红细胞生成素预处理脂肪间充质干细胞移植治疗大鼠糖尿病肾病

Erythropoietin preconditioned adipose-derived mesenchymal stem cell transplantation for treating diabetic nephropathy in rats

背景:研究证实,糖尿病肾病的发生、发展与肾小管上皮细胞转分化及肾间质纤维化密切相关。目的:探讨红细胞生成素干预的脂肪间充质干细胞对糖尿病肾病大鼠肾脏的保护作用及其机制。方法:获取和培养人脂肪间充质干细胞并进行多向分化能力鉴定;采用Transwell体外迁移体系,观察不同浓度红细胞生成素(0,5,20,50IU/m L)对脂肪间充质干细胞向高糖诱导大鼠肾小管上皮细胞系迁移的影响;将48只SD大鼠(徐州医学院动物实验中心提供)随机分为正常对照组、糖尿病模型组、脂肪间充质干细胞组、脂肪间充质干细胞+20IU/mL红细胞生成素组,每组12只。经尾静脉注射150μL细胞总数为3.5×105个脂肪间充质干细胞或20 IU/mL红细胞生成素干预的脂肪间充质干细胞,14周后采用Western blot检测肾组织中转化生长因子β1、α-平滑肌肌动蛋白、E-钙黏蛋白、基质金属蛋白酶9及基质金属蛋白酶抑制剂1的表达。结果与结论:(1)红细胞生成素干预使脂肪间充质干细胞的迁移数量明显增加且呈浓度依赖性(P<0.05),以20IU/mL浓度最为显著;NRK-52E细胞内基质细胞衍生因子1蛋白表达趋势与脂肪间充质干细胞迁移趋势基本一致;(2)与糖尿病模型组比较,脂肪间充质干细胞及脂肪间充质干细胞+20IU/mL红细胞生成素组转化生长因子β1、α-平滑肌肌动蛋白及基质金属蛋白酶抑制剂1蛋白表达均显著下降(P <0.05),E-钙黏蛋白、基质金属蛋白酶9蛋白表达显著上升(P <0.05),且脂肪间充质干细胞+红细胞生成素组变化更明显(P <0.05);(3)结果表明,红细胞生成素可以促进脂肪间充质干细胞的定向趋化,且在红细胞生成素作用下脂肪间充质干细胞可以更好地抑制肾小管上皮细胞转分化,减轻细胞外基质沉积,延缓糖尿病肾纤维化。

BACKGROUND:Studies have confirmed that the occurrence and development of diabetic nephropathy are related to tubular epithelial cell transdifferentiation and renal interstitial fibrosis.OBJECTIVE:To investigate the protective effect of erythropoietin preconditioned adipose-derived mesenchymal stem cells on diabetic nephropathy rat’s kidney and to evaluate its potential mechanisms.METHODS:Human adipose-derived mesenchymal stem cells were harvested and identified for cell multilineage differentiation.TranswelI migration system was used to observe the effects of erythropoietin of different concentrations(0,5,20,50 IU/mL)on the migration of adipose-derived mesenchymal stem cells to high glucose-induced rat renal tubular epithelial cell lines.Forty-eight Sprague-Dawley rats(provided by the Animal Experimental Center of Xuzhou Medical University in China)were randomly divided into normal control,diabetic model group,cell transplantation group and erythropoietin preconditioning group(n=12 per group).Adipose-derived mesenchymal stem cells(3.5×105,150μL)alone or erythropoietin(20 IU/mL)preconditioned adipose-derived mesenchymal stem cells were injected into the tail vein of rats in the latter two groups,respectively.The protein levels of transforming growthβ1,α-smooth muscle actin,E-cadherin,matrix metalloproteinase-9 and matrix metalloproteinase inhibitor-1 in the renal tissues were detected by western blot assay at 14 weeks after cell transplantation.RESULTS AND CONCLUSION:Erythropoietin preconditioning markedly increased the number of migrated adipose-derived mesenchymal stem cells in a concentration-dependent manner(P<0.05),and the ability peaked at a concentration of 20 IU/ml.The expression of stromal cell-derived factor-1 protein in NRK-52E cells was consistent with the migration trend of adipose-derived mesenchymal stem cells.Compared with the diabetic model group,the levels of transforming growthβ1,α-smooth muscle actin,and matrix metalloproteinase inhibitor-1 were increased dramatically,while the levels of E-cadherin and matrix metalloproteinase-9 decreased remarkably in the other groups(P<0.05),especially in erythropoietin preconditioning group(P<0.05).In conclusion,erythropoietin could promote the directional chemotaxis of adipose-derived mesenchymal stem cells,and moreover,adipose-derived mesenchymal stem cells could inhibit the transdifferentiation of renal tubular epithelial cells,reduce the deposition of extracellular matrix and delay the progression of diabetic renal fibrosis under the action of erythropoietin.