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目的:观察肾损伤分子-1(KIM-1)及缺氧诱导因子1α(HIF-1α)在人近端小管上皮细胞(HK-2)缺氧复氧过程中的表达及相互关系,探讨HIF-1α对KIM-1表达的调控作用及机制。方法:收集缺氧、缺氧复氧后不同时段培养的HK-2细胞,用RT-PCR和Western-Blot检测KIM-1及HIF-1α表达,同时观察HIF激动剂(CoCl2)及抑制剂(雷帕霉素)对KIM-1表达的影响;应用凝胶迁移实验(EMSA)和染色质免疫沉淀实验(ChIP)验证KIM-1启动子区和HIF-1结合情况。结果:HK-2细胞缺氧复氧过程中KIM-1和HIF-1在mRNA水平和蛋白水平表达相关,HIF-1激动剂CoCl2或抑制剂雷帕霉素可上调或下调KIM-1表达,并呈剂量依赖性;EMSA表明HIF-1α可以和KIM-1启动子区HRE结合位点为模板所设计的双链寡核苷酸探针特异性结合,而且这种特异性结合受HIF-1α调节,同时ChIP实验证实了KIM-1启动子区和HIF-1α的结合。结论:KIM-1在近端肾小管上皮细胞缺氧损伤时的表达受HIF-1α调控,KIM-1可能作为HIF-1的下游分子参与肾损伤及修复过程。
OBJECTIVE: To observe the expression and correlation of KIM-1 and HIF-1α during hypoxia-reoxygenation in human proximal tubular epithelial cells (HK-2), and to investigate the relationship between HIF-1α -1α on KIM-1 expression and its mechanism. Methods: HK-2 cells cultured in hypoxia and hypoxia-reoxygenation groups were collected. The expressions of KIM-1 and HIF-1α were detected by RT-PCR and Western-Blot. HIF agonists (CoCl2) and inhibitors Rapamycin) on the expression of KIM-1. The binding of KIM-1 promoter to HIF-1 was verified by EMSA and ChIP assays. Results: The mRNA and protein levels of KIM-1 and HIF-1 were correlated with hypoxia-reoxygenation in HK-2 cells. The expression of KIM-1 was up-regulated or down-regulated by HIF-1 agonist CoCl2 or rapamycin, EMSA showed that HIF-1αcan specifically bind to the double-stranded oligonucleotide probe designed by the HRE binding site of KIM-1 promoter region, and this specific binding is regulated by HIF-1α Regulation, at the same time ChIP experiment confirmed the KIM-1 promoter region and HIF-1α binding. CONCLUSION: The expression of KIM-1 is regulated by hypoxia in proximal tubular epithelial cells. KIM-1 may be involved in renal injury and repair process as a downstream molecule of HIF-1.