殖的抑制率；应用流式细胞仪Annexin-V-FITC/PI双染法检测HSCs凋亡率；Western Blot法分别检测HSCs细胞上Caspase-3蛋白和死亡受体5蛋白的表达；用SYBR荧光实时定量RT-PCR法检测HSCs死亡受体5mRNA的表达；收集各组上清液,ELISA法检测各组上清液中TRAIL的浓度。 结果：1BMSCs对HSCs的增殖具有抑制作用,在共培养组中,培养24后,肝星状细胞增殖抑制率升高,72h后明显升高,且呈现时间依赖性,在共培养的各个时间段与空白组、阴性对照组和实验组比较差异均有显著性统计学意义(P0.01)。2.Annexin-V-FITC/PI双染法检测BMSCs和HSCs共培养24,48,72h后HSCs的凋亡率分别为10.17%、15.15%和30.18%,与其他各组比较差异有显著性统计学意义(P0.01)。3ELISA法检测各组上清液中TRAIL的浓度：各组在培养48h时,TRAIL的分泌到达生理的增殖期高峰,72h后有所下降；共培养组的TRAIL浓度与BMSCs单独培养组比较差异有显著性统计学意义,在共培养各个时间段低于BMSCs单独培养组。4.共培养24h后,共培养组Caspase-3蛋白的表达显著升高,与空白组和阴性对照组比较差异有显著性统计学意义(P0.01),且随时间延长呈现递增趋势；实验组Caspase-3蛋白与共培养组比较差异有显著性统计学意义(P0.01),在共培养的各个时间段均低于共培养组。5.共培养组死亡受体5蛋白的表达随共培养时间的延长呈递增趋势,与空白组和阴性对照组比较差异有显著性统计学意义(P0.01),在共培养的各个时间段均高于以上两组；实验组死亡受体5蛋白在共培养的各个时间段均低于共培养组,二者比较差异有显著性统计学意义(P0.01)；空白组死亡受体5蛋白的表达和阴性组在共培养的各个时间段比较无差异(P0.05)6.共培养组BMSCs能明显上调HSCs中死亡受体5mRNA的表达,在共培养的各个时间段与其他各组比较差异有显著性统计学意义(P0.01)且表达高于以上各组；而空白组和阴性对照组死亡受体5mRNA的表达在共培养的各个时间段比较无差异(P0.05) 结论：BMSCs与HSCs共培养能抑制HSCs的增殖,促进HSCs的凋亡。其机制可能为BMSCs旁分泌的TRAIL通过上调Caspase-3和DR5蛋白的表达实现的。
[Abstract]:Objective: To observe the effect of rat bone marrow mesenchymal stem cells (BMSCs) on the expression of death receptor 5 and Caspase-3 protein in hepatic stellate cells (HSCs) and explore the apoptosis and its mechanism induced by BMSCs in HSCs.
Methods: the SD rat BMSCs was purified by the adherent wall screening method and passed to the third generation. The rat liver fibroblast cell line and the original HSCs cell were transmitted to the fourth generation after freezing and thawing. The liver fibroblasts and BMSCs cells were inoculated on the semi permeable membrane (Transwell insert), and the rat HSCs cells were inoculated on the 6 Hole culture plate, and the upper and lower bilayer co culture bodies were established. The experiment was divided into 4 groups: (1) blank control group: BMSCs and HSCs were cultured separately; (2) negative control group: co culture of HSCs and hepatic fibroblast (simulating the growth environment of hepatic stellate cells); (3) co culture group: co culture of HSCs and BMSCs; (4) experimental group: polyclonal antibody with tumor necrosis factor related apoptosis inducing ligand (TRAIL) After 6h was first acted with BMSCs, the liquid was not changed and then co cultured with HSCs. Under the inverted phase contrast microscope, the morphological changes of HSCs and BMSCs cells were observed dynamically at all time segments of 24h, 48h and 72h, and the inhibition rate of BMSCs to HSCs proliferation was detected by MTT method, and the apoptosis rate was detected by the method of flow cytometry. The expression of Caspase-3 protein and death receptor 5 protein on s cells. The expression of HSCs death receptor 5mRNA was detected by SYBR fluorescence real-time quantitative RT-PCR method, the supernatant of each group was collected, and the concentration of TRAIL in the supernatant of each group was detected by ELISA method.
Results: 1BMSCs had a inhibitory effect on the proliferation of HSCs. In the co culture group, the proliferation inhibition rate of hepatic stellate cells increased after 24, and 72h was significantly increased and showed time dependence. There was significant statistical significance (P0.01).2.Annexin-V-FITC/ between the negative control group and the experimental group in each time period of co culture. The apoptosis rate of HSCs was 10.17%, 15.15% and 30.18% respectively after BMSCs and HSCs were co cultured for 24,48,72h. There was significant statistical significance (P0.01).3ELISA method to detect the concentration of TRAIL in the supernatant of each group with the other groups (P0.01), and the.3ELISA method was used to detect the concentration of TRAIL in each group: each group reached the peak of the physiological proliferation period of the TRAIL, and decreased after 72h when the 48h was cultured. The concentration of TRAIL in the co culture group was significantly different from that in the BMSCs group, and the expression of Caspase-3 protein in the co culture group was significantly higher than that of the BMSCs single culture group.4. in the co culture group. The difference was statistically significant (P0.01) with the blank group and the negative control group (P0.01). The extension of Caspase-3 protein in the experimental group was significantly higher than that of the co culture group (P0.01). The expression of death receptor 5 protein in the co culture group was lower than that of the co culture group, and the expression of the 5 protein in the co culture group increased with the prolongation of the co culture time, and there was a significant difference from the blank group and the negative control group. The statistical significance (P0.01) was higher than that of the above two groups in all the time periods of co culture; the 5 protein of the death receptor in the experimental group was lower than that of the co culture group, and the difference between the two groups was significant (P0.01); the expression of the death receptor 5 egg white in the blank group and the negative group were in each time period of co culture. The P0.05 6. co culture group BMSCs can obviously increase the expression of death receptor 5mRNA in HSCs, and there is significant statistical significance (P0.01) in each time period of co culture with the other groups (P0.01), and the expression of 5mRNA in the blank group and negative control group is in each time period of co culture. No difference (P0.05)
Conclusion: the co culture of BMSCs and HSCs can inhibit the proliferation of HSCs and promote the apoptosis of HSCs. The mechanism may be that the TRAIL paracrine secreted by BMSCs can be realized by up regulation of the expression of Caspase-3 and DR5 protein.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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