体外培养兔脂肪源性间充质干细胞的成骨成脂分化

发布时间：2019-06-29 16:55

【摘要】：背景:脂肪源性间充质干细胞具有自我更新能力且在体外特定条件下具有多向分化潜能,在临床上具有广泛的应用前景。然而,脂肪源性间充质干细胞的分离培养仍存在诸多困难与不足。目的:体外分离培养获得兔脂肪源性间充质干细胞,对其形态学、生物学特性进行观察,并鉴定其向成骨、成脂分化的潜能。方法:切取兔颈背区皮下脂肪,用胶原酶消化法分离兔脂肪源性间充质干细胞,进行体外培养,流式细胞仪检测细胞表面标志物,CCK-8法检测细胞活性并绘制细胞生长曲线。第4代兔脂肪源性间充质干细胞向成脂、成骨诱导分化后进行油红O染色、茜素红染色、碱性磷酸酶染色,观察其成脂、成骨分化潜能。结果与结论:兔脂肪源性间充质干细胞呈长梭形漩涡样贴壁生长,能稳定传代至第10代,增殖能力强;流式细胞仪检测可高表达CD29、CD90及CD44,而CD34和CD45表达较低;成脂诱导后的兔脂肪源性间充质干细胞油红O染色呈阳性;成骨诱导后的兔脂肪源性间充质干细胞碱性磷酸酶和茜素红染色均呈阳性,以上结果显示实验成功分离培养出兔脂肪源性间充质干细胞,获得的细胞生长稳定,增殖活跃,高表达间充质干细胞的表面抗原以及具有向成骨、成脂等多向分化的潜能,有望为骨组织工程提供理想的种子细胞。
[Abstract]:Background: adipose derived mesenchymal stem cells have the ability of self-renewal and multidirectional differentiation potential under specific conditions in vitro, and have a wide range of clinical application prospects. However, there are still many difficulties and shortcomings in the isolation and culture of adipose-derived mesenchymal stem cells. Aim: rabbit adipose derived mesenchymal stem cells were isolated and cultured in vitro, their morphology and biological characteristics were observed, and their potential for osteogenic and adipogenic differentiation was identified. Methods: rabbit adipose cells were isolated from rabbit cervical dorsal region by collagenase digestion and cultured in vitro. Cell surface markers were detected by flow cytometry, cell activity was detected by CCK-8 assay and cell growth curve was drawn. The fourth generation of rabbit adipose-derived mesenchymal stem cells were adipogenic. After osteogenic differentiation, oil red O staining, alizalin red staining and alkaline phosphatase staining were performed to observe the adipogenesis and osteogenic differentiation potential of rabbit adipose-derived mesenchymal stem cells. Results and conclusion: rabbit adipose derived mesenchymal stem cells grew like long fusiform whirlpool adherent, which could stably pass to the 10th generation, and the proliferation ability was strong. Flow cytometry detected high expression of CD29,CD90 and CD44, but low expression of CD34 and CD45. Oil red O staining of rabbit adipose derived mesenchymal stem cells induced by fat was positive. The alkaline phosphatase and alizalin red staining of rabbit adipose-derived mesenchymal stem cells were positive after osteogenic induction. The above results showed that rabbit adipose-derived mesenchymal stem cells were successfully isolated and cultured. The obtained cells were stable in growth, active in proliferation, highly expressed in the surface antigen of mesenchymal stem cells and had the potential to differentiate into osteogenesis, adipogenesis and other multidirectional differentiation, which is expected to provide ideal seed cells for bone tissue engineering.
【作者单位】：复旦大学附属中山医院骨科;上海市第一人民医院宝山分院骨科;复旦大学附属中山医院青浦分院骨科;
【基金】：上海市卫计委资助项目(2010135)~~
【分类号】：R329.2

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