骨髓间充质干细胞对脂多糖诱导的足细胞损伤的影响及可能机制

发布时间：2019-01-13 09:56

【摘要】：目的观察骨髓间充质干细胞（bone mesenehymal stem cell, BMSC）对脂多糖（lipopolysaccharide, LPS）诱导的足细胞损伤的影响，探讨BMSC保护损伤足细胞的可能机制。方法全骨髓培养法分离培养BALB/C小鼠股骨的BMSC。免疫细胞化学染色鉴定肾小球足细胞。实验分为4组：正常条件培养的足细胞（Control组）、足细胞与BMSC共培养（BMSC组）、LPS诱导的足细胞（LPS组）、LPS诱导的足细胞与BMSC共培养（LPS+BMSC组）。各组在实验条件干预24小时后，倒置相差显微镜下观察足细胞的形态；RT-PCR测定足细胞nephrin、CD2AP、synaptopodin和TRPC6mRNA的表达；蛋白免疫印迹杂交（Western Blot）测定足细胞的nephrin、CD2AP、synaptopodin、TRPC6蛋白的表达。结果1、流式细胞仪显示体外分离培养的第3代小鼠BMSC表面抗原CD44阳性细胞表达率98.7%±0.9%，CD90阳性细胞表达率98.1%±1.4%，CD29阳性细胞表达率96.1%±2.4%，而CD11b/c阳性细胞表达率14.7%±0.6%，CD34阳性细胞表达率1.98%±0.5%。2、免疫细胞化学染色，未分化的足细胞，细胞核较小，呈细椭圆形，胞质中不表达足细胞特异性骨架蛋白synaptopodin；分化成熟的足细胞，细胞核变大变圆，胞质中可见大量表达synaptopodin。3、LPS组的足细胞胞体皱缩，细胞内砂砾样物质明显增多，空泡形成；LPS+BMSC组足细胞则仍可见原有细胞形态，细胞皱缩不明显，细胞内砂砾物质和空泡生成减少。4、与LPS组比较，LPS+BMSC组nephrin、CD2AP、synaptopodin mRNA的表达水平均明显升高（P0.05），，TRPC6mRNA的表达水平显著下降（P0.05）。5、与LPS组比较，LPS+BMSC组nephrin、CD2AP和synaptopodin蛋白的表达水平均明显升高（P0.05），TRPC6蛋白的表达水平显著下降（P0.05）。结论1、BMSC可以减轻LPS诱导的足细胞的损伤。2、BMSC对足细胞损伤的保护作用与其促进nephrin、CD2AP和synaptopodin的表达，抑制TRPC6的表达有关。
[Abstract]:Objective to observe the effect of bone marrow mesenchymal stem cell (bone mesenehymal stem cell, BMSC) on podocyte injury induced by lipopolysaccharide (lipopolysaccharide, LPS) and to explore the possible mechanism of BMSC protection against podocyte injury. Methods BMSC. of femur of BALB/C mice was isolated and cultured by whole bone marrow culture method. Glomerular podocytes were identified by immunocytochemical staining. The experiment was divided into four groups: podocyte cultured in normal condition (Control group), podocyte co-cultured with BMSC (podocyte induced by), LPS in BMSC group) (podocyte induced by), LPS in LPS group and (LPS BMSC group co-cultured with BMSC). The morphology of podocytes was observed under inverted phase contrast microscope and the expression of nephrin,CD2AP,synaptopodin and TRPC6mRNA in podocytes were measured by RT-PCR. The expression of nephrin,CD2AP,synaptopodin,TRPC6 protein in podocytes was detected by Western blot (Western Blot). Results 1. Flow cytometry showed that the expression rate of BMSC surface antigen CD44 positive cells was 98.7% 卤0.90.The expression rate of CD90 positive cells was 98.1% 卤1.4in vitro. The expression rate of CD29 positive cells was 96. 1% 卤2. 4%, while that of CD11b/c positive cells was 14. 7% 卤0. 6%. The expression rate of CD34 positive cells was 1. 98% 卤0. 5. 2. Immunocytochemical staining and undifferentiated podocytes were observed. The nucleus was small and oval, and no specific cytoskeleton protein synaptopodin; was expressed in the cytoplasm. In the differentiated mature podocytes, the nucleus became larger and rounded, and in the cytoplasm, the body of podocytes in the group of synaptopodin.3,LPS expressed a great deal of shrinkage, the sand and gravel like substances in the cells increased obviously, and the vacuoles were formed. In LPS BMSC group, the morphology of podocyte was still observed, the cell shrinkage was not obvious, and the formation of sand gravel and cavitation was decreased. 4. The expression of nephrin,CD2AP,synaptopodin mRNA in, LPS BMSC group was significantly higher than that in LPS group (P0.05). Compared with LPS group, the expression of nephrin,CD2AP and synaptopodin protein increased significantly in, LPS BMSC group (P0.05), and TRPC6 protein expression level decreased significantly (P0.05). Conclusion 1BMSC can attenuate the damage of podocyte induced by LPS. 2 the protective effect of BMSC on podocyte injury is related to its promoting the expression of nephrin,CD2AP and synaptopodin and inhibiting the expression of TRPC6.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.5

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