骨髓间充质干细胞源性微囊泡促进软骨细胞的增殖过程中P38-MAPK信号通路的调控机制

发布时间：2018-06-03 23:03

本文选题：BMSCs-MVs + 软骨细胞　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:(1)培养SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),探索及完善分离、提取及鉴定骨髓间充质干细胞源性微囊泡(bone marrow mesenchymal stem cells-derived microvesicle,BMSCs-MVs)系统方法;(2)通过BMSCs-MVs与软骨细胞共培养,通过RT-PCR技术检测目的信号转导途径上下游信号分子的含量变化。分析所得以期探索BMSCs-MVs发挥增殖作用中P38-MAPK的调控机制。方法:(1)2月龄SD大鼠BMSCs的分离、培养和鉴定;(2)BMSCs-MVs的分离和鉴定;(3)3月龄SD大鼠软骨细胞的获取;(4)选取多孔细胞培养板中的12孔,分为四组,每组3孔。首先接种软骨细胞。分组设置:A组:空白对照组;B组:BMSCs-MVs组;C组:P38-MAPK信号通路抑制剂组;D组:BMSCs-MVs+P38-MAPK信号通路抑制剂组,作用24小时后,提取各孔RNA,用RT-PCR分别检测P38-MAPK信号通路上游特异性基因MKK3、MKK6;靶基因(抗凋亡基因Bcl-2);下游特异性基因cREB-1;凋亡执行蛋白(Casp3)的表达。结果:(1)C组较A组上游特异性基因MKK3、MKK6较A组表达升高(P0.05)、下游特异性基因cREB-1表达下降(P0.05),证明P38-MAPK信号通道已被经典抑制剂SB203580阻断;(2)MKK3、MKK6在B组、C组、D组较A组表达均升高(P0.05);(3)下游特异性基因cREB-1在B组、C组、D组表达均下降(P0.05),以最后一组为最。(4)较之A组,B组、C组、D组靶基因(抗凋亡基因Bcl-2)表达升高(P0.05),凋亡执行蛋白(Casp3)表达高低趋势各有不同。结论:BMSCs-MVs对软骨细胞的促增值作用可通过对抗软骨细胞凋亡的层面保护软骨细胞、特异性阻断促凋亡的P38-MAPK信号通路实现,但不能排除多基因、多通道的共同作用。
[Abstract]:Objective to culture bone marrow mesenchymal stem cells from bone marrow mesenchymal stem cells (BMSCs) of SD rats, to explore and perfect the isolation, extraction and identification of bone marrow mesenchymal stem cells-derived micromesenchymal stem cells (BMSCs) from bone marrow mesenchymal stem cells (BMSCs-MVs2) and to co-culture them with chondrocytes by BMSCs-MVs. The changes of upstream and downstream signal molecules in target signal transduction pathway were detected by RT-PCR technique. The aim of this study was to explore the regulatory mechanism of P38-MAPK in the process of BMSCs-MVs proliferation. Methods BMSCs was isolated from 2-month-old Sprague-Dawley rats. The culture and identification of BMSCs-MVs of Sprague-Dawley rats at the age of 3 months were used to isolate and identify the chondrocytes of Sprague-Dawley rats at the age of 3 months. The 12 holes in the porous cell culture plate were selected and divided into four groups with 3 holes in each group. Chondrocytes were first inoculated. Group A: control group: group B: BMSCs-MVs, group C, group C: P38-MAPK signal pathway inhibitor group, group D, group D: BMSCs-MVs P38-MAPK signal pathway inhibitor group, 24 hours after exposure. RT-PCR was used to detect the expression of upstream specific gene MKK3mK6 and target genes (anti-apoptotic gene Bcl-2N; downstream specific gene cREB-1; apoptotic executive protein casp3) of P38-MAPK signaling pathway. Results compared with A group, the expression of MKK3MKK6 gene in group C was higher than that in group A (P 0.05), and the expression of downstream specific gene cREB-1 was decreased (P 0.05). It is proved that the signal channel of P38-MAPK has been blocked by SB203580, a classical inhibitor, in group B, group C, and group D is higher than group A (P 0.05). The expression of downstream specific gene cREB-1 in group B and C was significantly lower than that in group A (P 0.05). The expression of target gene (anti-apoptotic gene Bcl-2) in group A was significantly higher than that in group B (P 0.05), and the expression trend of apoptotic executive protein (AEP) was different. Conclusion the proliferation of chondrocytes can be stimulated by the P38-MAPK signaling pathway of chondrocytes, which can be blocked by the protection of chondrocytes against chondrocyte apoptosis, but the co-action of multi-gene and multi-channel can not be excluded.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R684.3

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