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

发布时间：2018-03-26 21:05

本文选题：糖尿病肾病　切入点：骨髓间充质干细胞　出处：《中国免疫学杂志》2017年05期

【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(P0.05);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(P0.05)。2组治疗前氧化应激ROS、MDA、SOD指标差异无统计学意义(P0.05);干细胞治疗组治疗后氧化应激ROS、MDA、SOD指标,低于对照组(P0.05)。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,植入骨髓间充质干细胞能保护机体肾脏,抑制糖尿病肾脏的氧化应激反应。
[Abstract]:Objective: to observe the isolation, identification and mechanism of bone marrow mesenchymal stem cells (BMSCs) in diabetic rats. Methods: bone marrow mesenchymal stem cells (BMSCs) were isolated from 31 rats by adherent culture. A diabetic rat model was established by intraperitoneal injection of streptozotocin (streptozotocin) and stem cell therapy. Rats in the control group were treated with insulin combined with probucol. Stem cell group was implanted with stem cell therapy. Before and after treatment, 24 h urinary protein excretion, creatinine clearance rate and weight ratio of kidney were detected in rats of two groups. The renal tissue was observed by PAS staining. Western blot was used to detect the level of genes and proteins related to renal tissue. Results: after 24 h culture of primary bone marrow mesenchymal stem cells, the distribution of cells was not uniform and the cells were clustered in culture flask. After 7 days of culture, the cells were in the shape of long spindle shape, elliptical shape, strong refraction, and the intercellular fusion was covered with bottle bottom, the morphology of bone marrow mesenchymal stem cells was uniform and fusiform after subculture. Outline clearance, strong refraction and rapid growth. The results of real-time PCR analysis of some cells showed that the positive expression of CD31 was negative and the urine protein excretion, creatinine clearance rate and weight of kidney were 24 h after treatment in the observation group. The results of PAS staining showed that glomerular structure was clear, morphology was regular, glomerular cell arrangement was regular, basement membrane and Bowman's sac were clear, and glomerular volume in control group was increased. The Mesangial area was widened and the matrix was increased obviously. The levels of Bcl-2 protein and Caspase-3 protein were significantly increased after treatment in stem cell treatment group, and Bcl-2 protein and Caspase-3 protein level in control group were significantly decreased after treatment. In the control group, the levels of Bcl-2 protein and Caspase-3 protein were increased significantly after treatment, and the oxidative stress rosier MDA-SOD finger was increased before treatment in the control group. There was no significant difference in the standard value (P 0.05). The oxidative stress rosier MDA-SOD index was found in the stem cell treatment group after treatment. Conclusion: rat bone marrow mesenchymal stem cells can be isolated by adherent culture, labeled with green fluorescent protein in vitro, and transplanted into bone marrow mesenchymal stem cells to protect kidney. Inhibition of oxidative stress in diabetic kidneys.
【作者单位】：河北医科大学第一医院;
【基金】：河北省卫生厅重点科技研究计划(160211K102)
【分类号】：R587.2;R692.9

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