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目的探讨人羊水来源c-kit~+间充质干细胞(c-kit~+human amniotic fluid-derived mesenchymal stemcells,c-kit~+HAFMSCs)的生物学特征及心肌诱导分化能力。方法通过产前诊断或自愿引产获取50份孕中期羊水标本,经流式细胞仪分选c-kit~+HAFMSCs,MTT法观察细胞增殖情况,并通过流式细胞仪、细胞免疫化学染色观察行细胞表型鉴定。在体外经成骨及成脂诱导分化,于诱导培养后21 d采用yon Kossa染色观察钙沉积颗粒,油红O染色观察脂滴形成情况。实时荧光定量PCR检测细胞经5氮-2’脱氧胞苷心肌诱导前后,NKx2.5、Tbx5、GATA-4、α-MHC 4种心肌特异性基因表达情况。结果经流式细胞仪分选,人孕中期羊水中c-kit~+HAFMSCs含量约为贴壁细胞的3.07%±1.03%;体外增殖迅速,表达MSCs表面标志CD29、CD44、CD73、CD90、CD105,不表达造血干细胞表面标志CD34、CD45;农达人类白细胞抗原(human leukocyte antigen,HLA)-ABC,不表达HLA-DR。细胞免疫化学染色示CD29、CD44、CD73、CD90、CD105、Oct-4染色呈阳性,CD34、CD45染色呈阴性。MTT检测示第5、10、15代c-kit~+HAFMSCs具有相似的生长曲线。成骨和成脂诱导培养后21 d,细胞内有钙盐沉积和脂滴形成。实时荧光定量PCR检测示,心肌诱导后14 d,NKx2.5、Tbx5、GATA-4、α-MHC 4种心肌特异性基因表达均较诱导前明显增加,差异有统计学意义(P<0.05)。结论通过流式细胞仪分选的c-kit~+HAFMSCs在体外可以诱导分化为心肌样细胞,可能成为心肌再生性治疗的种子细胞。
Objective To investigate the biological characteristics of c-kit ~ + human amniotic fluid-derived mesenchymal stem cells (c-kit ~ + HAFMSCs) and their ability to induce cardiomyocytes differentiation. Methods 50 preterm second trimester amniotic fluid samples were obtained by prenatal diagnosis or voluntary induction of labor. The c-kit ~ + HAFMSCs were sorted by flow cytometry. The cell proliferation was observed by MTT assay. The cell viability was analyzed by flow cytometry Cell phenotype identification. In vitro osteogenic and adipogenic differentiation, in the induction of culture 21 d after the use of yon Kossa staining calcium deposition particles, oil red O staining observed lipid droplets formation. Real-time fluorescence quantitative PCR was used to detect the expression of four kinds of cardiac-specific genes in NKx2.5, Tbx5, GATA-4 and α-MHC cells before and after their induction by 5-Aza-2’deoxycytidine. Results The percentage of c-kit ~ + HAFMSCs in amniotic fluid during interim pregnancy was about 3.07% ± 1.03% of that of adherent cells by flow cytometry. The proliferation of c-kit ~ + HAFMSCs in mid-second trimester was rapid and the surface markers of MSCs expressed CD29, CD44, CD73, CD90, CD105 , Do not express hematopoietic stem cell surface markers CD34, CD45; Nongda human leukocyte antigen (HLA) -ABC, do not express HLA-DR. Immunocytochemistry showed CD29, CD44, CD73, CD90, CD105, Oct-4 staining was positive, CD34, CD45 staining was negative. MTT assay showed that the growth curves of the 5th, 10th and 15th generation c-kit ~ + HAFMSCs had similar growth curves. At 21 days after osteogenic and adipogenic induction, calcium deposits and lipid droplets formed in the cells. Real-time quantitative PCR showed that cardiac muscle specific gene expressions of NKx2.5, Tbx5, GATA-4 and α-MHC were significantly increased at 14 d after myocardial induction, with significant difference (P <0.05) . Conclusion The c-kit ~ + HAFMSCs sorted by flow cytometry can induce the differentiation into cardiomyocyte-like cells in vitro and may be the seed cells for myocardial regenerative therapy.