红细胞生成素对模拟急性肾损伤微环境下培养骨髓间充质干细胞增殖的影响及机制探讨  

Effect of erythropoietin on mesenchymal stem cells proliferation in vitro under acute kidney injury microenvironment and its mechanism

作　　者：刘楠梅[1] 田军[1] 王巍巍[1] 程劲[1] 胡大勇[1] 张金元[1] 

机构地区：[1]解放军第四五五医院肾内科南京军区肾脏病专科中心,上海200052

出　　处：《中华肾脏病杂志》2011年第2期112-117,共6页Chinese Journal of Nephrology

基　　金：基金项目：南京军区122工程学科带头人培养基金;南京军区医药卫生基金（07M023）;南京军区科技创新重点项目基金（092006）;上海市卫生局科研课题青年基金（2009Y119）

摘　　要：目的观察经红细胞生成素（EPO）干预后，体外模拟急性肾损伤（AKI）微环境下培养骨髓间充质干细胞（MSC）的分裂增殖情况，并探讨产生这种变化的可能机制。方法抽取C57BL／6小鼠的骨髓，经Percoll密度梯度离心联合贴壁培养法分离纯化出小鼠MSC（mMSC），以流式细胞仪鉴定。夹闭雄性C57BL／6小鼠双侧肾蒂30min再开放30rain的方法制作AKI鼠模型，即刻取双侧肾脏皮质制作缺血再灌注（IR）肾脏匀浆上清。取扩增第3代的mMSC分组培养：A组：含10％胎牛血清的低糖DMEM培养基；B组：含10％胎牛血清的低糖DMEM培养基＋IR肾脏匀浆上清，体外模拟AKI微环境干预mMSC；C组：含10％胎牛血清的低糖DMEM培养基＋IR肾脏匀浆上清＋不同浓度EPO（1、5、10、50U／m1）。培养1、3、5、7d。CCK-8法检测各组培养mMSC的增殖，TUNEL法检测mMSC凋亡，Western印迹法检测mMSC表面EPO受体（EPOR）及增殖凋亡相关信号通路蛋白的表达。结果CCK-8法检测显示，经IR肾脏匀浆上清干预后，不同时间点mMSC的增殖效应显著减弱（P〈0．01），而TUNEL检测表明其胞核染色阳性细胞的百分比显著高于A组（P〈0．01）；给予EPO干预后，mMSC的增殖能力增强，胞核染色阳性细胞百分比降低，具有浓度依赖效应。Western印迹结果显示，第3代mMSC表面存在EPOR表达，培养5d后EPOR相对表达量为0．092±0．015。EPO以剂量和时间依赖性降低AKI微环境下凋亡相关蛋白caspase-3的表达，同时上调凋亡抑制蛋白Bcl-2的表达。用10U／mlEPO干预5d后，相对于B组，磷酸化蛋白酪氨酸激酶2及磷酸化信号转导和转录因子的表达均显著上调（均P〈0．01）。结论EPO能促进体外AKI微环境干预下培养mMSC的增殖，减轻其凋亡，该效应经EPOR介导，并与增殖凋亡相关信号通路蛋白有关。Objective To investigate the effect of erythropoietin （EPO） on mesenchymal stem cells （mMSCs） proliferation under acute kidney injury （AKI） microenvironment,and to study its possible mechanism. Methods C57BL/5 mice＇s MSCs （mMSCs） were isolated by Percoll density gradient centrifugation and adherence cultivation. Surface markers were identified by flowcytometry. AKI mice models were made by clamping bilateral renal pedicles for 30 minutes and reopening for 30 minutes. Then both renal cortex was drew immediately to make IR kidney homogenate superuatant. P3-mMSCs were divided into different groups： Group A： low glucose DMEM medium with 10% fetal bovine serum; Group B： low glucose DMEM medium with 10% fetal bovine serum plus IR kidney homogenate supernatant; Group C： low glucose DMEM medium with 10% fetal bovine serum plus IR kidney homogenate supernatant and different concentrations of EPO （1, 5, 10, 50 U/ml）. Each group was incubated for 1 d, 3 d, 5 d, 7 d. Proliferation of mMSCs was detected by CCK-8, and apoptosis was detected by TUNEL. The protein expression of erythropoietin receptor（EPOR） and the proteins of proliferation/apoptosis related signal pathway were examined by Western blotting. Results Under IR kidney homogenate supernatant, the proliferation abihty of mMSCs decreased significantly （P〈0.01）, while the apoptoic percentage was significantly higher than that of Group A （P〈0.01）. After intervention of EPO, mMSCs proliferation enhanced, at the same time, the apoptoic percentage decreased, in a dose-dependent manner. EPOR was positive in P3-mMSCs by Western blotting. EPO decreased the expression of caspase-3 in mMSCs under AKI microenvironment in a dose- and time-dependent manner, but increased the expression of Bcl-2. Cultured for 5 d, the expression of phosphor-Janus kinase2（p-JAK2） [（0.641± 0.028） vs （0.456±0.012）] and phosphor-signal transducer and activator of transcription（p-STAT5） [（0.398±0.016） vs （0.209±0.020）] was signific

关 键 词：红细胞生成素 间质干细胞移植 细胞增殖 细胞凋亡 急性肾损伤 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]