發(fā)布時(shí)間：2018-05-18 02:41

  本文選題：腎小管上皮細(xì)胞 + 缺氧　； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:觀察7,8-二羥基黃酮(7,8-dihydroxyflavone,7,8-DHF)對(duì)缺氧誘導(dǎo)的人近端腎小管上皮細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum stress,ERS)損傷的影響,并初步探討其可能的分子機(jī)制。方法:體外培養(yǎng)人近端腎小管上皮細(xì)胞(HK-2),采用缺氧培養(yǎng)箱建立細(xì)胞缺氧損傷模型,排除含血清培養(yǎng)基對(duì)缺氧的影響后,實(shí)驗(yàn)分為5組,分別為缺氧0 h、4 h、8 h、12 h、16 h組。實(shí)時(shí)熒光定量PCR(RT-PCR)法篩選缺氧誘導(dǎo)ERS的最佳缺氧時(shí)間。用不同濃度(50、100、150、200、250μmol/L)7,8-DHF處理HK-2細(xì)胞,采用細(xì)胞增殖與活性實(shí)驗(yàn)篩選7,8-DHF的安全濃度。用7,8-DHF預(yù)處理HK-2細(xì)胞缺氧模型,按如下處理因素分組:對(duì)照組、缺氧+DMSO組、缺氧+7,8-DHF組(包括100μmol/L組和150μmol/L組),通過(guò)檢測(cè)細(xì)胞增殖與活性篩選最佳給藥濃度。后將細(xì)胞隨機(jī)分為缺氧組、缺氧+7,8-DHF組(100μmol/L),Western印跡法檢測(cè)細(xì)胞蛋白激酶(Akt)、磷酸化蛋白激酶(p-Akt)、富含半胱氨酸蛋白61(Cysteine-rich protein61,Cyr61)、ERS相關(guān)促凋亡蛋白CCAAT增強(qiáng)子結(jié)合蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)的表達(dá)。用重組Cyr61慢病毒載體構(gòu)建穩(wěn)定表達(dá)Cyr61蛋白的Cyr61-HK-2細(xì)胞株進(jìn)行缺氧實(shí)驗(yàn),按如下處理因素分組:缺氧+空質(zhì)粒轉(zhuǎn)染組,缺氧+Cyr61過(guò)表達(dá)組,采用膜連蛋白V和碘化丙啶(Annexin V-FITC/PI)染色后采用流式細(xì)胞儀檢測(cè)細(xì)胞凋亡,Western印跡檢測(cè)Cyr61和CHOP蛋白的表達(dá)情況。結(jié)果:(1)與正常培養(yǎng)組細(xì)胞相比,RT-PCR實(shí)驗(yàn)結(jié)果顯示12 h為誘導(dǎo)ERS的最佳缺氧時(shí)間(P0.01)。(2)與對(duì)照組相比,細(xì)胞增殖與活性實(shí)驗(yàn)結(jié)果顯示100μmol/L的7,8-DHF組為最佳給藥濃度(P0.01)。(3)與缺氧+DMSO組相比,7,8-DHF預(yù)處理HK-2細(xì)胞,p-Akt、Cyr61表達(dá)量均明顯增高(P0.05),CHOP表達(dá)量明顯降低(P0.05);LY294002處理可抑制p-Akt的表達(dá),減少Cyr61及增加CHOP的表達(dá)(均P0.05)。(4)與缺氧+空質(zhì)粒轉(zhuǎn)染組相比,過(guò)表達(dá)Cyr61組細(xì)胞的凋亡率明顯降低(P0.01)。(5)與缺氧+空質(zhì)粒轉(zhuǎn)染組相比,過(guò)表達(dá)Cyr61組的Cyr61表達(dá)量明顯增高(P0.01),CHOP表達(dá)量明顯降低(P0.01)。結(jié)論:缺氧可誘導(dǎo)HK-2細(xì)胞發(fā)生ERS,7,8-DHF可能通過(guò)激活A(yù)kt通路上調(diào)Cyr61阻止ERS下游凋亡蛋白CHOP的活化,抑制細(xì)胞凋亡以及減輕缺氧誘導(dǎo)的HK-2細(xì)胞損傷,提示7,8-DHF可能對(duì)AKI發(fā)揮保護(hù)作用。
[Abstract]:Aim: to observe the effect of 78-dihydroxyflavone 78-DHF) on endoplasmic reticulum (ER) stress induced by hypoxia in human proximal tubular epithelial cells and to explore its possible molecular mechanism. Methods: human proximal tubular epithelial cells were cultured in vitro. The model of hypoxia injury was established by hypoxia incubator. After the effect of serum medium on hypoxia was excluded, the cells were divided into 5 groups, which were divided into 5 groups: 0 h, 4 h, 8 h, 12 h and 16 h, respectively. The best anoxic time for ERS induced by hypoxia was screened by real-time fluorescence quantitative PCR RT-PCR method. HK-2 cells were treated with different concentrations of 50100150200250 渭 mol / L ~ (78) DHF, and the safe concentration of 7 ~ (8-DHF) was screened by cell proliferation and activity test. The anoxic model of HK-2 cells was pretreated with 7-DHF and divided into four groups: control group, hypoxic DMSO group, anoxic 78-DHF group (including 100 渭 mol/L group and 150 渭 mol/L group). The cells were randomly divided into hypoxia group and hypoxia 78-DHF group. The expression of protein kinase, phosphorylated protein kinase and cysteine-rich protein 61(Cysteine-rich protein 61Cyr61ERS-associated apoptosis-promoting protein enhancer CCAAT enhancer, CCAATENHACE-BINING protein homologous protein (CHOP1) were detected by Western blot. The recombinant Cyr61 lentivirus vector was used to construct a stable Cyr61-HK-2 cell line expressing Cyr61 protein. The cells were divided into two groups according to the following factors: hypoxia empty plasmid transfection group, hypoxia Cyr61 overexpression group. The expression of Cyr61 and CHOP protein was detected by flow cytometry after the staining of annexin V and Annexin V-FITC / Pi. Results compared with normal cultured cells, the results of RT-PCR showed that 12h was the best hypoxia time to induce ERS. The results of cell proliferation and activity test showed that the best drug concentration was 7o 8-DHF (100 渭 mol/L). Compared with anoxic DMSO group, the expression level of p-Akttttiocyr61 in HK-2 cells pretreated with DMSO was significantly higher than that in anoxic DMSO group, and the expression of P0.05HY294002 could be inhibited by P0.05LY294002. Compared with the hypoxia empty plasmid transfection group, the apoptosis rate of the over-expressed Cyr61 group was significantly lower than that of the hypoxic empty plasmid transfection group, and the apoptosis rate of the overexpression Cyr61 group was significantly lower than that of the hypoxia empty plasmid transfection group. The expression of Cyr61 in Cyr61 group was significantly higher than that in control group. Conclusion: hypoxia may induce ERS78-DHF in HK-2 cells by up-regulating Cyr61 to inhibit the activation of ERS downstream apoptotic protein CHOP, inhibit cell apoptosis and attenuate HK-2 cell damage induced by hypoxia, suggesting that 78-DHF may play a protective role on AKI.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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2 徐巖;沈?qū)W飛;宋年華;;富半胱氨酸蛋白61對(duì)轉(zhuǎn)化生長(zhǎng)因子β1誘導(dǎo)腎小管上皮細(xì)胞凋亡的影響[J];中華腎臟病雜志;2012年10期

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本文編號(hào)：1904046