血管内皮细胞生长因子反义核糖核酸对肝癌细胞生长的抑制作用  

Study on inhibitory effect of antisense VEGF RNA on the growth of hepatocellular in vitro & in vivo

作　　者：郝继辉[1] 俞鸣[2] 史玉荣[1] 李强[1] 郝希山[1] 

机构地区：[1]天津肿瘤医院腹部外科,300060 [2]天津医科大学公共卫生学院

出　　处：《中华肝脏病杂志》2003年第12期725-727,共3页Chinese Journal of Hepatology

基　　金：天津市自然科学基金(013615611)

摘　　要：目的 探讨血管内皮细胞生长因子(VEGF)反义RNA转染人肝癌细胞后对细胞体内外生物学性状的影响。方法 将含正义、反义VEGFcDNA序列的质粒PCMV—VEGF、PCMV—FGEV及空载体质粒pcDNA3.1,在脂质体介导下导入SMMC—7721肝癌细胞,分别称为正义、反义及对照组,并通过G418筛选获得阳性克隆。细胞原位杂交和免疫组织化学方法检测转染后VEGF在肝癌细胞内的表达情况;MTT法和FCM检测转染后细胞在体外的增殖和凋亡情况;并制备裸鼠动物模型,观察转染后细胞的体内生长情况。结果 转染PCMV—FGEV后肝癌细胞内VEGF的转录及其蛋白的表达水平显著下降,但转染后体外细胞的增殖与凋亡情况均无明显变化。转染PCMV—FGEV后细胞在裸鼠体内的生长缓慢,反义组成瘤时间为(25.0±1.8)d,明显长于正义组(15.7±2.5)d和对照组(18.5±2.1)d,F=19.455,P<0.01;而平均瘤重以反义组最轻,为(0.96±0.28)g,F=21.501,P<0.01;同时反义组裸鼠肿瘤细胞发生明显的凋亡。结论 VEGF反义RNA转染人肝癌细胞可抑制肿瘤细胞VEGF的表达,在体外对细胞增殖和凋亡无影响,而体内可显著诱导细胞凋亡并抑制肿瘤生长。Objective To determine the effects of PCMV-FGEV transfection on the profile of SMMC-7721 hepatocellular in vitro & in vivo. Methods SMMC-7721 hepatocellular was transfected with PCMV-FGEV antisense, PCMV-VEOF sense and empty vector plasmid encapsulated by lipofectamine. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the SMMC-7721 hepatocellular were determined by in situ hybridization and immunochemical analysis. The effect of PCMV-FGEV transfection on SMMC-7721 hepatocellular proliferation was observed by MTT colorimetric assay. Flow cytometry was used to determine the effects of PCMV-FGEV transfection on cell apoptosis of SMMC-7721. The growth of transfected cells was also observed in nude mice. Results There was reduced VEGF expression in SMMC-7721 transfected with PCMV-FGEV confirmed by in situ hybridization and immunohistochemical analysis. There was no effect of PCMV-FGEV transfection on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cell with PCMV-FGEV transfected was slow in nude mice (vivo) and accompanied with obvious apoptosis. The latent time of tumor in the antisense mice group was 25.0 ±1.8 days, which was longer than that in sense and control group significantly (F = 19.455, P < 0.01). On the other hand, the average tumor weight in antisense group (0.96 g±0.28 g) was the smallest among the three groups (F = 21.501, P < 0.01). Conclusions The expression of VEGF was inhibited by PCMV-FGEV. There was no effect on cell proliferation and cell apoptosis of SMMC-7721 by transferring PCMV-FGEV gene into SMMC-7721 cells in vitro. But in vivo it can inhibit tumor growth and induce cell apoptosis.

关 键 词：血管内皮细胞生长因子 反义核糖核酸 肝癌 肿瘤抑制 肿瘤生物学 原位杂交 免疫组织化学 

分 类 号：R735.7[医药卫生—肿瘤]