兔骨髓单个核细胞体外向内皮细胞诱导的实验研究  

Research of endothelial cells induced from rabbit marrow mononuelear cells in vitro

作　　者：谭洪波[1] 杨柳[1] 段小军[1] 张洪鑫[1] 左镇华[1] 金旭红[1] 李忠[1] 王富友[1] 

机构地区：[1]第三军医大学西南医院关节外科中心,重庆400038

出　　处：《中国矫形外科杂志》2006年第22期1737-1740,I0003,共5页Orthopedic Journal of China

基　　金：国家自然科学基金资助项目(30300079)

摘　　要：[目的]研究兔骨髓单个核细胞（marrow mononuclear cells，MNCs）向内皮方向诱导的简捷有效方法，进行科学的计量分析并观察体外传代培养条件下的主要生物学特点。[方法]梯度离心分离兔MNCs，直接诱导培养，以每10^6个MNCs收获第1、2代内皮细胞的数量计量内皮细胞的收获效率；传代培养，倒置显微镜观察细胞形态及生长特点，免疫组化及内皮细胞功能鉴定。[结果]以1×10^6／cm^2的密度接种MNCs，经7～8d培养可收获第1代内皮细胞；第1、2代内皮细胞的收获总量和MNCs的收获量呈显著的正相关，每10^6个MNCs平均可收获第1代内皮细胞约（7．086±0．75）×10^4个，平均收获第2代细胞（53．20±6．47）×10^4个。诱导的内皮细胞为铺路石样，呈单层生长，第2代细胞CD31、八因子相关抗原免疫组化均为阳性，摄取低密度脂蛋白和结合凝集素实验均阳性。[结论]以1×10^6／cm^2的MNCs接种密度，收获第1、2代内皮细胞所需时间较短，收获效率高，在体外培养条件下生物学特性稳定，有望作为骨组织工程血管化的种子细胞。[ Objective ] To research the efficient way of isolating and proliferating of induced endothelial cells （ECs） , measure the number of the ECs and observe the main biological characteristic of ECs m vitro during the passage cultivation. [ Method] The marrow mononuclear cells（MNCs） in rabbit marrow were isolated by the gradiem centrifugation and cultivated to isolate and induce ECs. The efficiency of isolating and harvesting ECs was measured by the number of the first passage ECs from every 106 of the MNCs. The changes of ECs morphology, growth and proliferation were observed during the passage cultivation. The immunohistochemical staining of CD31 and vWF and function of ECs were checked. [ Result] After cultivated at 1 ×10^6/cm^2 MNCs for 7 -8 d, the first passage ECs were harvested. The number of the first and the second passage ECs had a remarkable statistical positive correlation with the number of the MNCs from one rabbit. After cultivated for 7 - 8 d, the first passage ECs of 7. 086 ± 0.75 × 10^4 in number and the second passage ECs of 53.20 ± 6.47 ×10^4 in number were harvested from every 106 MNCs on average ; ECs, which were monolayer arrayed to form cobblelike shape,The cells of 2nd passage showed positive staining of CD31 and vWF, and positive labeling of DiI-LDL and UEA. [ Conclusion] The period from cultivating to harvesting ECs is shortened at the density of 1 × 10^6/cm2 MNCs. It is feasible that the efficiency of isolating and harvesting ECs could be measured by the number of the first or the second passage ECs from every 10^6 MNCs. The induced ECs can act as seeding cells in bone tissue engineering in future.

关 键 词：内皮细胞 组织工程 细胞培养 种子细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]