内质网应激—自噬反应减轻NRK-52E细胞过蛋白负荷损伤

发布时间：2018-02-14 21:40

本文关键词： 内质网应激自噬蛋白尿细胞损伤凋亡　出处：《吉林大学》2014年硕士论文　论文类型：学位论文

【摘要】：各种以蛋白尿为主要表现的肾病的病理和病理生理的发展是一个复杂且缓慢的过程，最后变化均表现为肾小球硬化症或肾小管间质纤维化，然而近年来的研究表明小管间质病变与肾损伤有更为紧密的关系。已有研究证实蛋白尿水平和慢性肾衰的进展密切相关。大量动物实验和临床研究发现过蛋白负荷直接导致肾小管间质受损，但是其机制尚未完全阐明，对蛋白尿导致的肾小管间质损伤也没有有效的治疗手段。为了研究尿蛋白对肾小管上皮细胞（NRK-52E）损伤的作用机制，提出以下推测：胞内蛋白蓄积，引起内质网应激；同时，蓄积蛋白拥塞溶酶体降解通路，抑制自噬反应，因内质网应激与自噬的关联性，未折叠蛋白反应加剧，导致细胞损伤甚至死亡。因此，设计以下思路：实验目的：本研究通过阿霉素（adriamycin，ADM）和去脂无内毒素白蛋白（Bovine SerumAlbumin，BSA）致NRK-52E细胞过蛋白负荷来模拟体内蛋白尿环境，研究过蛋白负荷致肾小管上皮细胞凋亡的机制，并利用自噬抑制剂氯喹（chloroquine，CQ）来抑制自噬的终末阶段，观察肾小管上皮细胞的凋亡情况，为进行性肾脏损伤治疗靶点的研究奠定理论基础。实验方法： 1.MTT法检测ADM、BSA及联合CQ对NRK-52E细胞存活率的影响；2.倒置显微镜观察细胞形态变化；3. Annexin V/propidium iodide双染后，流式细胞仪检测NRK-52E细胞凋亡；4.western blot法检测GRP78、CHOP、LC3、Beclin1和p-PERK的表达情况。实验结果： MTT结果显示ADM和BSA引起NRK-52E细胞损伤呈时间和剂量依赖性；倒置显微镜下观察细胞发生凋亡时，细胞变圆，细胞间隙增加；流式细胞术显示ADM、BSA及联合CQ能够显著增加NRK-52E细胞凋亡；western blot结果显示ADM+BSA组GRP78、CHOP及LC3的表达高于对照组及单独ADM、BSA组，Beclin1的相对表达量降低；CQ抑制自噬后内质网应激相关蛋白GRP78、CHOP及凋亡因子p-PERK表达增高。实验结论： 1.阿霉素及去脂无内毒素BSA致NRK-52E细胞发生的过蛋白负荷导致细胞内质网应激，，继而诱导细胞凋亡。 2.抑制自噬导致内质网应激增强，明显增加过蛋白负荷对肾小管上皮细胞的损伤，可以为治疗蛋白尿造成肾小管损伤寻找有效靶点提供理论依据。
[Abstract]:The pathological and pathophysiological development of various nephropathy characterized by proteinuria is a complex and slow process, and the final changes are glomerulosclerosis or tubulointerstitial fibrosis. However, recent studies have shown that tubulointerstitial lesions are more closely related to renal injury. It has been shown that the level of proteinuria is closely related to the progression of chronic renal failure. The kidney tubulointerstitium is damaged directly by the charge. However, its mechanism has not been fully elucidated, and there is no effective treatment for tubulointerstitial injury caused by proteinuria. In order to study the mechanism of urinary protein on NRK-52E damage in renal tubular epithelial cells, the following hypotheses were proposed: intracellular protein accumulation caused endoplasmic reticulum stress, and accumulation protein blocked lysosomal degradation pathway and inhibited autophagy. Because of the association between endoplasmic reticulum stress and autophagy, the unfolded protein response is aggravated, leading to cell damage and even death. Objective:. In order to simulate the proteinuria environment of NRK-52E cells induced by adriamycin adriamycin (ADM) and lipoprotein free albumin (BSA), the mechanism of apoptosis of renal tubular epithelial cells induced by protein overload was studied. The terminal stage of autophagy was inhibited by chloroquineuron (CQ), and the apoptosis of renal tubular epithelial cells was observed, which laid a theoretical foundation for the study of therapeutic targets for progressive renal injury. Experimental methods:. 1. The effect of NRK-52E cell survival was detected by MTT assay and combined with CQ. The morphological changes of NRK-52E cells were observed by inverted microscope. After double staining of Annexin Vapordium iodide, apoptosis of NRK-52E cells was detected by flow cytometry and the expression of GRP78 CHOPLC3 Beclin1 and p-Perk was detected by western blot assay. Experimental results:. MTT results showed that ADM and BSA induced NRK-52E cell damage in a time-and dose-dependent manner, and the cells became round and intercellular space increased when apoptosis was observed under inverted microscope. Flow cytometry showed that ADMN BSA and CQ could significantly increase apoptosis of NRK-52E cells. The results showed that the expression of GRP78 chop and LC3 in ADM BSA group was significantly higher than that in control group and ADMN BSA group. The relative expression of Beclin1 in ADM BSA group was significantly lower than that in control group and ADMN BSA group alone. CQ inhibited endoplasmic reticulum stress after autophagy. The expression of GRP78 chop and p-PERK were increased. The experimental conclusions are as follows:. 1. The overload of NRK-52E cells induced by adriamycin and lipid-free BSA induced endoplasmic reticulum stress and then induced apoptosis. 2. Inhibition of autophagy led to increased endoplasmic reticulum stress and significantly increased the damage of renal tubular epithelial cells caused by protein overload, which could provide a theoretical basis for finding effective targets for the treatment of renal tubular injury caused by proteinuria.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.6

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