腺病毒介导的鼠内皮抑素基因转染肺癌细胞体外抑制血管内皮细胞活性的实验研究

Mouse endostatin gene transfected lung cancer cells inhibit proliferation of endothelial cells in vitro

目的测定携带小鼠内皮抑素(mES)基因的腺病毒(Ad-mES)在肿瘤细胞中的表达,观察mES对体外培养的血管内皮细胞的抑制作用。方法分别以0.01、0.1、1、10及100共5个不同感染复数(MOI)的Ad-mES转染Lewis肺癌细胞株(LLC)。采用免疫组织化学法及Westernblot检测Ad-mES转染LLC48h后mES蛋白的表达;应用四甲基偶氮唑蓝比色(MTT)分析法测定不同MOI值Ad-mES转染上清对基础状态下及经碱性成纤维细胞生长因子(bFGF)刺激的人脐静脉内皮细胞(ECV304)的增殖抑制作用。结果Ad-mES转染LLC48h后,LLC细胞浆内可见棕黄色mES阳性表达颗粒,培养上清可表达位于相对分子质量20000的mES阳性条带,而未转染对照组呈阴性。随转染Ad-mESMOI值的增高,培养上清基础条件下及经bFGF刺激的内皮细胞的抑制作用越显著(与基础条件下比较,P<0.05)。结论构建的Ad-mES转染LLC后能够表达分泌mES;表达分泌于培养上清中的mES对血管内皮细胞(尤其是bFGF刺激)具有明显的增殖抑制作用。

Objective To observe the expression of adenovirus vector coding for mouse endostatin gene（Ad-mES） in lung cancer cells and its antiangiogenic activity on human umbilical vein endothelial cells（ECV304） in vitro.Methods Lewis lung cancer（LLC） cells were transfected with Ad-mES at different multiplicity of infection（MOI）.The expression of mES in LLC cells and supernatant after 48 hours was detected by immunohistochemical staining and Western blot respectively.The inhibitory effect of supernatant at different MOI on ECV304 non-stamulated and stimulated by basic fibroblast growth factor（bFGF） was measured by methyl thiazolyl tetrazolium（MTT） assay.Results After transfected for 48 hours,endostatin was identified in the cell plasma of infected LLC and negative result was founded in non-infected LLC.Western blot revealed band of endostatin in 20 kDa in culture supernatant of infected LLC and negative results in non-infected LLC.The inhibitory effects on ECV304 cell proliferation were stronger at higher MOI,and the difference was significant between stimulated and non-stimulated cells by bFGF（P〈0.05）.Conclusion Ad-mES can transfect and express endostatin effectively in LLC with biological activity.