Toll样受体9（TLR9）在足细胞损伤中的作用

发布时间：2018-10-22 16:30

【摘要】：目的：Toll样受体(TLRs)及其信号是机体对抗病原微生物的重要机制,是联系先天免疫和后天免疫的重要桥梁。相关研究表明,TLR4在足细胞中表达,并且能够被激活从而介导足细胞损伤：此外,已有研究发现,TLR9在某些肾小球疾病的足细胞中表达,但它在足细胞损伤中的作用尚不清楚。本研究利用足细胞嘌呤霉素氨基核苷(puromycin aminonucleoside, PAN)损伤模型,试图确定TLR9是否参与足细胞的损伤过程。方法：1.用PAN诱导体外培养的足细胞损伤,以定量逆转录PCR (qRT-PCR)和免疫印迹(Western Blotting, WB)检测足细胞TLR9以及该通路其它组分的变化,包括IRAKI, TRAF6, NF-κ Bp65, p38MAPK和IL12,用免疫荧光染色检测NF-K B p65核转移,以及用流式细胞术检查足细胞凋亡；此外,用PAN诱导大鼠足细胞损伤,以qRT-PCR和免疫组化法检测大鼠肾小球中TLR9表达变化。2.将TLR9干扰RNA (siRNA)表达质粒和对照质粒分别转染足细胞,用WB比较:NF-κ Bp65和p38 MAPK磷酸化、qRT-PCR比较IL12的mRNA变化,以及流式细胞仪比较足细胞凋亡情况,从而确定TLR9信号通路是否参与PAN诱导的足细胞损伤。结果：1.与对照相比,经PAN刺激的足细胞,其TLR9、IRAK1、TRAF6、IL12的mRNA水平明显上调,磷酸化p65和磷酸化p38显著增加,细胞凋亡增加,NF-κ Bp65发生核转移。PAN大鼠模型的肾小球中TLR9 mRNA表达上调,免疫组化显示出TLR9蛋白在足细胞中显著增加。2.足细胞转染TLR9干扰质粒后,TLR9的mRNA和蛋白表达水平降低；经PAN处理后,该细胞与转染对照siRNA的细胞相比,其磷酸化p65和磷酸化p38的水平降低,IL12的mRNA表达降低,以及足细胞凋亡减少。结论：TLR9信号通路参与了嘌呤霉素氨基核苷诱导的足细胞损伤过程,其机制可能是促进炎症因子产生以及p38MAPK依赖的细胞凋亡。本研究提示TLR9信号通路在某些肾小球疾病中的作用。
[Abstract]:Objective: Toll-like receptor (TLRs) and its signal are important mechanisms of organism against pathogenic microorganisms and an important bridge between innate and acquired immunity. Studies have shown that TLR4 is expressed in podocytes and can be activated to mediate podocyte injury. In addition, it has been found that TLR9 is expressed in podocytes of some glomerular diseases, but its role in podocyte injury is not clear. In this study, the (puromycin aminonucleoside, PAN) damage model of podocyte purinomycin aminonucleoside was used to determine whether TLR9 was involved in the damage of podocyte. Method 1: 1. The damage of podocyte was induced by PAN in vitro. The changes of TLR9 and other components of podocyte, including IRAKI, TRAF6, NF- 魏 Bp65, p38MAPK and IL12, were detected by quantitative reverse transcription PCR (qRT-PCR) and Western blotting (Western Blotting, WB), and the nuclear metastasis of NF-K B p65 was detected by immunofluorescence staining. The apoptosis of podocyte was detected by flow cytometry, and the damage of rat podocyte was induced by PAN, and the expression of TLR9 in rat glomeruli was detected by qRT-PCR and immunohistochemistry. 2. TLR9 interference RNA (siRNA) expression plasmid and control plasmid were transfected into podocyte, respectively. The phosphorylation of NF- 魏 Bp65 and p38 MAPK was compared by WB, mRNA of IL12 was compared by qRT-PCR, and apoptosis of podocyte was compared by flow cytometry. So as to determine whether the TLR9 signaling pathway is involved in PAN induced podocyte injury. The result is 1: 1. Compared with the control group, the mRNA level of TLR9,IRAK1,TRAF6,IL12 in podocytes stimulated by PAN was upregulated, the phosphorylation p65 and phosphorylated p38 were significantly increased, apoptosis was increased, and nuclear metastasis occurred in NF- 魏 Bp65. The expression of TLR9 mRNA was up-regulated in the glomerulus of PAN rat model. Immunohistochemistry showed that TLR9 protein increased significantly in podocytes. 2. 2. The expression of mRNA and protein in TLR9 decreased after transfection of podocyte TLR9 interference plasmid, and the levels of phosphorylated p65 and phosphorylated p38 and mRNA expression of IL12 decreased after PAN treatment. And the decrease of podocyte apoptosis. Conclusion: TLR9 signaling pathway is involved in the damage of podocyte induced by purine mycin aminonucleoside. The mechanism may be to promote the production of inflammatory factors and the apoptosis of p38MAPK dependent cells. This study suggests the role of TLR9 signaling pathway in some glomerular diseases.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692

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