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背景:脐带组织依赖于母体免疫系统的保护,且胚胎自身免疫系统相对不发育、MHC表达低下,故来源于脐带的间充质干细胞的生物学特性已成为目前研究热点。目的:拟在体外分离培养人脐带组织间充质干细胞,并观察其多向分化能力。设计、时间及地点:细胞学体外观察,于2006-10/2007-05在四川大学组织工程重建实验室完成。材料:脐带来源于胎龄37～40周的健康新生儿,由四川大学华西第二医院产房提供。方法:取脐带沿血管长轴切开,去掉血管,再将脐带重新缝合形成环状,灌入胶原酶悬液,6～8h后灌洗离心,获取细胞后贴壁法分离培养、扩增,细胞呈集落生长后传代。取传至第5代细胞,分别加入成骨、成脂诱导分化培养基。主要观察指标:脐带间充质干细胞的形态、生长状况、表面抗原分子的表达、多向分化潜能。结果:去除血管后获取脐带组织细胞的方法可获得贴壁生长的细胞,呈短棒状或梭形样细胞,易扩增和形成集落;高表达基质细胞抗原CD29,CD51,CD71,而不表达CD34,CD45及HLA-DR等造血干细胞抗原分子;成骨诱导后茜素红染色胞浆中有大量的钙沉积,碱性磷酸酶钙钴法染色胞质呈灰黑色,阳性细胞率>85%;成脂诱导后油红O染色示胞浆充满油滴空泡。结论:脐带组织存在具有分化能力的间充质干细胞,并可在体外进行培养扩增形成集落细胞传代,传代细胞表达基质细胞表面抗原,能够向成骨细胞、成脂肪细胞方向分化。
BACKGROUND: Umbilical cord tissue depends on the protection of the mother’s immune system, and the embryo’s autoimmune system is relatively undeveloped and MHC expression is low. Therefore, the biological characteristics of umbilical cord-derived mesenchymal stem cells have become the current research hotspots. OBJECTIVE: To isolate and culture human umbilical cord mesenchymal stem cells (MSCs) in vitro and observe their multilineage differentiation ability. DESIGN, TIME AND SETTING: The cytology in vitro observation was performed at the tissue engineering reconstruction laboratory of Sichuan University from October 2006 to May 2007. MATERIALS: The umbilical cord was derived from healthy neonates aged 37- 40 weeks and was provided by the Ward of West China Second Hospital of Sichuan University. Methods: The umbilical cord was cut along the long axis of the blood vessel to remove the blood vessel. The umbilical cord was re-stitched to form a ring. The collagenase suspension was infused into the collagenase suspension. After 6 to 8 hours, the cells were centrifuged and the cells were isolated and cultured. Cell colonies grow after passage. The cells were passaged to passage 5 and added into osteogenic and adipogenic differentiation medium. MAIN OUTCOME MEASURES: Morphology and growth of umbilical cord mesenchymal stem cells, expression of surface antigen molecules, multidirectional differentiation potential. Results: The method of obtaining umbilical cord tissue cells after removal of blood vessels could obtain adherent growth cells with short rod-like or spindle-like cells, which were easy to expand and form colonies. High expression of stromal cell antigens CD29, CD51 and CD71 but not CD34 , CD45, and HLA-DR. After albino-induced osteosynthesis, alizarin red staining showed a large amount of calcium deposition in the cytoplasm. The alkaline phosphatase calcium-cobalt staining showed a gray-black cytoplasm with a positive rate of> 85% After adipogenic induction, oil red O staining showed that the cytoplasm was filled with droplets of vacuoles. Conclusion: The umbilical cord tissue has the ability to differentiate into mesenchymal stem cells, which can be subcultured and cultured in vitro to subculture colony cells. The subcultured cells express stromal cell surface antigen and can differentiate into osteoblasts and adipocytes.