人外周血单个核细胞向心肌样细胞分化的研究

目的通过体外直接培养人外周血单个核细胞(MNCs),并应用5-氮杂胞苷诱导其分化,探讨MNCs经过培养和诱导能否向心肌样细胞分化。方法以Ficoll密度梯度离心法从人外周血中分离出MNCs,于含20%FBS的DMEM培养液中培养,第3天以10μmol/L的5-氮杂胞苷诱导24 h。第14天,对于所培养的贴壁细胞行肌球蛋白、α-肌节肌动蛋白、结蛋白和肌钙蛋白T的免疫细胞化学染色;逆转录聚合酶链式反应(RT-PCR)检测细胞是否表达-αMHC和-βMHC等基因;同时作CD34、AC133和α-肌节肌动蛋白的流式细胞计数分析(FACS)。结果培养第14天开始纺锤形贴壁细胞数量明显增多,并聚集成簇。免疫细胞化学染色法显示肌球蛋白、α-肌节肌动蛋白、结蛋白和肌钙蛋白T均呈阳性。RT-PCR结果显示经5-氮杂胞苷诱导分化后的MNCs存在α和-βMHC基因的表达。FACS显示,CD34阳性或AC133阳性率为(53.04±5.60)%、α-肌节肌动蛋白阳性率为(42.70±11.73)%。结论体外培养并经5-氮杂胞苷诱导后的MNCs既具有心肌样细胞的部分特点,又具有干细胞的部分特征。在适当的条件下,MNCs有向心肌样细胞分化的潜能。

In vitro study of cardiomyocyte-like differentiation of human peripheral blood-derived mononuclear cells

Objective To observe if peripheral blood-derived MNCs can differentiate into cadiomyocyte-like cells after cultivation and stimulation by 5-azacytidine.Methods MNCs isolated from human femoral arterial anticoagulated blood sample by Ficoll density gradient centrifugation were cultured with DMEM containing 20% fetal bovine serum in 2% gelatin coated flasks in incubator system(37℃、5%CO2).5-azacytidine(10 μmol/L) was used to stimulate MNCs for 24 hours on day 3.Immunocytochemical examinations of myosin,α-sarcomeric actin,desmin and troponin T were performed on day 14.α-MHC and β-MHC gene expression was also tested by RT-PCR on day 14.In addition,flow cytometry analysis of CD34,AC133 and α-sarcomeric actin was performed after 14-day culture.Results The attaching cells began to elongate after 5-azacytidine stimulation,and manifested as spindle-like appearance and colony-forming tendency on day 14.Myosin,α-sarcomeric actin,troponin T and desmin were positively stained in cytoplasm in quite a number of attaching cells.RT-PCR results revealed α-MHC and β-MHC.Flow cytometry analysis demonstrated that the attaching cells were positive in CD34,AC133 and α-sarcomeric actin.Conclusion The cultured and induced peripheral blood-derived mononuclear cells have characteristics of both cardiomyocytes and stem cells.under appropriate conditions, these MNCs have the potentiality of cardiomyocyte-like differentiation.