大鼠椎间盘巢源性干细胞的体外分离、培养和特性鉴定

In vitro isolation, culture and characteristics identification of rat stem cells derived from potential stem cell niches of the intervertebral disc

目的:对大鼠椎间盘干细胞巢来源的干细胞(ISN-SCs)进行体外分离、培养和特性鉴定。方法:以10周龄雄性SD大鼠作为研究对象,按照解剖区域分离椎间盘干细胞巢组织(骺板外周软骨膜部分),用Ⅱ型胶原酶消化获取细胞后进行体外培养,选用第四代细胞进行干细胞相关特性的鉴定:采用流式细胞技术测定细胞周期;MTT法测定增殖曲线;流式细胞技术检测干细胞相关表型;q PCR检测干细胞相关基因的表达水平;细胞三系诱导分化培养后采用茜素红染色及钙钴染色检测成骨分化能力,阿利新蓝染色检测成软骨分化能力,油红O染色检测成脂肪分化能力,q PCR检测多向分化相关基因的表达水平。结果:大鼠ISN-SCs呈成纤维样细胞,多触角,具有贴壁能力,是一种慢周期细胞,高表达干细胞相关阳性表面抗原分子CD29、CD90、CD44,低表达干细胞相关阴性表面抗原分子CD34、CD45、CD19、CD11b;成骨诱导分化后茜素红染色和钙钴染色均为阳性,成软骨分化后阿利新蓝染色阳性,成脂肪分化后油红O染色阳性,且各分化相关基因(成骨:Runx2、OPN、OCN;成软骨:SOX-9、COL2a1、ACAN;成脂肪:PPARγ、C/EBPα)表达水平较对照组显著增高,其与骨髓间充质干细胞(BMSCs)具有相似的干细胞相关基因(NANOG、SOX-2、OCT-4)表达水平。结论:ISN-SCs具备干细胞的生物学特性,在体外经诱导后可以向软骨细胞及脂肪细胞转化,可为椎间盘的自体生物学修复研究提供良好的细胞来源。

Objectives： To investigate the isolation, culture and identification of rat stem cells derived from potential stem cell niches of the intervertebral disc（ISN-SCs）. Methods： Sprague Dawley rats（male, 10 weeks old） were used as the experimental animals in this study. Tissues of potential stem cell niches of the intervertebral disc（ISN） were separated from the anatomical regions（the perichondrium areas outside of the epiphyseal plate） before being digested by type Ⅱ collagenase. After being harvested, the cells were cultured and expanded in vitro, and passage 4 populations were used for further analysis. The flow cytometry was used to detect the cell cycle and immunophenotypes of ISN-SCs. The MTT method was used to determine proliferation curve of the cells. q PCR analysis was used to determine the relative m RNA expression level of stem cell-related genes. After being cultured in the differentiation medium, the cells were stained by alizarin red and calcium cobalt for osteogenesis, alcian blue for chondrogenesis, and oil red O for adipogenesis. Also,the relative expression of multipotential differentiation-related genes were determined by q PCR. Results： Rat ISN-SCs were fibroblast-like cells with multi-tentacles forming vortexes partially. They were slow-cycling cells with the ability of adherence to plastic. Meanwhile, high expression levels of stem cell-related positive antigen molecules（CD29, CD90, CD44） and low expression levels of negative antigen molecules（CD34, CD45, CD19,CD11b） were observed. Besides, positive results were detected in alizarin red and calcium cobalt staining for osteogenesis, alcian blue staining for chondrogenesis, and oil red O staining for adipogenesis. Also, the differentiation-related gene（osteogenesis： Runx2, OPN and OCN; chondrogenesis： SOX-9, COL2a1 and ACAN;adipogenesis： PPARγ and C/EBPα） expression levels significantly increased in the corresponding induction group compared with the controls. Moreover, ISN-SCs expressed comparable stem cell-related genes（NANOG,SOX-2, OCT-4） with bone marrow mesenchymal stem cells（BMSCs）. Conclusions： ISN-SCs belong to the MSC family and possess multipotent differentiation capacities. These findings will contribute to further studies on the migration of ISN-SCs into intervertebral disc（IVD） and its self-repair effect. Meanwhile, a new perspective on biological strategy for IVD self-regeneration is proposed.