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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。
OBJECTIVE: To isolate, culture and characterize ISN-SCs isolated from rat intervertebral disc stem cells. Methods: Ten-week-old male Sprague-Dawley rats were used as research objects. The tissue of the stem cells in the periapical plate was separated according to the anatomic region. The cells were digested with type Ⅱ collagenase and then cultured in vitro. Identification of stem cell-related traits: Cell cycle was determined by flow cytometry; proliferation curves were determined by MTT assay; stem cell-related phenotypes were detected by flow cytometry; q-PCR was used to detect expression levels of stem cell related genes; The ability of osteogenic differentiation was detected by elemental red staining and calcium-cobalt staining. The cartilage differentiation ability was detected by Alizarin Blue staining. The adipogenic differentiation ability was detected by Oil Red O staining. The expression of multidirectional differentiation-related genes was detected by q PCR. Results: ISN-SCs were fibroblast-like, multi-antennae and adherent. They were a kind of slow-circulating cells with high expressed CD29, CD90, CD44 and low expression of stem cell-associated negative surface antigen CD34, CD45, CD19 and CD11b. Alizarin red staining and calcium and cobalt staining were all positive after osteogenic differentiation. Alizarin blue staining was positive after chondrogenic differentiation. Oil red O staining was positive after adipogenic differentiation. (Osteogenic: Runx2, OPN, OCN; into cartilage: SOX-9, COL2a1, ACAN; adipose tissue: PPARγ, C / EBPα) were significantly higher than that of the control group, which was similar to BMSCs Of stem cell related gene (NANOG, SOX-2, OCT-4) expression levels. CONCLUSIONS: ISN-SCs possess the biological characteristics of stem cells and can be transformed into chondrocytes and adipocytes after being induced in vitro. ISN-SCs can provide a good source of cells for the study of autologous biomechanical repair of discs.