脐带全层源间充质干细胞分离培养及向成软骨细胞分化的实验研究

Study on culture of mesenchymal stem cells isolated from whole human umbilical cord and its differentiation into chondrocyte in vitro

目的从人脐带全层中分离培养间充质干细胞(MSCs),并进行成软骨诱导分化,为组织工程软骨和软骨损伤后修复提供种子细胞。方法采用胶原酶消化法从脐带全层中分离培养间充质干细胞,显微镜下观察细胞形态,细胞计数法绘制细胞生长曲线,采用流式细胞仪检测细胞周期及细胞表型,采用微团细胞培养在软骨诱导液中向软骨细胞分化,阿尔辛蓝及甲苯胺蓝染色检测细胞分化情况,RT-PCR法检测诱导后细胞表达聚集蛋白聚糖(ACAN)基因情况。结果人脐带全层来源的MSCs呈成纤维样形态漩涡状贴壁生长,细胞高表达HLA-I类分子、CD73、CD90、CD166及CD105,不表达CD34、CD45、CD14、CD31、CD80、CD86及HLA-DR。细胞诱导分化21d后,阿尔辛兰及甲苯胺蓝染色阳性;RT-PCR检测诱导后的细胞表达ACAN,而对照组无表达。结论人脐带全层为成体MSCs提供一种新而方便的来源,人脐带间充质干细胞(hucMSCs)体外培养能够向软骨细胞分化。

Objective To culture the mesenchymal stem cells isolated from the whole human umbilical cord and explore its differentiation into the chondrocyte in vitro and offer the resources of seeding cells in cartilage tissue engineering. Methods The whole human umbilical cord mesenchymal stem cells （ hucMSCs） were isolated by using a collagenase digestion method and expanded in vitro. The growth curve was drawn by cell counting, cell cycle and the phenotypes were evaluated by flow eytometry. HucMSCs were induced to differentiate into ehondro- cytes in special differentiation condition using the technology of micromass culture. Alcian blue staining and toluid- ine blue staining were used to determine the differentiation. The chondrocytes related gene aggrecan （ ACAN ） expression was detected by RT-PCR. Results The hucMSCs isolated from the whole human umbilical cord had the characteristics of plastic adherence and fibroblast-like morphology. The adherent cells displayed an abundant presence of HLA-I,CD73 ,CD90,CD166,CD105 and absence of CD34,CD45,CD14,CD31 ,CDS0,CD86 and HLA-DR. After 21 days induction, alcian blue staining and toluidine blue staining were positive. RT-PCR reactions confirmed that the induced hucMSCs expressed ACAN gene. Conclusion The whole human umbilical cord can be considered as a convenient source of MSCs and hucMSCs can be induced into chondrocytes in vitro.