陈芳,刘红,胡韬韬,陈丹
CHEN Fang,LIU Hong,HU Taotao,CHEN Dan

• 1:武汉市中西医结合医院肾病内科

摘要(Abstract)：

目的 探讨缺氧诱导因子-2α(HIF-2α)在肾小管上皮细胞缺氧损伤中的作用及机制。方法 体外培养人近曲小管上皮细胞(HK-2细胞),以2.5μmol/L抗霉素A作用24 h构建缺氧HK-2细胞模型，采用qRT-PCR法和Western blot法检测HIF-2α在正常HK-2细胞和缺氧HK-2细胞中的表达；将HIF-2α干扰序列(siRNA-HIF-2α)及其阴性对照(siRNA-NC)、NIMA相关激酶-1(NEK1)过表达质粒(pcDNA3.0-NEK1)及空载体质粒(pcDNA3.0-NC)转染至缺氧HK-2细胞后，采用CCK-8法检测缺氧HK-2细胞增殖活性，Annexin V-FITC/PI双染法检测缺氧HK-2细胞凋亡率，qRT-PCR法和Western blot法检测缺氧HK-2细胞中NEK1表达。结果 与正常HK-2细胞比较，缺氧HK-2细胞中HIF-2α mRNA和蛋白表达量均升高，差异均有统计学意义(P<0.05);与Control组比较，Hypoxia组细胞增殖活性降低、细胞凋亡率升高、细胞中NEK1 mRNA和蛋白表达量均升高，差异均有统计学意义(P<0.05);与Hypoxia组比较，si-NC组细胞增殖活性、细胞凋亡率、细胞中NEK1 mRNA和蛋白表达量均无明显变化，差异无统计学意义(P>0.05);与si-NC组比较，si-HIF-2α组细胞增殖活性降低、细胞凋亡率升高、细胞中NEK1 mRNA和蛋白表达量降低，差异均有统计学意义(P<0.05);与si-HIF-2α组比较，si-HIF-2α+pcDNA3.0-NC组细胞增殖活性、细胞凋亡率均无明显变化，差异无统计学意义(P>0.05);与si-HIF-2α+pcDNA3.0-NC组细胞比较，si-HIF-2α+pcDNA3.0-NEK1组细胞增殖活性升高、细胞凋亡率降低，差异均有统计学意义(P<0.05)。结论 HIF-2α可能通过调控NEK1表达促进缺氧HK-2细胞增殖、抑制细胞凋亡，从而减轻HK-2细胞缺氧损伤。
Objective To investigate the role of Hypoxia-inducible factors-2α(HIF-2α) in hypoxia injury of renal tubular epithelial cells and its related molecular mechanism. Methods Human proximal convoluted tubule epithelial cells(HK-2 cells) were cultured in vitro and anoxic HK-2 cells were induced by 2.5 μmol/L antibiotic a for 24h, the expression of HIF-2α in normal HK-2 cells and hypoxic HK-2 cells were detected by qRT-PCR and Western blot. After transfection of HIF-2α interference sequence(siRNA-HIF-2α) and its negative control(siRNA-NC), NIMA-related kinase 1(NEK1) overexpression plasmid(pcDNA3.0-NEK1), and empty vector plasmid(pcDNA3.0-NC) into HK-2 cells, the proliferative activities of hypoxic HK-2 cells were measured by CCK-8 method, and the apoptosis rate of hypoxic HK-2 cells was detected by Annexin V-FITC/PI double staining, and the expression of NEK1 in hypoxic HK-2 cells was detected by qRT-PCR and Western blot. Results Compared with normal HK-2 cells, the expression of HIF-2α mRNA and protein in hypoxic HK-2 cells increased, and the difference was statistically significant(P<0.05). Compared with the Control group, the cell proliferation activity in Hypoxia group decreased, while the apoptosis rate increased. The expression of NEK1 mRNA and protein also increased, and the difference was statistically significant(P<0.05); Compared with Hypoxia group, there was no significant change in the cell proliferation activity, the apoptosis rate, the expression of NEK1 mRNA and protein in si-NC group(P>0.05); Compared with si-NC group, the cell proliferation activity in si-HIF-2α group decreased, while the apoptosis rate increased, and the expression of NEK1 mRNA and protein decreased, the difference was statistically significant(P<0.05); Compared with si-HIF-2α group, there was no significant change in the proliferation activity and apoptosis rate in si-HIF-2α + pcDNA3.0-NC group(P>0.05); Compared with the si-HIF-2α + pcDNA3.0-NC group, the cell proliferation activity in si-HIF-2α+pcDNA3.0-NEK1 group increased, while the apoptosis rate decreased, and the difference was statistically significant(P<0.05). Conclusion HIF-2α may promote the proliferation and inhibit apoptosis of hypoxic HK-2 cells by regulating the expression of NEK1, so as to attenuate the hypoxia injury of HK-2 cells.

关键词(KeyWords)： HIF-2α;肾小管上皮细胞;NEK1;缺氧损伤;细胞增殖;细胞凋亡
hypoxia inducible factors-2α(Hif-2α);renal tubular epithelial cells;NIMA-related kinase 1(NEK1);hypoxia injury;cell proliferation;cell apoptosis

Abstract:

Keywords:

基金项目(Foundation): 湖北省卫生计生委中医药科研项目(ZY2019Q024);; 武汉市卫生和计划生育委员会科研项目(WX16C36)

作者(Author): 陈芳,刘红,胡韬韬,陈丹
CHEN Fang,LIU Hong,HU Taotao,CHEN Dan

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