7,8-二羟基黄酮对缺氧诱导下肾小管上皮细胞内质网应激的抑制作用

发布时间：2018-05-18 02:41

本文选题：肾小管上皮细胞 + 缺氧　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:观察7,8-二羟基黄酮(7,8-dihydroxyflavone,7,8-DHF)对缺氧诱导的人近端肾小管上皮细胞内质网应激(Endoplasmic reticulum stress,ERS)损伤的影响,并初步探讨其可能的分子机制。方法:体外培养人近端肾小管上皮细胞(HK-2),采用缺氧培养箱建立细胞缺氧损伤模型,排除含血清培养基对缺氧的影响后,实验分为5组,分别为缺氧0 h、4 h、8 h、12 h、16 h组。实时荧光定量PCR(RT-PCR)法筛选缺氧诱导ERS的最佳缺氧时间。用不同浓度(50、100、150、200、250μmol/L)7,8-DHF处理HK-2细胞,采用细胞增殖与活性实验筛选7,8-DHF的安全浓度。用7,8-DHF预处理HK-2细胞缺氧模型,按如下处理因素分组:对照组、缺氧+DMSO组、缺氧+7,8-DHF组(包括100μmol/L组和150μmol/L组),通过检测细胞增殖与活性筛选最佳给药浓度。后将细胞随机分为缺氧组、缺氧+7,8-DHF组(100μmol/L),Western印迹法检测细胞蛋白激酶(Akt)、磷酸化蛋白激酶(p-Akt)、富含半胱氨酸蛋白61(Cysteine-rich protein61,Cyr61)、ERS相关促凋亡蛋白CCAAT增强子结合蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)的表达。用重组Cyr61慢病毒载体构建稳定表达Cyr61蛋白的Cyr61-HK-2细胞株进行缺氧实验,按如下处理因素分组:缺氧+空质粒转染组,缺氧+Cyr61过表达组,采用膜连蛋白V和碘化丙啶(Annexin V-FITC/PI)染色后采用流式细胞仪检测细胞凋亡,Western印迹检测Cyr61和CHOP蛋白的表达情况。结果:(1)与正常培养组细胞相比,RT-PCR实验结果显示12 h为诱导ERS的最佳缺氧时间(P0.01)。(2)与对照组相比,细胞增殖与活性实验结果显示100μmol/L的7,8-DHF组为最佳给药浓度(P0.01)。(3)与缺氧+DMSO组相比,7,8-DHF预处理HK-2细胞,p-Akt、Cyr61表达量均明显增高(P0.05),CHOP表达量明显降低(P0.05);LY294002处理可抑制p-Akt的表达,减少Cyr61及增加CHOP的表达(均P0.05)。(4)与缺氧+空质粒转染组相比,过表达Cyr61组细胞的凋亡率明显降低(P0.01)。(5)与缺氧+空质粒转染组相比,过表达Cyr61组的Cyr61表达量明显增高(P0.01),CHOP表达量明显降低(P0.01)。结论:缺氧可诱导HK-2细胞发生ERS,7,8-DHF可能通过激活Akt通路上调Cyr61阻止ERS下游凋亡蛋白CHOP的活化,抑制细胞凋亡以及减轻缺氧诱导的HK-2细胞损伤,提示7,8-DHF可能对AKI发挥保护作用。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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