骨髓间充质干细胞干预对缺血再灌注诱导肾小管上皮细胞凋亡的影响  

Effects of Mesenchymal Stem Cells on Renal Tubular Epithelial Cells' Apoptosisin Mice Under Ischemia/reperfusion 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455医院肾内科,南京军区肾脏病专科中心,上海200052

出　　处：《中国中西医结合肾病杂志》2010年第7期581-585,I0001,共6页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：中国人民解放军南京军区医药卫生基金资助项目(No.07M023);中国人民解放军南京军区122工程学科带头人培养基金资助项目;上海市卫生局科研基金资助项目(No.2009Y119)

摘　　要：目的:观察骨髓间充质干细胞(mesenchymal stemcells,MSCs)对缺血再灌注(ischemia/reperfusion,I/R)诱导的急性肾损伤模型小鼠肾小管上皮细胞(renal tubular epithelial cells,RTECs)凋亡的保护作用及机制。方法:Percoll密度梯度离心结合贴壁培养法有效分离出C57BL/6小鼠的骨髓间充质干细胞(mMSCs)。雄性C57BL/6小鼠45只,分为正常对照组(15只)、I/R组(15只、夹闭双侧肾蒂30min开放)、I/R+mMSCs组(15只、夹闭双侧肾蒂30min开放的同时尾静脉注射mMSCs)。造模并干预后,于1d、2d、3d、7d、14d分别处死部分小鼠,留取动脉血及肾组织,检测血尿素氮(BUN)及肌酐(Scr),制作肾组织切片行HE染色进行肾小管损伤程度评分,TUNEL法检测RTECs的凋亡,Western Blot法检测建模后第2天肾小管组织中Caspase-3、Bcl-2蛋白水平的表达。结果:I/R后小鼠的BUN及Scr均显著高于正常对照组,但同一时间点I/R+mMSCs组小鼠的BUN及Scr水平较I/R组为低,以术后第7天差异最为显著(P<0.01),肾小管损伤程度评分也有着显著降低。TUNEL检测表明I/R可诱导RTECs发生明显的凋亡,以术后第2天凋亡指数(apoptosis index,AI)最高(46.71±11.20)(P<0.01),而mMSCs干预可显著降低各时间点RTECs的凋亡水平(P<0.05或P<0.01),至14d时AI基本恢复至对照组水平。Western blot进一步显示:I/R+mMSCs组小鼠的肾组织中Caspase-3蛋白水平明显下降(1.16±0.33,P<0.01),而Bcl-2水平却有明显增高(0.94±0.27,P<0.01)。结论:移植的MSCs对I/R诱导的肾损害小鼠RTECs凋亡具有抑制作用,从而有利于肾小管损伤的早期恢复,其保护机制可能与抑制Caspase-3表达,上调Bcl-2表达有关,但其细胞学和分子机制还有待进一步研究。Objective：To observe exogenous mesenchymal stem cells （MSCs）intervening apoptosis in renal tubular epithelial cells （RTECs）and to explore the mechanism by ischemia/reperfusion induced of acute renal injury in mice models.Methods：C57BL/6 mice＇s mMSCs were successfully isolated by Percoll density gradient centrifugation and adherence cultivation.Forty-five healthy male C57BL/6 mice were scattered into the control group（15）,the I/R group（15,clamping bilateral renal pedicles and then reopening after 30 minutes）,and the I/R＋mMSCs group（15,clamping bilateral renal pedicles and then reopening after 30 minutes,meanwhile,mMSCs were injected through caudal vein into the body of model mice）.On the 1st day,2nd day,3rd day,7th day and 14th day after model established,mice were sacrificed（3/each group） and blood and kidneys samples were collected.The blood sample was for the detection of serum urea nitrogen（BUN） and creatinine （Scr）.The kidneys samples were dyed with HE for observation of the pathological changes.In situ RTECs＇ apoptosis were detected by TUNEL,the protein level of Caspase-3,Bcl-2 in renal tubules on the 2nd day after model established were detected by Western blot.Results：The level of BUN and Scr in the I/R group and the I/R＋BrdU-mMSCs group were much higher than those in the control group.While comparing with that in the I/R group,the level of BUN and Scr in I/R＋mMSCs were much lower.ATN score are also significantly lower.TUNEL result showed that I/R could induce RTECs＇ apoptosis,the level of apoptotic index reached the peak on the 2nd day （46.71±11.20）（P0.01）.While mMSCs＇ intervention could significantly relieve RTECs＇ apoptosis（P0.05 or P0.01）,the apoptotic index of this group was almost same to the control group after 14 days.Western blot analysis revealed：contrasted with those in the I/R group,the level of Caspase-3 in the I/R＋mMSCs group decreased notably（1.16±0.33,P0.01）,while the level of Bcl-2 increased significantly（0.94±0

关 键 词：急性肾损伤 骨髓间充质干细胞 缺血再灌注 肾小管上皮细胞 

分 类 号：R692.6[医药卫生—泌尿科学]