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【目的】以腺病毒携带的人内皮抑素基因 Endostatin基因 (Ad/hEnd)感染舌癌细胞株Tca8113和人脐静脉内皮细胞株ECV30 4,观察其在舌癌细胞和静脉内皮细胞株中的表达。【方法】用 2 93细胞扩增Ad/hEnd ,并抽提、纯化。以 10MOI的Ad/GFP感染ECV细胞 ,观察细胞中表达绿色荧光蛋白的细胞所占的比例。采用免疫组化法检测Endostatin蛋白在感染了Ad/hEnd的Tca细胞和ECV细胞中的表达。Ad/hEnd感染Tca细胞后收集细胞培养液上清 ,ELISA检测上清中Endostatin蛋白含量。Western blot检测Endostatin蛋白在Tca和ECV细胞中的表达。【结果】扩增的Ad/hEnd通过空斑形成试验准确测定其滴度可达 1× 1 0 1 0 ～ 8× 1 0 1 0 PFU/mL。表达绿色荧光蛋白的细胞所占的比例为 31 %。实验显示感染Ad/hEnd后 ,在Tca细胞和ECV细胞胞浆内有Endostatin蛋白合成。细胞培养液上清中Endostatin蛋白表达浓度呈时间剂量依赖关系 ,最高达到 5 97μg/L。Western blot检测显示Endostatin蛋白可在两种细胞中高效表达 ,相对分子质量与自然Endo statin蛋白一致 ,无杂合蛋白 ,呈剂量依赖关系 ,且持续到第 7天仍呈明显的Endostatin蛋白质条带。【结论】本实验制备的重组腺病毒Ad/hEnd能在舌癌细胞和血管内皮细胞中有效表达Endostatin蛋白 ,可用于抗
【Objective】 The purpose of this study was to investigate the expression of Tca8113 and human umbilical vein endothelial cell line ECV30 4 in adenovirus-bearing human endostatin gene (Ad / hEnd) in tongue cancer cells and vein endothelial cells . 【Method】 Ad / hEnd was amplified by 2 93 cells, extracted and purified. ECV cells were infected with 10 MOI of Ad / GFP and the proportion of cells expressing green fluorescent protein was observed. The expression of Endostatin protein in Tca cells and ECV cells infected with Ad / hEnd was detected by immunohistochemistry. Tca cells infected with Ad / hEnd cell culture supernatant was collected, ELISA detection of supernatant Endostatin protein content. Western blot was used to detect the expression of Endostatin protein in Tca and ECV cells. 【Result】 The titer of amplified Ad / hEnd was 1 × 10 11 ~ 8 × 10 10 PFU / mL by plaque formation assay. The proportion of cells expressing green fluorescent protein was 31%. Experiments show that after infection with Ad / hEnd, there is Endostatin protein synthesis in the cytoplasm of Tca cells and ECV cells. Endostatin protein expression in the cell culture supernatant in a time-dependent manner, up to 5 97μg / L. Western blot showed that Endostatin protein was highly expressed in both cells. The relative molecular mass was consistent with the native Endo statin protein and no hybrid protein in a dose-dependent manner. Endostatin protein bands remained evident on the 7th day. 【Conclusion】 The recombinant adenovirus Ad / hEnd prepared in this experiment can effectively express Endostatin protein in tongue cancer cells and vascular endothelial cells,