间充质干细胞对2型糖尿病小鼠糖尿病肾病进展的影响及其机制

Effects of mesenchymal stem cells on the progression of diabetic nephropathy in type 2 diabetic mice and its mechanism

目的 探讨脐带间充质干细胞(UC-MSCs)早期注射对2型糖尿病(T2DM)小鼠糖尿病肾病进展的影响及其可能机制。方法 选取20只5周龄雄性db/db小鼠构建T2DM小鼠模型,随机分为干细胞治疗组(MSCs组,n=10)和T2DM组(n=10);另设置鼠龄匹配的db/m小鼠为正常对照组(NC组,n=10)。MSCs组小鼠行连续6周的人UCMSCs(1×106/0.2 ml生理盐水)尾静脉输注治疗,NC组及T2DM组尾静脉输注等体积生理盐水,每周1次,连续输注6次。每周监测各组小鼠的血糖和体重。第6次治疗结束后,心脏灌流处死各组小鼠并采集血液、留取肾脏组织,行生化和组织病理学检查。采用Western blotting检测各组肾脏沉默信息调节因子1(SIRT1)蛋白以及紧密连接蛋白1(Claudin-1)的表达情况,并采用免疫组化检测SIRT1、Claudin-1、Ⅰ型胶原蛋白(Col Ⅰ)、Col Ⅳ的表达情况。电镜观察各组小鼠肾小球的形态。取对数生长期HK-2细胞,将细胞分为对照组、高糖诱导模型组(HG组)、高糖诱导siRNA组(HG+siRNA组)、MSCs组(HG+MSC组)、SIRT-1 siRNA组(HG+MSC+siRNA组),采用Western blotting检测各组凋亡相关蛋白的表达水平。结果 第6次治疗后,与NC组[(7.66±0.37) mmol/L]比较,T2DM组、MSCs组血糖水平[分别为(32.54±0.36)、(29.74±1.04) mmol/L]均明显升高(P<0.01);而与T2DM组比较,MSCs组血糖水平明显降低(P<0.05)。病理检查结果显示,与NC组比较,T2DM组出现明显的肾小球硬化,基底膜弥漫性增厚,肾脏间质纤维化程度明显增加;与T2DM组比较,MSCs组肾小球硬化程度减轻,系膜外基质沉积延缓,肾脏间质纤维化程度改善。Western blotting及免疫组化结果显示,与T2DM组比较,MSCs组SIRT1蛋白表达水平明显增高(P<0.01),Claudin-1蛋白表达水平明显降低(P<0.01),α-SMA蛋白表达水平明显降低(P<0.05)。此外,免疫组化结果显示,MSCs组Col Ⅰ、Col Ⅳ阳性面积百分比较T2DM组明显降低(P<0.01)。电镜结果显示,与NC组比较,T2DM组出现明显的肾小球滤过屏障破坏,基底膜弥漫性增厚,足突广泛融合和消失;与T2DM组比较,MSCs组肾小球足突结构规则、完整。Western blotting结果显示,与对照组比较,HG组SIRT-1蛋白含量明显降低(P<0.01),凋亡相关的细胞色素C蛋白含量明显增高(P<0.01);与HG组比较,HG+MSC组SIRT-1蛋白含量明显增高(P<0.01),凋亡相关的Bax和细胞色素C蛋白含量明显降低(P<0.01);与HG+MSC组比较,HG+MSC+siRNA组SIRT-1蛋白含量降低(P<0.01),凋亡相关的Bax和细胞色素C蛋白含量明显增高(P<0.01)。结论 UC-MSCs治疗可能通过上调SIRT1的表达、下调Claudin-1的表达,对T2DM小鼠发挥肾脏保护功能并且延缓糖尿病肾病的发生发展。

Objective To investigate the effect of early injection of umbilical cord mesenchymal stem cells(UC-MSCs)on the progression of diabetic nephropathy(DN)in type 2 diabetes mellitus(T2DM)mice and its mechanism.Methods Twenty 5-week-old male db/db mice were used to establish T2DM model and randomly divided into stem cell treatment group(MSCs group,n=10)and T2DM group(DM group,n=10);In addition,age-matched db/m mice were set as normal control group(NC group,n=10).Human UC-MSCs(1×106/0.2 ml saline)were infused into the tail vein of mice in MSCs group for 6 weeks,while the same volume of saline was infused into the tail vein of mice in NC and T2DM groups once a week for 6 consecutive times.Blood glucose and body weight of mice in each group were monitored weekly.At the end of the sixth treatment,the mice in each group were killed by heart perfusion,and the blood and kidney tissues were collected for biochemical and histopathological examination.Western blotting was used to detect the expression of silent information regulator 2 homolog 1(SIRT1)and Claudin-1,and immunohistochemistry was used to detect the expression of SIRT1,Claudin-1,type 1 collagen(ColⅠ)and ColⅣ.The morphology of glomeruli was observed by electron microscope.HK-2 cells in logarithmic growth phase were divided into control group,high glucose induced model group(HG group),high glucose induced siRNA group(HG+siRNA group),MSC groups(HG+MSC group)and SIRT-1 siRNA group(HG+MSC+siRNA group).The expression levels of apoptosis-related proteins were detected by Western blotting.Results After the sixth treatment,compared with NC group[(7.66±0.37)mmol/L],the levels of blood glucose increased significantly(P<0.01)in T2DM group and MSCS group[(32.54±0.36)mmol/L,(29.74±1.04)respectively];Compared with T2DM group,the level of blood glucose in MSCs group decreased significantly(P<0.05).Pathological results showed that compared with NC group,glomerular sclerosis,diffuse thickening of basement membrane and renal interstitial fibrosis were significantly increased in T2DM group;compared with T2DM group,glomerular sclerosis,deposition of mesangial extracellular matrix and renal interstitial fibrosis were alleviated in MSCs group.The results of Western blotting and immunohistochemistry showed that compared with T2DM group,the expression level of SIRT1 protein in MSCs group increased significantly(P<0.01),the expression of Claudin-1 protein decreased significantly(P<0.01),and ofα-smooth muscle actin(α-SMA)protein decreased significantly in MSCs group(P<0.05).In addition,the immunohistochemical results showed that the percentage of ColⅠand ColⅣpositive area in MSCs group was significantly lower than that in T2DM group(P<0.01).Compared with NC group,T2DM group showed marked destruction of glomerular filtration barrier,diffuse thickening of basement membrane,and extensive fusion and disappearance of foot processes.Compared with T2DM group,the structure of glomerular foot process in MSCs group was regular and complete.Western blotting results showed that SIRT-1 protein content was significantly lower in HG group than that in control group(P<0.01),while apoptosis-related Cytochrome-C protein contents were significantly higher in HG group than that in control group(P<0.01).Compared with HG group,the protein content of SIRT-1 in HG+MSC group was significantly higher(P<0.01),and the content of apoptosis-related Bax and Cytochrome-C protein decreased significantly(P<0.01).Compared with HG+MSC group,the protein content of SIRT-1 in HG+MSC+siRNA group was significantly decreased(P<0.01),while the protein content of apoptosis-related Bax and Cytochrome-C increased significantly(P<0.01).Conclusion UC-MSCs treatment may play a protective role for kidney of T2DM mice and delay the development of DN by up-regulating the expression of SIRT1 and down-regulating the expression of Claudin-1.