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目的探讨体外培养的骨髓基质细胞的部分生物学特性、成内皮细胞分化能力及在缺血区存活能力。方法2005年1月至11月,第三军医大学新桥医院全军心血管内科中心分离5~6周龄的小鼠胫骨、股骨,预冷的DMEM/F12培养基冲洗出骨髓,密度梯度离心分离出骨髓单核细胞,接种后12~16d形成单层贴壁的成纤维样细胞。体外诱导分化鉴定分离的细胞。建立下肢缺血模型,荧光标记的体外扩增的骨髓基质细胞被移植入缺血组织,移植后1周,荧光显微镜及HE染色,检查荧光强弱分布与HE染色密度的时空关系。结果体外传代培养的基质细胞诱导后分泌NO,移植1周在缺血区检测到大量荧光阳性细胞存活。结论在本实验条件下,体外培养的骨髓基质细胞生长稳定,传代后的细胞增殖较快,表现出较早期细胞特点,在体外能分化为血管内皮细胞,可望用作缺血性心脏病的细胞治疗的供体细胞。
Objective To investigate the biological characteristics of cultured bone marrow stromal cells in vitro and their ability to differentiate into endothelial cells and survive in the ischemic area. Methods From January 2005 to November 2005, the tibia and femur of 5-6-week-old mice were isolated from the cardiovascular center of Xinqiao Hospital of the Third Military Medical University. The bone marrow was washed out from the pre-cooled DMEM / F12 medium and density gradient centrifugation Bone marrow mononuclear cells were isolated and monolayer adherent fibroblast-like cells formed 12 to 16 days after inoculation. Induction of differentiation in vitro Identification of isolated cells. Establish a model of lower limb ischemia. Fluorescently labeled bone marrow stromal cells expanded in vitro were transplanted into ischemic tissue. Fluorescence microscopy and HE staining were performed one week after transplantation to examine the spatio-temporal relationship between fluorescence intensity distribution and HE staining density. Results After passaged in vitro, stromal cells secreted NO, and a large number of fluorescent-positive cells were detected in the ischemic area one week after transplantation. Conclusion Under this experimental condition, the cultured bone marrow stromal cells in vitro grew stably, the passaged cells proliferated rapidly and showed earlier cell characteristics, which could differentiate into vascular endothelial cells in vitro and could be used as ischemic heart disease Cell-treated donor cells.