不同提取方法对人脂肪间充质干细胞生物学活性影响

EFFECTS OF DIFFERENT EXTRACTION METHODS ON THE BIOLOGICAL ACTIVITY OF HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELLS

目的探讨使用不同方法分离提取的脂肪间充质干细胞(ADMSCs)是否存在细胞生物学活性的差异。方法分别使用直接消化法和干细胞基质胶消化法分离提取ADMSCs,进行细胞表型鉴定、增殖活性检测,应用Real-time PCR方法检测诱导分化后成脂标志基因过氧化物酶体激活受体γ(PPAR-γ)和成骨标志基因runt转录因子2(RUNX-2)mRNA的表达,Western blot方法检测PPAR-γ和RUNX-2蛋白表达。结果基质胶消化法得到的ADMSCs相较于直接消化法得到的ADMSCs在培养第3天的增殖活力更高(F=727.139,P<0.05)。与直接消化法比较,成脂诱导分化6~12 d后基质胶消化法得到ADMSCs的PPAR-γmRNA表达增高(F=40.260~157.532,P<0.05),PPAR-γ蛋白表达在诱导分化6 d后增高(F=1501.475,P<0.05);成骨诱导分化3~12 d基质胶消化法得到ADMSCs的RUNX-2 mRNA表达较直接消化法增高,诱导分化3 d蛋白表达增高(F=58.018~2402.049,P<0.05)。结论使用基质胶消化法得到的ADMSCs成脂、成骨分化潜力优于直接消化法。

Objective To compare the biological activity of adipose-derived mesenchymal stem cells(ADMSCs)between different extraction methods.Methods ADMSCs were isolated using the direct digestion method and the Matrigel digestion method separately.The phenotype and proliferative activity of ADMSCs were determined.Real-time PCR was used to measure the mRNA expression of the adipogenic marker gene peroxisome proliferator-activated receptorγ(PPAR-γ)and the osteogenic marker gene runt-related transcription factor 2(RUNX-2).Western blot was used to determine the protein expression of PPAR-γand RUNX-2.Results Compared with those obtained by the direct digestion method,ADMSCs obtained by the Matrigel digestion method had significantly higher proliferative activity on day 3 of culture(F=727.139,P<0.05),significantly increased PPAR-γmRNA expression levels after 6-12 d of adipogenic induction(F=40.260-157.532,P<0.05),a significantly increased PPAR-γprotein expression level after 6 d of adipogenic induction(F=1501.475,P<0.05),and significantly increased RUNX-2 mRNA levels after 3-12 d of osteogenic induction and RUNX-2 protein expression after 3 d of osteogenic induction(F=58.018-2402.049,P<0.05).Conclusion ADMSCs obtained by the Matrigel digestion method show better potential of adipogenic and osteogenic differentiation than those obtained by the direct digestion method.