脂肪基质干细胞诱导分化为心肌样细胞的实验研究

Experimental Study of Differentiation of Adipose-derived Stem Cells into Cardiomyocyte-like Cells

目的研究脂肪基质干细胞(ASCs)的分离、纯化、扩增以及在体外定向分化为心肌样细胞的能力。方法获取并培养正常人的ASCs,倒置显微镜下观察其形态;流式细胞仪检测其免疫表型。5-氮胞苷诱导ASCs在体外分化为心肌样细胞,倒置显微镜观察心肌样细胞的形态,逆转录-聚合酶链反应(RT-PCR)检测心肌相关基因ANP、cTnT、cT-nI和αMHC的表达;免疫荧光法检测cTnT和结蛋白的表达;Western blot法检测Connexin 43的表达。结果ASCs呈纤维样贴壁生长,体外培养易扩增;ASCs表达相关抗原CD29和CD105,不表达CD31、CD34、CD45和HLA-DR;ASCs经5-氮胞苷的作用可呈现典型的心肌样细胞形态,ANP、cTnT、cTnI和αMHC基因及cTnT、结蛋白和Connexin 43均呈阳性表达。结论脂肪组织中分离培养的ASCs具有体外大量扩增并保持低分化状态的特性,以及定向分化为心肌样细胞的能力,有望成为治疗心肌梗死的种子细胞。

Objective To study the isolation, purification, expansion and in vitro differentiation potential into cardiomyoeyte-like cells of adipose-derived stem cells （ASCs） from human adipose tissue. Methods ASCs were isolated and cultured, their morphology was observed under a converted microscope and their phenotypes detected by using flow cytometry. ASCs were induced by 5 Azacytidine to differentiate into eardiomyoeyte-like cells in vitro. Reverse transcription PCR was used to detect the expression of ANP, cTnT, cTnI and αMHC after 28 days. Immunofluorescence and Western blot were used to detect the expression of cTnT, Desmin and Connexin43, respectively. Results ASCs displayed a fibroblast-like morphology adhering to the culture plate. They expressed several surface antigens such as CD29 and CD105, while did not express CD31, CD34, CD45 and HLA-DR. Under suitable conditions, ASCs had the capacity of differentiation into eardiomyoeyte-like cells. They expressed ANP, cTnT, cTnI, αMHC, Desmin and Connexin43 28 days after adding 5-Aza in the culture system. Conclusion ASCs can be isolated from adipose tissue. They can proliferate and differentiate into cardiomyocyte-like cells in vitro. ASCs can be used as an alternative source of cells for myocardial infarction therapy.