一种体外快速分离脂肪来源干细胞的方法

目的: 以小鼠为模型, 建立一种基于流式细胞仪为检测手段的快速分离脂肪来源干细胞的方法, 解决间充质干细胞进入实际应用过程中遇到的难题。方法: 取BALB/c小鼠腹股沟内侧的皮下脂肪组织, 采用Ⅰ型胶原酶消化等系列措施, 获取脂肪干细胞(adipose-derived stem cells, ADSCs)。所得细胞分离培养4代后, 流式细胞仪分选出CD73+CD45- ADSCs后成骨诱导分化。碱性磷酸酶染色和实时荧光定量PCR检测其分化情况。结果: 刚分离培养的ADSCs细胞普遍呈圆形或椭圆形, 传至第三代的细胞, 非MSCs细胞逐渐被淘汰, 剩余的细胞形态逐渐变得一致, 细胞形态呈梭形。流式细胞仪检测发现ADSCs细胞表面抗原标记CD73+CD45- 为20.7%。所得的ADSCs成骨诱导分化后碱性磷酸酶(alkaline phosphatase, ALP)染色呈阳性, 实时荧光定量PCR检测成骨标志基因发现它们表达上调, 其中ALP的表达高达22倍。结论: 本方法可以获得纯度较高的ADSCs, 且耗时少成本低; 且提示可采用该方法来获得大量的人源ADSCs用于组织工程修复。

Abstract

Objective: Using mouse as model, to establish an easy method to isolate adipose derived stem cells (ADSCs) based on flow cytometry (FCM) assay for further development. Methods: Adipose-derived stem cells (ADSCs) were isolated from BALB/c mice’s subcutaneous adipose tissue with a series of treatments contains collagenase I digestion and so on. FCM was used to identify ADSCs. Alkaline phosphatase staining and real-time fluorescence quantitative PCR were used to detect the cells’ differentiation. Results: After cultured 4 passages, 20.7% CD73+CD45- cells population were identified as ADSCs in flow cytometry assay. After inducing culture, alkaline phosphatase staining verified that the isolcated ADSCs could differentiate to osteoblasts positively. Results of quantitative PCR suggested the symbolize genes of osteogenic differentiation were up-regulated compared with the control group, and the expression of ALP was as high as 22 times especially. Conclusion: The method is an easy way to obtain the higher purity ADSCs but less cost. It also could be used to get a large number of ADSCs in tissue engineering repair.

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郭淑军, 刘伟, 金媛, 王强, 李雅丹, 陈小佳. 一种体外快速分离脂肪来源干细胞的方法[J]. 激光生物学报, 2017, 26(1): 91. GUO Shujun, LIU Wei, JIN Yuan, WANG Qiang, LI Yadan, CHEN Xiaojia. One Easy Method to Isolate Adipose-Derived Stem Cells in vitro[J]. Acta Laser Biology Sinica, 2017, 26(1): 91.

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