【摘要】：目的觀察骨髓間充質干細胞（bone mesenehymal stem cell, BMSC）對脂多糖（lipopolysaccharide, LPS）誘導的足細胞損傷的影響，探討B(tài)MSC保護損傷足細胞的可能機制。 方法全骨髓培養(yǎng)法分離培養(yǎng)BALB/C小鼠股骨的BMSC。免疫細胞化學染色鑒定腎小球足細胞。實驗分為4組：正常條件培養(yǎng)的足細胞（Control組）、足細胞與BMSC共培養(yǎng)（BMSC組）、LPS誘導的足細胞（LPS組）、LPS誘導的足細胞與BMSC共培養(yǎng)（LPS+BMSC組）。各組在實驗條件干預24小時后，倒置相差顯微鏡下觀察足細胞的形態(tài)；RT-PCR測定足細胞nephrin、CD2AP、synaptopodin和TRPC6mRNA的表達；蛋白免疫印跡雜交（Western Blot）測定足細胞的nephrin、CD2AP、synaptopodin、TRPC6蛋白的表達。 結果1、流式細胞儀顯示體外分離培養(yǎng)的第3代小鼠BMSC表面抗原CD44陽性細胞表達率98.7%±0.9%，CD90陽性細胞表達率98.1%±1.4%，CD29陽性細胞表達率96.1%±2.4%，而CD11b/c陽性細胞表達率14.7%±0.6%，CD34陽性細胞表達率1.98%±0.5%。2、免疫細胞化學染色，未分化的足細胞，細胞核較小，呈細橢圓形，胞質中不表達足細胞特異性骨架蛋白synaptopodin；分化成熟的足細胞，細胞核變大變圓，胞質中可見大量表達synaptopodin。3、LPS組的足細胞胞體皺縮，細胞內砂礫樣物質明顯增多，空泡形成；LPS+BMSC組足細胞則仍可見原有細胞形態(tài)，細胞皺縮不明顯，細胞內砂礫物質和空泡生成減少。4、與LPS組比較，LPS+BMSC組nephrin、CD2AP、synaptopodin mRNA的表達水平均明顯升高（P0.05），，TRPC6mRNA的表達水平顯著下降（P0.05）。5、與LPS組比較，LPS+BMSC組nephrin、CD2AP和synaptopodin蛋白的表達水平均明顯升高（P0.05），TRPC6蛋白的表達水平顯著下降（P0.05）。 結論1、BMSC可以減輕LPS誘導的足細胞的損傷。2、BMSC對足細胞損傷的保護作用與其促進nephrin、CD2AP和synaptopodin的表達，抑制TRPC6的表達有關。
[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% 鹵0.90.The expression rate of CD90 positive cells was 98.1% 鹵1.4in vitro. The expression rate of CD29 positive cells was 96. 1% 鹵2. 4%, while that of CD11b/c positive cells was 14. 7% 鹵0. 6%. The expression rate of CD34 positive cells was 1. 98% 鹵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.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692.5

【參考文獻】

相關期刊論文 前7條

1 楊煥丹;董晨;關鳳軍;高莉莉;趙彤;豐炳峰;;骨髓間充質干細胞移植對嘌呤霉素氨基核苷腎病大鼠足細胞修復作用的研究[J];中國當代兒科雜志;2010年06期

2 譚會斌;傅淑霞;楊林;;纈沙坦聯合苯那普利對糖尿病大鼠足細胞損傷的影響及腎臟保護機制的研究[J];中國中西醫(yī)結合腎病雜志;2008年04期

3 陳朝紅;劉志紅;孫驊;姚根宏;秦衛(wèi)松;黎磊石;;雷公藤甲素干預足細胞病變的體外觀察[J];腎臟病與透析腎移植雜志;2007年02期

4 李煥萍;劉春蓉;張永亮;;骨髓間充質干細胞在醫(yī)學領域中的研究與應用[J];中國組織工程研究與臨床康復;2007年28期

5 萬建新;郭琦;潘陽彬;崔炯;傅檳檳;許艷芳;;骨髓間充質干細胞修復損傷腎小管上皮細胞:可能與可行?[J];中國組織工程研究與臨床康復;2010年32期

6 徐亞光;劉佳;劉艷春;趙魯米;許雪強;趙秀芬;錢軍;邢昌贏;;氟伐他汀對高糖誘導人足細胞Nephrin和血管緊張素Ⅱ表達的影響[J];江蘇醫(yī)藥;2012年15期

7 趙巧萍;楊汝春;;腎小球足細胞骨架蛋白研究進展[J];浙江中西醫(yī)結合雜志;2012年08期

本文編號：2408336