论文部分内容阅读
[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] The research aimed to explore the efficient separation method of ASC, and to label, detect and trace the separated ASC by fluorescent gene labeling method. [Method] By optimizing digestion conditions of adipose tissue, collagenase, trypsin and combination were respectively used for digestion of adipose tissue. Cell yield was detected and identified by flow cytometry. Afterwards, the ASC markers isolated and cultured in vitro were labeled with GFP-Fluc double reporter gene to identify a number of differentiation potential. [Result] Collagenase and trypsin enzyme digestion could significantly improve the separation efficiency of ASC, and the cell yield increased 4 ~ 5 times than the single enzyme digestion. In vitro expansion of ASC to express CD29, CD90 and other mainly. The ASCs labeled with GFP-Fluc gene have adipogenic and osteogenic potential and can be used for quantitative tracking after living stem cell transplantation. [Conclusion] Collagenase and trypsin complex enzymolysis method can significantly improve the primary separation efficiency of adipose stem cells by 4 ~ 5 times.