中文摘要：

目的 探讨沉默脯氨酸羟化酶2（PHD2）在人肾小管上皮细胞（HK-2）低氧损伤中对自噬的调控作用及相关机制.方法 采用氯化钴（200μmol/L）建立体外HK-2细胞的化学低氧模型,观察低氧培养后的不同时间点（0、6、12、24、36、48 h）电镜下的细胞超微结构改变,Alamar Blue法测定细胞存活率并评估细胞凋亡和自噬水平.同时转染HIF-1 siRNA、PHD2 siRNA,探讨PHD2在低氧情况下对自噬的调控机制.结果 PHD2在常氧下也有表达,随着低氧刺激的持续其蛋白水平逐渐上调,24 h时出现明显变化（P＜0.01）,48 h表达最为显著（P＜0.01）.PHD2 siRNA转染后HK-2细胞在低氧培养24h时HIF-1的蛋白表达显著升高（P＜0.01）,HIF-2α表达无明显改变.与阴性对照siRNA组相比,PHD2 siRNA转染组Bcl-xl蛋白水平上调（P＜0.01）,Bax及LC3-Ⅱ/LC3-Ⅰ比值下调（均P＜0.01）,同时细胞活力上升[（37.04±3.25％比（28.32±2.41）％,P＜0.01].氯化钴处理前加入3-甲基腺嘌呤（3-MA,5mmol/L）后,HK-2细胞LC3-Ⅱ/LC3-Ⅰ比值变化以及细胞活力与PHD2 siRNA转染结果相似,同时电镜下显示自噬体较单纯低氧组减少,细胞的超微结构也相对完整.同时沉默PHD2和HIF-1α则消除了由单独沉默PHD2带来的保护作用.结论 沉默PHD2可能通过稳定HK-2细胞的HIF-1 α活性、上调Bcl-xl蛋白水平,从而抑制细胞凋亡和自噬并减轻化学低氧诱导的细胞损伤.

英文摘要：

Objective To test the hypothesis of autophagy that silencing PHD2 gene could increase hypoxia inducible factor （HIF）-1α levels in the renal medulla and attenuate hypoxia injury in cultured human renal proximal tubular epithelial cell （HK-2） under cobalt dichloride （CoCl2） exposure.Methods HK-2 cells were harvested at hour 0,6,12,24,36 and 48 after exposure to CoC12 （200 μmol/L）.The role of HIF/PHD pathway in CoCl2-induced cell apoptosis/autophagy was studied by employing small-interfering RNA （siRNA）.Dynamic profiles of apoptosis markers （Bax,Bcl-xl） and autophagy marker （LC3） of HK-2 cells within 48 h after exposing to CoCl2 were recorded.Alamar Blue assay was used for quantitative analysis of cellular growth and viability.Electron microscopy analysis was employed to evaluate the changes in autophagic structures.Results The protein expressions of PHD2 were gradually increased after exposing to CoCl2 （200 μmol/L）,with statistics significance at 24 h and reached the peak at 48 h （both P ＜ 0.01）.PHD2 siRNA reduced PHD2 levels by ＞ 60％ and significantly increased HIF-1α protein levels （P ＜ 0.01）,but had little effect on HIF-2α.The protein expression of Bcl-xl was significantly up-regulated,while the level of Bax and LC3-Ⅱ/LC3-Ⅰ were down-regulated in PHD2 siRNA group （all P ＜ 0.01）,compared with the negative control group.Meanwhile,either 3-Methyladenine （an autophagy inhibitor） treatment or PHD2 knockdown rescued cell death and increased cell viability through autophagy inactivation.The ratio of LC3-Ⅱ/LC3-Ⅰ and the quantity of autophagosomes were decreased,and the cell ultrastructure was also relatively intacter than the negative control group.Of interest,co-administration of HIF-1α siRNA with PHD2 siRNA abrogated renoprotective effect conveyed by PHD2 siRNA alone,suggesting that activation of endogenous HIF-1α-dependent pathways mediated the autophagy inactivation effects of PHD2 silencing.Conclusions Direct inhibition of PHD2 promotes renal