糖尿病肾病患者尿源性干细胞的分离鉴定及与健康人尿源性干细胞的细胞生物学比较研究

Isolationand Identification of Urine Derived Stem Cells from Patients with Diabetic Nephropathy and Comparison with Urine Derived Stem Cells from Healthy Peoplein Biological Characteristics

干细胞移植是糖尿病肾病新的治疗途径,但异种干细胞存在伦理及免疫排斥等问题。该研究从糖尿病肾病患者尿液中提取出一种类似干细胞的细胞,称之为糖尿病病人尿源性干细胞(USCs from patients with diabetic nephropathy,D-USCs),并比较了与健康人尿源性干细胞(urine derived stem cells,USCs)生物学特性的差异。通过观察细胞形态,流式细胞术检测细胞表面标志物,茜素红及油红O染色分别检测成骨和成脂分化潜能,以鉴定其干细胞特性。通过绘制生长曲线比较D-USCs与USCs增殖能力,人促血管生成因子检测试剂盒检测细胞外分泌因子的表达情况,流式细胞术检测细胞凋亡率的差异。结果表明,此类细胞可以在体外分离培养,连续传代生长,细胞形态保持米粒状,表达间充质干细胞标志物(包括CD 24、CD 29、CD 73、CD 90、CD 105)、周细胞标志物(CD 146),不表达造血干细胞标志物(包括CD 31、CD 34、CD 45),具有成骨和成脂分化的潜能。与USCs相比,D-USCs的增殖能力受到损伤,分泌因子种类基本保持一致,但数量上有所减少,细胞凋亡率升高。综上所述,该研究成功从糖尿病肾病病人尿液中提取出尿源性干细胞,为干细胞移植治疗肾损害提供了可能的新的细胞来源,避免了异体免疫排斥反应。

Stem cell transplantation is a new therapy for diabetic nephropathy, but there are some problems such as ethical and immune rejection of xenograft. In present study, we extracted a type of cell which was similar to stem cells from the urine of diabetic nephropathy patients, called urinary stem cell from patients with diabetic nephropathy（D-USCs）. And we compared the biological characteristics between D-USCs and urine derived stem cells（USCs） of healthy people. The potential differentiation ability of the cells was examined by the analysis of cell morphology and surface markers via flow cytology and the staining of alizarin red and oil red O, respectively. We also mapped the growth curve to compare D-USCs and USCs of healthy people proliferative capacity. Human angiogenic factor assay kit was applied to examine the expression of extracellular secretion factor, performed flow cytology to detect cell apoptosis. Our results showed that the cells could be cultured in vitro continuously, and that their morphology remained the size of the grains. These cells were shown to expressing mesenchymal stem cell markers（including CD 24, CD 29, CD 73, CD 90, CD 105） and weakly cell markers（CD 146） in a lower level. And these cells did not express hematopoietic stem cell markers（including CD 31, CD 34 and CD 45）. It also had bone and lipid differentiation potential. Compared with USCs, the proliferative ability of D-USCs was damaged, the secretion factor was basically consistently, the number was decreased and the apoptosis rate was increased. In summary, our results showed that D-USCs were successfully extracted from urine of patients with diabetic nephropathy. Our findings showed that D-USCs might provide a new source of cells for the treatment of renal damage by stem cell transplantation with avoiding allogeneic immune rejection.