本文關(guān)鍵詞： 內(nèi)質(zhì)網(wǎng)應(yīng)激 自噬 蛋白尿 細(xì)胞損傷 凋亡　出處：《吉林大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：各種以蛋白尿為主要表現(xiàn)的腎病的病理和病理生理的發(fā)展是一個復(fù)雜且緩慢的過程，最后變化均表現(xiàn)為腎小球硬化癥或腎小管間質(zhì)纖維化，然而近年來的研究表明小管間質(zhì)病變與腎損傷有更為緊密的關(guān)系。已有研究證實蛋白尿水平和慢性腎衰的進(jìn)展密切相關(guān)。大量動物實驗和臨床研究發(fā)現(xiàn)過蛋白負(fù)荷直接導(dǎo)致腎小管間質(zhì)受損，但是其機制尚未完全闡明，對蛋白尿?qū)е碌哪I小管間質(zhì)損傷也沒有有效的治療手段。 為了研究尿蛋白對腎小管上皮細(xì)胞（NRK-52E）損傷的作用機制，提出以下推測：胞內(nèi)蛋白蓄積，引起內(nèi)質(zhì)網(wǎng)應(yīng)激；同時，蓄積蛋白擁塞溶酶體降解通路，抑制自噬反應(yīng)，因內(nèi)質(zhì)網(wǎng)應(yīng)激與自噬的關(guān)聯(lián)性，未折疊蛋白反應(yīng)加劇，導(dǎo)致細(xì)胞損傷甚至死亡。因此，設(shè)計以下思路： 實驗?zāi)康模?本研究通過阿霉素（adriamycin，ADM）和去脂無內(nèi)毒素白蛋白（Bovine SerumAlbumin，BSA）致NRK-52E細(xì)胞過蛋白負(fù)荷來模擬體內(nèi)蛋白尿環(huán)境，研究過蛋白負(fù)荷致腎小管上皮細(xì)胞凋亡的機制，并利用自噬抑制劑氯喹（chloroquine，CQ）來抑制自噬的終末階段，觀察腎小管上皮細(xì)胞的凋亡情況，為進(jìn)行性腎臟損傷治療靶點的研究奠定理論基礎(chǔ)。 實驗方法： 1.MTT法檢測ADM、BSA及聯(lián)合CQ對NRK-52E細(xì)胞存活率的影響；2.倒置顯微鏡觀察細(xì)胞形態(tài)變化；3. Annexin V/propidium iodide雙染后，流式細(xì)胞儀檢測NRK-52E細(xì)胞凋亡；4.western blot法檢測GRP78、CHOP、LC3、Beclin1和p-PERK的表達(dá)情況。 實驗結(jié)果： MTT結(jié)果顯示ADM和BSA引起NRK-52E細(xì)胞損傷呈時間和劑量依賴性；倒置顯微鏡下觀察細(xì)胞發(fā)生凋亡時，細(xì)胞變圓，細(xì)胞間隙增加；流式細(xì)胞術(shù)顯示ADM、BSA及聯(lián)合CQ能夠顯著增加NRK-52E細(xì)胞凋亡；western blot結(jié)果顯示ADM+BSA組GRP78、CHOP及LC3的表達(dá)高于對照組及單獨ADM、BSA組，Beclin1的相對表達(dá)量降低；CQ抑制自噬后內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白GRP78、CHOP及凋亡因子p-PERK表達(dá)增高。 實驗結(jié)論： 1.阿霉素及去脂無內(nèi)毒素BSA致NRK-52E細(xì)胞發(fā)生的過蛋白負(fù)荷導(dǎo)致細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激，，繼而誘導(dǎo)細(xì)胞凋亡。 2.抑制自噬導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激增強，明顯增加過蛋白負(fù)荷對腎小管上皮細(xì)胞的損傷，可以為治療蛋白尿造成腎小管損傷尋找有效靶點提供理論依據(jù)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692.6

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相關(guān)期刊論文 前1條

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