两种不同的体外诱导人脐带间充质干细胞成软骨细胞方法的比较

Comparison of Two Different Methods Induced Human Umbilical Cord Mesenchymal Stem Cells to Differentiate into Chondrocytes In Vitro

该课题探讨了3D悬滴培养与贴壁培养体外诱导人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)在成软骨分化特性上的差异。采用机械匀浆法和组织块贴壁培养法体外分离培养获得第5代h UC-MSCs,流式细胞术检测干细胞表面免疫标记物,经3D悬滴培养与贴壁培养诱导应用番红O和阿利辛蓝染色及荧光定量检测富含透明质酸糖胺聚糖的细胞外基质形成情况以及不同时间点(3天、7天、14天)成软骨相关基因ACAN、MIA、COL1A2、COL2A1和COL10A1表达,并用免疫组化染色的方法检测II型胶原的表达情况。结果显示,流式细胞术检测的第5代h UC-MSCs间充质干细胞表面标记物结果符合国际细胞疗法协会制定的鉴定标准;3D悬滴培养的h UC-MSCs可形成致密的细胞聚合物,贴壁培养的细胞形态成长梭形;番红O和阿利辛蓝染色结果显示,3D悬滴培养和贴壁培养的h UC-MSCs均能形成软骨细胞;但荧光实时定量PCR结果显示,3D悬滴培养的h UC-MSCs形成软骨细胞的成软骨相关基因的表达明显多于贴壁培养细胞形成软骨细胞的成软骨相关基因;II型胶原免疫组化染色的阳性细胞染色及统计结果显示,3D悬滴培养比贴壁培养的诱导的h UC-MSCs成软骨能力更强。3D悬滴培养法是一种理想的h UC-MSCs诱导成软骨培养方法。

The purpose of this study is to study the chondrogenic properties capacity of human umbilical cord mesenchymal stem cells(hUC-MSCs)after three-dimensional hanging drop culture method and adherent culture method.The hUC-MSCs were isolated from Wharton’s jelly and cultured by mechanical homogenization in vitro.The morphology of 3D spheroid under optical microscope,and surface antigens were examined by flow cytometric analysis were compared.The hUC-MSCs were induced to differentiate into chondrocytes in vitro,and expression levels of chondrocyte related genes ACAN,MIA,COL1 A2,COL2 A1 and COL10 A1 were detected by fluorescence quantitative PCR on the 3 rd,7 th and 14 th day.The microspheres were induced for a chondro-like appearance and visualized by Saffron O staining and Alcian blue staining.Immunohistochemistry was used to detect the expression of type II collagen.Flow cytometry data showed the hUC-MSCs at passage 5 are positive for CD105,CD90,CD44,CD73,negative for CD19,CD34,CD45,HLA-DR,which were in line with The morphological characteristics and surface immune markers of mesenchymal stem cells developed by the International Society for Cellular Therapy(ISCT)in 2006.The hUC-MSCs cultured in 3D hanging drop culture can form a dense cell polymer,and the cell morphology of adherent culture can grow into a spindle shape.The results of Saffron O and Alcian blue staining showed that both the hUC-MSCs cultured in 3D hanging drop culture and adherent culture could form chondrocytes,but the results of fluorescence real-time quantitative PCR showed that the number of chondrogenic genes related to the formation of chondrocytes in the hUC-MSCs cultured in 3D hanging drop culture were more than that in the adherent culture.The semi-quantitative analysis result of type II collagen with immunohistochemistry showed that the chondrogenic ability of hUC-MSCs induced by 3D hanging drop culture was stronger than that by adherent culture.3D hanging drop cultured technology is an ideal method to culture hUC-MSCs.