骨髓间充质干细胞分离鉴定及在糖尿病大鼠肾脏保护中的机制研究  

Mesenchymal stem cell isolation,identification and protection mechanism in kidney of diabetic rats

作　　者：康岩[1] 李志杰[1] 王丽娜[1] 郭玉卿[1] 刘璠[1] 张洁[1] KANG Yan LI Zhi-Jie WANG Li-Na GUO Yu-Qing LIU Pan ZHANG Jie.(The First Affiliated Hospitol of Hebei Medical University, Shijianzhuang 05031, China)

机构地区：[1]河北医科大学第一医院,石家庄050031

出　　处：《中国免疫学杂志》2017年第5期673-678,共6页Chinese Journal of Immunology

基　　金：河北省卫生厅重点科技研究计划(160211K102)

摘　　要：目的:观察骨髓间充质干细胞分离、鉴定方法及在糖尿病大鼠肾脏保护中的机制。方法:取大鼠31只,1只大鼠采用贴壁培养法分离大鼠骨髓间充质干细胞,利用相差显微镜观察细胞形态,Real-time PCR检测培养细胞表面分子量。30只大鼠采用链脲佐菌素腹腔注射构建糖尿病大鼠模型。根据处理方法不同分为对照组(n=15)和干细胞治疗组(n=15)。对照组大鼠采用胰岛素联合普罗布考治疗,干细胞治疗组植入干细胞治疗,治疗前、后检测2组大鼠空腹血糖、24 h尿蛋白排泄、肌酐清除率、肾重比体重;采用PAS染色观察2组肾组织情况;采用Western blot检测肾脏组织相关基因和蛋白水平。结果:原代骨髓间充质干细胞培养24 h后换液时可见细胞分布不均匀,在培养瓶呈分片聚集,形态不规则。培养7 d后细胞集落多,细胞规则,呈长梭形、椭圆形,折光性强,细胞间相互融合铺满瓶底;骨髓间充质干细胞传代培养后细胞形态均一,呈长梭形,轮廓清除,折光性强,生长迅速,部分细胞呈漩涡状;Real-time PCR检测结果显示:SH2、CD44、CD31呈阳性表达,CD34呈阴性表达;观察组治疗后空腹血糖、24 h尿蛋白排泄、肌酐清除率、肾重比体重,显著低于对照组(P<0.05);干细胞治疗组大鼠肾脏组织PAS染色结果显示:肾小球结构清晰,形态规则,肾小球内细胞排列规则,基底膜和包曼氏囊清晰。对照组大鼠肾小球体积增大,系膜区增宽,基质明显增多;干细胞治疗组治疗后Bcl-2蛋白水平得到显著提高,Bax和Caspase-3蛋白水平得到显著下降。对照组Bcl-2蛋白水平得到显著降低,Bax和Caspase-3蛋白水平得到提高(P<0.05)。2组治疗前氧化应激ROS、MDA、SOD指标差异无统计学意义(P>0.05);干细胞治疗组治疗后氧化应激ROS、MDA、SOD指标,低于对照组(P<0.05)。结论:利用贴壁培养法能分离大鼠骨髓间充质干细胞并利用绿色荧光蛋白进行体外标记,植入骨�Objective：To observe the bone marrow mesenchymal stem cell isolation,identification methods and protection mechanisms in the kidney of diabetic rats.Methods： Thirty-one rats were used to isolate bone marrow mesenchymal stem cells（BMSCs） by adherent culture method.Cell morphology was observed by phase contrast microscope.The surface molecular weight of cultured cells was detected by Real-time PCR.30 rats were induced by intraperitoneal injection of streptozotocin to establish diabetic rat model （n =15）.And stem cell treatment group （n =15） according to the different treatment methods.Rats in the control group were treated with insulin combined with probucol.Stem cell treatment group was implanted with stem cells.Before and after treatment,fasting blood glucose,24 h urinary protein excretion,creatinine clearance rate and kidney weight were measured.PAS staining.And the renal tissue was detected by Western blot.Results： The primary bone marrow mesenchymal stem cells （MSCs） were cultured for 24 hours, and the cells were irregularly distributed in the culture flask.After 7 days of culture,the cells were regular,elongated,elliptical,and strong refraction,and the cells were fused with each other.The morphology of BMSCs was uniform and showed a long fusiform shape,CD44,CD31 and CD34 were observed by RT-PCR.The levels of fasting blood glucose,24 h urine protein excretion and creatinine clearance rate in the observation group were significantly higher than those in the control group （P〈0.05）.The results of PAS staining showed that the glomerular structure was clear,the morphology was regular,the arrangement rules of glomerular cells,the basement membrane and the encapsulation of the glomerular membrane were significantly higher than those of the control group Man忆s capsule clear （P〈0.05）.In the control group,the glomerular volume increased,the mesangial area widened and the stroma increased signifycantly.The Bcl-2 protein level and the Bax and Caspase-3 protein levels were significantly

关 键 词：糖尿病肾病 骨髓间充质干细胞 肾脏保护 相链脲佐菌素 PAS染色 Real-time PCR法 WESTERN BLOT 

分 类 号：R392[医药卫生—免疫学]