人脐带间充质干细胞分离培养及鉴定的相关研究

目的：原代培养并鉴定人脐带间充质干细胞(human umbilical cord mesenchymal stem cells，hUCMSCs)，对hUCMSCs的生物学性状、表面抗原、多向分化潜能等进行研究，并初步检测其内源性Hedgehog(Hh)信号通路主要因子的RNA水平。方法：取足月健康剖宫产胎儿脐带，提取hUCMSCs，贴壁培养。绘制生长曲线，检测细胞周期。进行体外成骨、成脂及成软骨诱导。流式细胞仪检测细胞表面标志物。Real-time PCR检测原代细胞内源性Hh信号通路主要因子的mRNA水平。结果：脐带剪碎经胶原酶消化后24 h细胞即可贴壁，7 d可生长达培养皿底面积85%融合。 P3代hUCMSCs的生长曲线呈上升趋势，传代后第3 d进入倍数生长期。并具有成骨分化、成脂分化及成软骨分化的能力。流式细胞仪检测99.04％的细胞高表达整合素分子CD29，98.80％的细胞高表达粘附分子CD44，85.09％的细胞高表达内皮糖蛋白分子CD105，只有0.19％的细胞表达造血干细胞标记物CD45，0.58%的细胞表达血管内皮分子CD34。脐带来源的间充质干细胞内能检测到Hh信号通路中主要因子的内源性表达，其中Shh水平相对较高。结论：原代培养的hUCMSCs为进一步探索Hh信号通路在颅颌骨缺损修复中的作用机制提供可靠的细胞来源。

The study of isolatating and identificating the human umbilical cord mesenchymal stem cells

Objective:The aim of this study was to isolate and identify the human umbilical cord mesenchymal stem cells (hUCMSCs). Method:Umbilical cord was cut into pieces, and then digested with collagenaseII. The mesenchymal stem cells from human umbilical cord were cultured. After that, the multipotent differentiation characteristics were demonstrated using standard in vitro conditions. Furthermore,the biological properties of hUCMSCs were investigated by flow cytometry. And the relevant mRNAs levels of Hedgehog signaling pathway were detected by real-time PCR. Result:Monoptychial het-erogeneous cells were obtained.The cells were able to differentiate into osteogenic, adipogenic and cartilage cells. A total of 98.4 % of cells at passage 3 expressed cluster of differentiation CD29 and 98.80% of CD44,85.09% of CD105, but only 0.19%of CD45 and 0.58%of CD34.And expression of the factors of hedgehog signaling pathway were able to be detected by real-time PCR. Conclusion:hUCMSCs that are isolated from the human umbilical cord and exhibit the identified character-istics may be used as seed cells in jaw bone defect reconstruction.