大鼠ASC体外分离培养、鉴定及其双报告基因标记示踪

Isolation,culture and characterization of ASCs and its dual reporter gene labeling for tracking

[目的]探索ASC高效分离方法,采用荧光基因标记法对分离的ASC进行标记、功能检测和示踪研究。[方法]通过优化脂肪组织的酶解消化条件,分别采用胶原酶、胰蛋白酶及组合对脂肪组织消化,检测细胞产量,并用流式细胞术进行鉴定。之后利用GFP-Fluc双报告基因对体外分离培养的ASC进行标记,鉴定其多项分化潜能。[结果]胶原酶、胰蛋白酶复合酶消化能够显著提高ASC分离效率,细胞产量较单一酶消化提高4-5倍。体外扩增培养ASC以表达CD29、CD90等为主。GFP-Fluc基因标记后的ASCs具有成脂肪、成骨分化潜能,可用于活体干细胞移植后的定量追踪。[结论]胶原酶、胰蛋白酶复合酶解法可以显著提高脂肪干细胞的原代分离效率4~5倍。

[ Objective] Adipose tissue- derived stem cells （ASCs） are widely used in tissue engineering and regenerative medicine. Fluorescence labeling and high efficiency of isolation play important role in the application and evaluation of ASCs. The present study aims to develop a new method for the efficient isolation of ASCs by combining different enzymes. [ Methods] The isolation of ASC by collagenase digestion, tryptic digestion, the combination of collagenase and tryptic ＂digestion, were evalu- ated with flow cytometry. Then the ASCs were labeled with GFP - Fluc dual - luciferase reporter gene and the multi - potential differentiation ability as well as the proliferation ability were evaluated. [ Results] The combination of 0.1% collagenase and 0. 1% tryptic digestion for 45 minutes could significantly increase the efficient harvest of ASC for 4 - 5 times. Flow cytometry re- sults indicated that most of the ASCs were positively for the expression of CD29, CD90 and negative for CD34 and CD 45. What＇ s more, similar with ASCs, GFP - Fluc dual - luciferase reporter gene system labeled ASC also had the multi - potential differentiation ability as well as the proliferation ability. The in vitro and in vivo imaging results showed that ASC could be used for the quantitative tracking of the cell implantation therapy. [ Conclusion] The combination of collagenase and tryptic digestion could increase the isolation efficiency of ASC and be beneficial for the application of it in the regenerative medicine.