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

doi:10.3969/j.issn.2095-4344.1601

Abstract

Abstract:

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, 150 μL) 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 growth β1, 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 growth β1, 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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Diabetic Nephropathies, Adipose Tissue, Mesenchymal Stem Cells, Erythropoietin, Tissue Engineering

CLC Number:

Wang Disheng, Kong Liusha, Wang Jia, Li Xia. Erythropoietin preconditioned adipose-derived mesenchymal stem cell transplantation for treating diabetic nephropathy in rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(9): 1370-1376.

Figures/Tables 2

2.1 ADMSCs的形态及多向分化能力体外培养的第3代ADMSCs为贴壁细胞，呈成纤维细胞样梭形，见图1A；成骨诱导培养基诱导21 d，茜素红染色后倒置相差显微镜下可见红色钙盐沉积颗粒，见图1B；成脂诱导培养基诱导21 d，油红O染色后倒置相差显微镜下可见红色脂滴，见图1C。 2.2 ADMSCs体外趋化及NRK-52E细胞内基质细胞衍生因子1表达水平显微镜下紫色为迁移的ADMSCs，模拟的高糖环境对ADMSCs有趋化效应，EPO干预使ADMSCs的迁移数量明显增加且呈浓度依赖性(P < 0.05)，以20 IU/mL浓度最为显著，但进一步增加EPO浓度(50 IU/mL)，则对ADMSCs迁移数无明显影响(P > 0.05)，见图2；NRK-52E细胞内基质细胞衍生因子1蛋白表达趋势与ADMSCs迁移趋势基本一致，见图3。 2.3 免疫印迹法检测肾组织中相关蛋白的表达正常对照组的转化生长因子β1蛋白表达微弱；糖尿病肾病组转化生长因子β1的蛋白表达明显增加(P < 0.05)，ADMSCs组和ADMSCs+EPO组与糖尿病肾病组相比，转化生长因子β1蛋白表达下降(P < 0.05)，且ADMSC+EPO组下降更为显著(P < 0.05)。α-平滑肌肌动蛋白表达趋势与转化生长因子β1蛋白表达趋势相似。正常对照组表达一定量的E-钙黏蛋白，糖尿病肾病组E-钙黏蛋白表达水平明显下降(P < 0.05)，而在ADMSCs和ADMSCs+EPO干预下E-钙黏蛋白表达上调(P < 0.05)，且ADMSCs+EPO组上调更为明显(P < 0.05)，见图4。基质金属蛋白酶抑制剂1在糖尿病肾病组表达较正常对照组明显增多(P < 0.05)，而在ADMSCs组及ADMSCs+ EPO组表达较糖尿病肾病组显著减少(P < 0.05)，且在ADMSCs+EPO组减少更为明显(P < 0.05)。糖尿病肾病组基质金属蛋白酶9表达较正常对照组显著下降(P < 0.05)，ADMSCs组及ADMSCs+EPO组基质金属蛋白酶9表达有所上调(P < 0.05)，且ADMSCs+EPO组上调更明显(P < 0.05)，见图5。"

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