【摘要】：目的觀察骨髓間充質(zhì)干細(xì)胞（bone mesenehymal stem cell, BMSC）對(duì)脂多糖（lipopolysaccharide, LPS）誘導(dǎo)的足細(xì)胞損傷的影響，探討B(tài)MSC保護(hù)損傷足細(xì)胞的可能機(jī)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(xì)胞化學(xué)染色鑒定腎小球足細(xì)胞。實(shí)驗(yàn)分為4組：正常條件培養(yǎng)的足細(xì)胞（Control組）、足細(xì)胞與BMSC共培養(yǎng)（BMSC組）、LPS誘導(dǎo)的足細(xì)胞（LPS組）、LPS誘導(dǎo)的足細(xì)胞與BMSC共培養(yǎng)（LPS+BMSC組）。各組在實(shí)驗(yàn)條件干預(yù)24小時(shí)后，倒置相差顯微鏡下觀察足細(xì)胞的形態(tài)；RT-PCR測(cè)定足細(xì)胞nephrin、CD2AP、synaptopodin和TRPC6mRNA的表達(dá)；蛋白免疫印跡雜交（Western Blot）測(cè)定足細(xì)胞的nephrin、CD2AP、synaptopodin、TRPC6蛋白的表達(dá)。 結(jié)果1、流式細(xì)胞儀顯示體外分離培養(yǎng)的第3代小鼠BMSC表面抗原CD44陽(yáng)性細(xì)胞表達(dá)率98.7%±0.9%，CD90陽(yáng)性細(xì)胞表達(dá)率98.1%±1.4%，CD29陽(yáng)性細(xì)胞表達(dá)率96.1%±2.4%，而CD11b/c陽(yáng)性細(xì)胞表達(dá)率14.7%±0.6%，CD34陽(yáng)性細(xì)胞表達(dá)率1.98%±0.5%。2、免疫細(xì)胞化學(xué)染色，未分化的足細(xì)胞，細(xì)胞核較小，呈細(xì)橢圓形，胞質(zhì)中不表達(dá)足細(xì)胞特異性骨架蛋白synaptopodin；分化成熟的足細(xì)胞，細(xì)胞核變大變圓，胞質(zhì)中可見大量表達(dá)synaptopodin。3、LPS組的足細(xì)胞胞體皺縮，細(xì)胞內(nèi)砂礫樣物質(zhì)明顯增多，空泡形成；LPS+BMSC組足細(xì)胞則仍可見原有細(xì)胞形態(tài)，細(xì)胞皺縮不明顯，細(xì)胞內(nèi)砂礫物質(zhì)和空泡生成減少。4、與LPS組比較，LPS+BMSC組nephrin、CD2AP、synaptopodin mRNA的表達(dá)水平均明顯升高（P0.05），，TRPC6mRNA的表達(dá)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(dá)水平均明顯升高（P0.05），TRPC6蛋白的表達(dá)水平顯著下降（P0.05）。 結(jié)論1、BMSC可以減輕LPS誘導(dǎo)的足細(xì)胞的損傷。2、BMSC對(duì)足細(xì)胞損傷的保護(hù)作用與其促進(jìn)nephrin、CD2AP和synaptopodin的表達(dá)，抑制TRPC6的表達(dá)有關(guān)。
[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(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.5

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