儿童原发性肾病综合征患者尿源干细胞分离培养技术研究

Method of Isolation and Culture of Urine-Derived Stem Cells from Pediatric Patients with Primary Nephrotic Syndrom

该研究探讨建立儿童原发性肾病综合征(primary nephrotic syndrome,PNS)尿源干细胞(urine-derived stem cells from pediatric patients with PNS,p-USCs)分离培养技术。体外分离培养细胞,进一步观察细胞形态、分析细胞生长曲线及细胞周期、检测细胞表面标志物表达。采用茜素红及油红O染色检测细胞成骨和成脂分化潜能。结果显示,通过离心、贴壁方法从PNS患儿尿液中分离出的细胞,外观米粒样并呈对数生长;细胞表达间充质干细胞表面标志物CD24(cluster of differentiation 24)、CD29、CD73、CD90,但少数细胞表达CD105,周细胞标志物CD146表达阳性,造血干细胞标志物CD34表达阴性;细胞经成骨成脂诱导后,茜素红和油红O染色均呈阳性。以上结果表明,该研究成功建立了PNS患儿p-USCs分离培养的方法,为探讨p-USCs用于儿童PNS早期肾损伤的诊断及难治性肾病的治疗研究提供前期技术方法。

The aim of this study was to establish a method for isolation and culture of urine-derived stem cells （USCs） from pediatric patients with primary nephrotic syndrome （PNS） （p-USCs）. Cells were separated by centrifugation and adherent culture. The morphology, growth curve and cell cycle of cells were observed. The expression of surface markers of cells was analysed by flow cytometry. Osteogenic and adipogenic differentiation potential of cells were detected by alizarin red and Oil Red-O dye staining. We found that cells were grain-like appearance and logarithmic growth. Cells expressed surface markers associated with pericytes （CD 146） and mesenchymal stem cells, such as CD24 （cluster of differentiation 24）, CD29, CD73 and CD90, but a few cells expressed CD105, while didn＇t express hematopoietic stem cell surface marker CD34. Both of osteogenic-differentiated and adipogenic-differentiated cells were positive by alizarin red staining and Oil Red-O staining, respectively. These results showed that the method of isolation and culture USCs from pediatric patients with PNS was established successfully, and provided an initial methods for further investigating the potential application of p-USCs in the diagnosis of early renal injury of PNS patients and the treatment of refractory nephrotic syndrome.