【摘要】：目的：Toll樣受體(TLRs)及其信號是機體對抗病原微生物的重要機制,是聯(lián)系先天免疫和后天免疫的重要橋梁。相關(guān)研究表明,TLR4在足細胞中表達,并且能夠被激活從而介導足細胞損傷：此外,已有研究發(fā)現(xiàn),TLR9在某些腎小球疾病的足細胞中表達,但它在足細胞損傷中的作用尚不清楚。本研究利用足細胞嘌呤霉素氨基核苷(puromycin aminonucleoside, PAN)損傷模型,試圖確定TLR9是否參與足細胞的損傷過程。方法：1.用PAN誘導體外培養(yǎng)的足細胞損傷,以定量逆轉(zhuǎn)錄PCR (qRT-PCR)和免疫印跡(Western Blotting, WB)檢測足細胞TLR9以及該通路其它組分的變化,包括IRAKI, TRAF6, NF-κ Bp65, p38MAPK和IL12,用免疫熒光染色檢測NF-K B p65核轉(zhuǎn)移,以及用流式細胞術(shù)檢查足細胞凋亡；此外,用PAN誘導大鼠足細胞損傷,以qRT-PCR和免疫組化法檢測大鼠腎小球中TLR9表達變化。2.將TLR9干擾RNA (siRNA)表達質(zhì)粒和對照質(zhì)粒分別轉(zhuǎn)染足細胞,用WB比較:NF-κ Bp65和p38 MAPK磷酸化、qRT-PCR比較IL12的mRNA變化,以及流式細胞儀比較足細胞凋亡情況,從而確定TLR9信號通路是否參與PAN誘導的足細胞損傷。結(jié)果：1.與對照相比,經(jīng)PAN刺激的足細胞,其TLR9、IRAK1、TRAF6、IL12的mRNA水平明顯上調(diào),磷酸化p65和磷酸化p38顯著增加,細胞凋亡增加,NF-κ Bp65發(fā)生核轉(zhuǎn)移。PAN大鼠模型的腎小球中TLR9 mRNA表達上調(diào),免疫組化顯示出TLR9蛋白在足細胞中顯著增加。2.足細胞轉(zhuǎn)染TLR9干擾質(zhì)粒后,TLR9的mRNA和蛋白表達水平降低；經(jīng)PAN處理后,該細胞與轉(zhuǎn)染對照siRNA的細胞相比,其磷酸化p65和磷酸化p38的水平降低,IL12的mRNA表達降低,以及足細胞凋亡減少。結(jié)論：TLR9信號通路參與了嘌呤霉素氨基核苷誘導的足細胞損傷過程,其機制可能是促進炎癥因子產(chǎn)生以及p38MAPK依賴的細胞凋亡。本研究提示TLR9信號通路在某些腎小球疾病中的作用。
[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.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692

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相關(guān)期刊論文 前1條

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本文編號：2287693