摘要
背景:目前血管内皮细胞原代获取方法中,使用较多的就是大动脉血管内皮细胞培养及微动脉血管内皮细胞培养,传统多采用酶消化法及组织贴壁法,所获得血管内皮细胞数量和纯度已经不能满足科研实验的需要。目的:探讨兔主动脉血管内皮细胞的原代的改良提取及鉴定方法。方法:切取一段兔主动脉,甲组采用改良提取法培养血管内皮细胞,显微器械剥离出血管内层,再用酶消化法获得单个原代细胞,以内皮细胞培养液培养;乙组采用组织贴壁法生长,整个血管内面贴于培养皿中,同时于体积分数5%CO_2,37℃温箱中孵育1 h。分别获得2组细胞沉淀并进行体外培养。采用显微镜观察细胞形态,通过免疫组织化学染色检测CD31、Ⅷ因子及Vimentin蛋白进行血管内皮细胞鉴定,并与组织贴壁生长法培养对比。结果与结论:采用改良提取法得出的内皮细胞纯度比组织贴壁生长高,数量更多。免疫组织化学鉴定结果CD31(+),Ⅷ因子(+),Vimentin蛋白(-)。结果表明此法可以更容易获得纯度较高,数量更多的血管内皮细胞,可操作性及实用性较强。
BACKGROUND： Primary vascular endothelial cells are mostly harvested through aorta endothelial cell cultures and micro-artery endothelial cell cultures using enzyme digestion and tissue adhesion methods, and the quality and purity of harvested cells cannot meet the need for current scientific research. OBJECTIVE： To investigate an improved extraction of primary vascular endothelial cells and the relevant identification method. METHODS： A segment of rabbit aorta was cut to culture vascular endothelial cells using the improved extraction method in group A or using adhesion method in group B. In the group A, the vascular intima was striped out with microsurgical instruments, and digested enzymatically to acquire single primary cells followed by culture in endothelial cell culture medium. In the group B, the whole vascular intima was adhered to the culture dish that was incubated in a 5%CO_2, 37 ℃ incubator for 1 hour. Cell pellets in the two groups were cultured in vitro. Cell morphology was observed using a microscope; immunohistochemical staining was used to detect CD31, VIII factor and Vimentin protein for identification of vascular endothelial cells. RESULTS AND CONCLUSION： The purity and number of vascular endothelial cells extracted by the improved method were higher than those by the adhesion method. Immunohistochemical findings showed positive expression of CD31 and VIII factor, but negative expression of Vimentin. These findings indicate that the improved extraction method can obtain more vascular endothelial cells with higher purity, which is of strong operability and practicality.
出处
《中国组织工程研究》
CAS
北大核心
2016年第7期1019-1024,共6页
Chinese Journal of Tissue Engineering Research
基金
湖北省自然科学基金重点项目(2014CFA084)~~
