人肝癌血管内皮细胞分离培养、鉴定及比较  

Isolation, identification and comparison of tumor-derived endothelial cells from human hepatocellular carcinoma

作　　者：孔令群[1] 朱小东[1] 徐华祥[1] 陶中华[1] 王文权[1] 孙惠川[1] 

机构地区：[1]复旦大学附属中山医院肝癌研究所,上海200032

出　　处：《中华实验外科杂志》2011年第2期236-238,共3页Chinese Journal of Experimental Surgery

摘　　要：目的 建立人肝癌血管内皮细胞（TEC）分离培养方法并鉴定比较.方法 采用CD105、CD31抗体交联免疫磁珠方法优化分选TEC并传代培养;通过形态观察、功能实验、流式检测及实时聚合酶链反应（real-time PCR）等验证比较.结果 CD105＋TEC呈细长＂纤维条索状＂,CD31＋TEC则呈＂梭形＂;流式检测TEC表达CD68和α-平滑肌肌动蛋白（α-SMA）低于5%,排除巨噬细胞和成纤维细胞污染,PCR检测CD105＋TEC、CD31＋TEC甲胎蛋白mRNA显著低于肿瘤细胞HepG2（P＜0.01）,表达水平低于HepG2的1/8,排除肿瘤细胞污染;95%以上阳性分选细胞呈乙酰化低密度脂蛋白摄取实验阳性;增殖实验检测CD105＋TEC和CD31＋TEC生长48 h的吸光度值分别为0.45±0.04和0.35±0.02（P＜0.05）,CD105＋TEC增殖能力强于CD31＋TEC;TEC在Matrigel胶中可形成毛细管网状结构,CD31＋TEC强于CD105＋TEC.结论 CD105、CD31抗体交联免疫磁珠分选法均可分离得到高纯度人肝癌TEC.Objective To isolate, identify and compare vascular endothelial cells derived from human hepatocellular carcinoma （HCC）. Methods Modified immunomagnefic methods using magnetic beads conjugated with anti-CD105 and anti-CD31 antibody were used to isolate tumor-derived endothelial cells （TEC）, namely CD31 ＋TEC and CD105 ＋TEC. CD31 ＋TEC and CD105 ＋TEC were cultured in vitro,identified and compared by morphological appearance, functional characterization, flow cytometry and realtime polymerase chain reaction （PCR） analyses. Results The isolated CD105 ＋ TEC presented ＂fibrous bands＂ appearance while CD31 ＋ TEC presented ＂fusiformis＂ appearance. Macrophages, fibrocytes, and tumor cells contamination was excluded. Surface CD68 and α-SMA expression was less than 5%. Alpha fetoprotein expression was significantly lower in CD31 ＋ TEC and CD105 ＋ TEC than that in HepG2 cells （ P 〈0. 01 ）, and the expression level was less than 1/8 folds of HepG2 cells. Internalization of acetylated lowdensity lipoprotein was positive in more than 95% of isolated cells. The absorbance （A） values of CD105 ＋TEC and CD31 ＋ TEC grown for 48 h were 0. 45 ± 0. 04 and 0. 35 ± 0. 02 respectively （ P 〈 0. 05 ）. Proliferation of CD105 ＋ TEC was significantly more active than CD31 ＋ TEC when cultured in the serum supplemented with medium for 24, 48, and 72 h. The isolated cells could form capillary-like tubes on Matrigel matrix, stronger capability of CD31 ＋ TEC than CD105 ＋ TEC. Conclusion CD31 ＋ TEC and CD105 ＋ TEC can be conveniently purified by modified immunomagnetic methods.

关 键 词：癌 肝细胞 内皮细胞 血管生成 

分 类 号：R735.7[医药卫生—肿瘤]