脐带间充质干细胞无血清培养的实验研究

Studies on culturing of umbilical cord mesenchymal stem cells under serum-free condition

目的建立一种简单的体外无血清培养扩增人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)的方法。方法利用酶消化法分离hUCMSCs,在无血清干细胞培养体系下培养扩增,进行形态学观察,CCK-8法分析细胞生长曲线,流式细胞仪分析细胞表面抗原表达及细胞周期,进行成骨、成脂诱导分化。结果 hUCMSCs呈典型的成纤维细胞形态,具有较强的增殖能力。CD29、CD44、CD90、CD105均呈阳性表达,而CD34、CD45呈阴性表达。3周可诱导分化为脂肪细胞和成骨细胞。结论利用酶消化法和无血清的培养体系能够快速分离培养出大量hUCMSCs,该方法扩增得到的间充质细胞具备稳定的细胞标记物和生长状态,可用于各种治疗的临床试验。

Objective To establish a simple method for isolation and expansion of mesenchymal stem cells from human umbilical cord under serum-free condition, for potential clinical application. Methods The primary mesenchymal stem cells was isolated from human umbilical cord by enzyme digestion. The morphology of human umbilical cord mesenchymal stem ceils （hUCMSC） was observed with phase contrast microscope, and human umbilical cord mesenchymal stem ceils growth curve was analyzed by CCK-8 assay, hUCMSC cells cycle and surface markers were ana- lyzed by flow cytometry instrument.The muhilineage differentiation capability of hUCMSC was examined by culturing ceils under conditions favorable for adipogenic and osteogenic differentiation. Results The morphology of hUCMSC is the same as typical fibroblast, hUCMSC kept a capability of multiplication. The result of flow cytometry showed that hUCMSC is positive for specific surface markers such as CD29,CD44,CD90 and CD105, but negative for CD34 and CD45. hUCMSC could be induced toward osteoblast and adipocyte under 3 weeks＇ appropriate experimental condi- tions. Conclusion Using human mesenchymal stem cell specific growth factors conditioned medium, hUCMSC can be effectively isolated and expanded from human umbilical cord.hUCMSC prepared in this protocol has stable phenotypic characteristics, probably qualified for future clinical application.