重组人促红细胞生成素对人肾小管上皮细胞缺血再灌注损伤的保护机制  

Protective Effect of rHuEPO on Ischemia-Reperfusion-Induced Apoptosis in Cultured Renal Tubular Epithelial Cells

作　　者：周文祥 杨永丽 夏章晖[3] 杨晓[4] 聂祥智 董骏武 徐翠玲 周国梅 

机构地区：[1]武汉市一医院肾内科,湖北武汉430022 [2]湖北省中山医院肾内科,湖北武汉430033 [3]华中科技大学同济医学院附属梨园医院,湖北武汉430070 [4]华中科技大学同济医学院附属协和医院肾内科,湖北武汉430022

出　　处：《武汉大学学报（医学版）》2010年第5期565-570,共6页Medical Journal of Wuhan University

基　　金：湖北省自然科学基金资助项目(编号:2007ABA290)

摘　　要：目的:探讨重组人促红细胞生成素(rHuEPO)在人肾小管上皮细胞(HK-2)缺血再灌注损伤保护机制。方法:体外培养HK-2细胞,随机分为5组:①正常对照组;②缺血再灌注损伤组;③预先给药组:缺血损伤30min前预先给予rHuEPO;④即时给药组:再灌注损伤5min前给予rHuEPO;⑤延迟给药组:再灌注损伤30min后给予rHuEPO。用AnnexinV/PI染色结合流式细胞仪技术检测细胞凋亡;用RT-PCR及Western印迹法分别检测蛋白激酶B(Akt)、糖原合成酶激酶3β(GSK-3β)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)mRNA及蛋白活性水平。结果:缺血再灌注诱导细胞损伤后,细胞内Akt(Ser473)、GSK-3β(Ser9)水平下降,Caspase-3基因及蛋白水平均上调,与正常对照组相比,差异有统计学意义(P<0.05),而Akt、GSK-3β基因水平不变(P>0.05);rHuEPO各干预组与缺血再灌注损伤组相比,细胞内Akt(Ser473)、GSK-3β(Ser9)表达水平上调,Caspase-3基因及蛋白水平均下调,预先给药组调节幅度最大,即时给药组次之,延迟给药组上调幅度最小,差异均有统计学意义(P<0.05);同时预先给药组(9.17%±0.35%)、即时给药组(12.47%±0.86%)和延迟给药组(15.63%±0.75%)细胞凋亡率明显低于缺血再灌注损伤模型组(17.97%±1.06%),差异均有统计学意义(P<0.05)。结论:rHuEPO可能通过增强Akt活性,抑制其下游因子GSK-3β蛋白活性,从而抑制其下游凋亡蛋白酶Caspase-3活性的变化,减轻了抗霉素A诱导的人肾小管上皮细胞的缺血再灌注损伤。Objective：To explore the protective effects and mechanisms of recombinant human erythro-poietin（rHuEPO）on renal tubular epithelial cell apoptosis induced by ischemia-reperfusion inju-ry.Methods：The model of ischemia-reperfusion（I/R）injury was established through ischemia for one hour and reperfusion for two hours in cultured renal tubular epithelial（HK-2）cells.HK-2cells were cultured and then divided into five groups as following：①control group,②I/R group：receiving antimycin A 10μmol/L and A23187 1μmol/L,③ pre-dosage group：I/R ＋ rHuEPO 30min before ischemia,④instant-dosage group：I/R＋rHuEPO 5min before reperfu-sion,and⑤delayed-dosage group：I/R＋rHuEPO 30min following reperfusion.The apoptotic ratio of HK-2was monitored by FCM.The expression of Akt,GSK-3βand caspase-3mRNA were assessed by RT-PCR and activities of Akt,GSK-3βand caspase-3 were assessed by Western blot analyses.Results：① Compared with control group,the apoptosis ratio of HK-2 increased, expression of phospho-Akt and phospho-GSK-3β markedly decreased and activities of Caspase-3 increased in I/R group（P0.05）.②Compared with I/R group,the administration of rHuEPO before ischemia or just after ischemia significantly reduced the extent of apoptotic cells,and was associated with maintenance of improved Akt（Ser473）,GSK-3β（Ser9）levels and decreased Caspase-3 activity（P0.05）.Conclusion：The early administration of rHuEPO at ischemia could protect HK-2 from apoptosis induced by ischemia-reperfusion injury through Akt/GSK-3β/caspase-3signaling pathway.

关 键 词：重组人促红细胞生成素 缺血再灌注损伤 蛋白激酶B 糖原合成酶激酶3Β 肾小管上皮细胞 细胞凋亡 

分 类 号：R692.6[医药卫生—泌尿科学]