人胎肺间充质干细胞的分离

Isolation of human fetal lung mesenchymal stem cells

目的探索分离人胎肺间充质干细胞(MSCs)的有效方法。方法在无菌条件下采集人工流产胎儿的肺组织,分别以组织块培养法和酶消化法分离细胞;通过相差显微镜观察细胞形态,流式细胞学方法检测细胞表型、成脂和成骨分化的多向分化潜能并证实其MSCs特征。结果两种分离方法所得细胞,在原代培养初期的形态有所不同,但培养后期和传代后均为长梭形的贴壁细胞。流式细胞学检测显示,两种方法分离的细胞均表达MSCs标志物CD13、CD29、CD44、CD90、CD105、CD166及HLA-ABC,但不表达造血细胞系的标志物CD45、CD34、CD14、CD38、CD133和内皮相关抗原CD31,也不表达CD41a、CD42b、CD49d、CD106、CD61和HLA-DR。成脂诱导3～5 d后,部分细胞转变为肥大、扁平的多角形细胞;1周后可见细胞内有囊泡状脂滴积聚;2周后脂滴增多、变大,并可被红O着色。成骨诱导者细胞内逐渐出现钙盐沉着,经诱导2周后大部分细胞内可见因钙盐沉着被茜素红S着色。结论组织块培养法和胶原酶消化法均为获得人胎肺MSCs的有效方法。

Objective To investigate the effective methods to isolate human fetal lung mesenchymal stem cells （MSCs）.Methods The fetal lung tissues were collected after abortion under aseptic conditions.The cells were isolated by explant culture and enzyme digestion respectively,followed by cell morphology observation under phase contrast microscopy; cell phenotype was detected by flow cytometry,and multipotent differentiation potential via adipogenic and osteogenic induction,Results The morphology of cells isolated by two methods was different in the initial stage of primary culture.However,they became homogeneous fibroblast-like cells in later stage of primary culture and after passages.Flow cytometry analysis showed that both cell populations isolated by two different methods expressed MSCs markers CD13,CD29,CD44,CD90,CD105,CD166 and HLA-ABC,but not hematopoietic markers （CD45,CD34,CD14,CD38 and CD133）,endothelialmarkers CD31,and other surface markers （CD41a,CD42b,CD49d,CD61,CD106 and HLA-DR）.Some of the MSCs changed into large,flattened cells after 3-5 days of being exposed to adipogenic differentiation medium.One week after adipogenic induction,accumulated lipid vacuoles were observed in the cytoplasm.After another week of induction,more and larger lipid vacuoles which were stained red with Oil O were observed in the cytoplasm.After osteogenic induction,calcium deposits were gradually observed in the cytoplasm.Two weeks after osteogenic induction,calcium deposits stained red by Alizarin Red S were found in most of the cells.Conclusion Both tissue culture and collagenase digestion methods are effective methods to isolate human fetal lung mesenchymal stem cells.