表达抗-VEGF发夹状核酶腺病毒载体的构建及其对结肠癌生长的抑制作用  

Construction of adenovirus expressing anti-vascular endothelial growth factor hairpin ribozyme and its inhibition of growth of colorectal cancer HT-29 cells

作　　者：常树建[1] 陈卫昌[1] 杨吉成[2] 谢宇峰[2] 盛伟华[2] 

机构地区：[1]苏州大学附一院消化内科,江苏苏州215006 [2]苏州大学基础医学院细胞与分子生物学教研室,江苏苏州215123

出　　处：《癌症》2009年第4期373-378,共6页Chinese Journal of Cancer

基　　金：江苏省135医学重点人才基金(No.RC2007076)~~

摘　　要：背景与目的:VEGF过表达提示结肠癌预后不良。本实验构建带有抗-VEGF发夹状核酶的复制缺陷型腺病毒,观察其对结肠癌VEGF表达及肿瘤生长的抑制作用。方法:将人工合成的抗-VEGF发夹状核酶DNA定向克隆至转移质粒pAdTrack-CMV多克隆位点,然后与病毒骨架质粒pAdEasy-1在细菌BJ5183中重组,在293a细胞中包装成完整病毒(命名为Ad-Rz)并复制扩增、纯化。RT-PCR检测病毒感染后抗-VEGF发夹状核酶在HT-29细胞中的表达、real-time PCR及ELISA检测其对VEGF mRNA及蛋白表达的抑制作用。观察Ad-Rz对HT-29细胞和裸鼠移植瘤生长的抑制作用,CD34标记检测肿瘤组织微血管密度(MVD)。结果:抗-VEGF发夹状核酶成功克隆至复制缺陷型腺病毒载体上。抗-VEGF发夹状核酶可在HT-29细胞中表达,Ad-Rz感染的HT-29细胞中VEGFmRNA的相对表达量大约为PBS组的45%(P<0.05)。ELISA结果显示Ad-Rz感染后的细胞上清液中蛋白含量(A值)为0.455±0.35/百万细胞,低于PBS组(P<0.05)。Ad-Rz对HT-29细胞的生长抑制无统计学意义(P>0.05),但可显著抑制肿瘤组织内血管的形成(P<0.05),Ad-Rz治疗的移植瘤增殖率低于PBS组,但差异无统计学意义(P>0.05)。结论:腺病毒载体介导的抗-VEGF发夹状核酶可有效抑制HT-29细胞VEGF的表达及移植瘤组织内血管的生成。Background and Objective： Oversexpression of vascular endothelial growth factor （VEGF） is correlated to poor prognosis of colorectal cancer. This study was to construct replication deficient adenovirus carrying anti- VEGF hairpin ribozyme and investigate its inhibitory effects on VEGF expression and growth of colorectal cancer HT-29 cells. Methods： Anti-VEGF hairpin ribozyme was cloned into the transfer vector pAdTrack-CMV, and recombined with adenoviral backbone vector pAdEasy-1 in BJ5183 bacteria. The recombinant plasmids were packaged and amplified in 293 cells （named Ad-Rz）. The expression of anti-VEGF ribozyme in HT-29 cells was examined by RT-PCR. Inhibition of Ad-RZ on VEGF mRNA and protein expressions were detected by real-time PCR and ELISA, respectively. Inhibition of cell growth was measured by MTT assay. HT-29 cell xenografts were established in nude mice and the microvessel density （MVD） was determined by CD34 staining. Results：The anti-VEGF ribozyme was successfully inserted into the adenoviral vector. Anti-VEGF ribozyme was effectively expressed in HT-29 cells. After infection by Ad-RZ, the relative expression of VEGF mRNA was decreased to about （45± 0.01 ） % of that of PBS control group in HT-29 cells （P〈0.05） the amount of VEGF protein in the supernatant of Ad-Rz treated cells E（OD= （0.46±0.35）/million cells] was lower than that of PBS treated cells [（OD= （0.80±0.35）/million cells] （P〈0.05）. Although Ad-Rz did not significantly inhibit cell growth of HT-29 cells （P〉0.05）, it significantly inhibited tumor angiogenesis of HT-29 cell xenografts in nude mice, compared with PBS group （P〈0.05）. The proliferation rate of xenografts treated by Ad-Rz was lower than that treated by PBS, but the difference was not significant （P〉 0.05）. Conclusion. Anti-VEGF hairpin ribozyme mediated by adenovirus could inhibit the expression of VEGF and the tumor angiogenesis in colorectal cancer HT-29 cells.

关 键 词：抗-VEGF发夹状核酶 腺病毒载体 构建 VEGF 结肠肿瘤 

分 类 号：R735.35[医药卫生—肿瘤]