反义VEGF_(165)基因对肝癌细胞生长的抑制作用  

Inhibition of Proliferation on Hepatocarcinoma Cells by Antisense VEGF_(165) Gene

作　　者：顾松[1] 刘长建[1] 乔彤[1] 孙雪梅[2] 

机构地区：[1]南京大学医学院附属鼓楼医院血管外科,南京210008 [2]南京大学医学院附属鼓楼医院细胞及分子生物学实验室,南京210008

出　　处：《南京大学学报（自然科学版）》2004年第5期613-622,共10页Journal of Nanjing University（Natural Science）

基　　金：江苏省自然科学基金(BK2003010)

摘　　要：人肝癌细胞株SMMC-7721体外转染反义VEGR_(165)基因,观察反义VEGF_(165)基因对肝癌细胞生长的作用。用分子克隆的方法构建反义VEGF_(165)基因其核表达载体 pcDNA_3-as-VEGF_(165),用阳离子脂质体介导的基因转染技术,将反义基因转入SMMG-7721肝癌细胞株,观察反义VEGF_(165)基因抑制肝癌细胞 VEGF蛋白表达,抑制细胞增殖,促进细胞凋亡的作用。RT-PCR结果显示转染反义VEGF_(165)基因后肝癌细胞内 VEG mRNA水平显著下降;ELISA方法显示转染反义基因 2 d后培养液上清 VEGF蛋白分泌显著下降((142.01±7.95)vs(1 625.52±64.46)pg/mL,n=4,P<0.01);免疫组织化学染色显示在培养板中转染及义VEGF_(165)基因的肝癌细胞细胞数显著减少,仅有少数细胞表达VEGF蛋白。流式细胞仪结果显示转染反义VEGF_(165)基因2 d后肝癌细胞的存活率下降了 12.53％(n=4,P<0.05),而凋亡率则上升了 13.12％(n=4,P<0.01)。MTT法显示反义VEGF_(165)基因能显著抑制肝癌细胞的增殖。反义VEGF_(165)基因能显著的抑制肝癌细胞VEGF蛋白的表达,抑制细胞的增殖,促进肝癌细胞凋亡。因此反义VEGF_(165)基因治疗有望成为一种新的治疗肝癌的方法。This paper is to study the effect in vitro on the proliferation of hepatocarcinoma SMMC-7721 by antisense VEGF_(165) eukaryotic expression vector pcDNA3-as-VEGF_(165). The vector was transferred into human hepatocarcinoma cells SMMC-7721 with cation lipofectamine 2000(LF2000) mediated methods to study the suppression of VEGF protein expression and the promotion of apoptosis on hepatocarcinoma cells. The detection indicated VEGF mRNA expression was notably decreased in SMMC-7721 cells by RT-PCR after pcDNA_3-as- VEGF_(165) transfection. The expression of VEGF protein was dramatically inhibited (( 142.01±7.95) vs (1625.52±64.46) pg/mL, P<0.01) 2 days after transfection, which correlated with the dose of antisense VEGF_(165) gene. VEGF protein was mostly expressed in pcDNA_3-β-gal transferred SMMC-7721 cells while few in pcDNA_3-as-VEGF_(165) transferred cells by immunohistochemical staining. Besides, the apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98 ±0.86% vs 4.86±0, 27 %, P<0.01) and the survival rate was notably decreased (80.99 ±3.20 % vs 93.52 ±3.93 %, P<0.05) due to antisense VEGF_(165) by flow cytometry (FCM). The transfection of antisense VEGF_(165) gene resulted in the inhibitory effects on the proliferation of hepatocarcinoma cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). It is confirmed in our study that antisense VEGF_(165) can inhibit the expression of VEGF protein, interfere with the proliferation, and induce the apoptosis of hepatocarcinoma cells. Antisense VEGF_(165) gene therapy may play an important role in the treatment of human hepatocarcinoma.

关 键 词：血管内皮细胞生长因子 VEGF 肝癌 反义 基因治疗 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]