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目的:原代培养并鉴定人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,h UCMSCs),对h UCMSCs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h UCMSCs,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m RNA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。P_3代h UCMSCs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h UCMSCs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。
OBJECTIVE: To primary culture and identify human umbilical cord mesenchymal stem cells (hUCMSCs), to study the biological characteristics, surface antigen and multidirectional differentiation potential of h UCMSCs and to detect their endogenous RNA level of Hedgehog (Hh) signaling pathway. Methods: Fetal umbilical cord from full-term healthy cesarean section was collected, h UCMSCs were extracted and cultured adherently. Draw a growth curve to detect cell cycle. In vitro osteogenesis, adipogenic and chondrogenic induction. Flow cytometry to detect cell surface markers. Real-time PCR was used to detect the m RNA level of the major factor of endogenous Hh signaling pathway in primary cells. Results: Umbilical cord cells could be adhered 24 h after collagenase digestion, and reached the basal level of 85% after 7 days of culture. The growth curve of hMSCs on P 3 generation showed an upward trend, and entered the multiple growth phase on the 3rd day after passage. And with osteogenic differentiation, adipogenic differentiation and cartilage differentiation ability. Flow cytometry detected 99.04% cells highly expressing integrin molecule CD29, 98.80% cells highly expressing adhesion molecule CD44, 85.09% cells highly expressing endoglin molecule CD105, only 0.19% cells expressing hematopoietic stem cell marker CD45 , 0.58% of cells expressed vascular endothelial molecule CD34. Umbilical cord-derived mesenchymal stem cells can detect the endogenous expression of major factors in the Hh signaling pathway, in which the Shh level is relatively high. CONCLUSION: The primary cultured hUCMSCs provide a reliable source of cells for exploring the mechanism of Hh signaling in the repair of cranial and mandibular defects.