重组腺相关病毒介导的RNAi靶向治疗作用研究

Study of Recombinant Adeno-associated Virus Mediated RNAi Targeting Therapy

目的:采用前期成功构建的靶向沉寂CDK2基因的重组腺相关病毒r AAV-sh RNA-CDK2转染人肝癌Hep G2细胞,研究其对人肝癌细胞增殖的抑制作用。方法:取人肝癌Hep G2细胞于裸鼠前肢腋下接种,构建裸鼠皮下移植瘤模型,将成瘤裸鼠随机分为三组:肿瘤组、NC对照组、r AAV-sh RNA-CDK2给药组。各试验组均通过尾静脉注射给药,每隔五天用游标卡尺测量肿瘤的长径(a)、短径(b),计算肿瘤体积。根据每组裸鼠移植瘤体积的平均值,绘制移植瘤生长曲线。于给药24 h后处死,称取瘤重,计算抑瘤率,应用实时荧光定量PCR和Western blot方法检测各组肝癌组织中CDK2基因m RNA和蛋白的表达量,观察r AAV-sh RNA-CDK2对肝癌组织CDK2表达的影响;结果:r AAV-sh RNA-CDK2能够显著抑制肝癌Hep G2细胞的增殖,其抑瘤率为72.18%;并能够下调肝癌组织中CDK2基因m RNA与蛋白表达量;结论:r AAV-sh RNA-CDK2实现了体内靶向治疗肝癌的目的,并确定静脉定量给药方式。

Objective： To study the inhibitory effect on human hepatoma cells, the recombinant adenovirus r AAV-sh RNA-CDK2,a target silenced CDK2 gene, was successfully transfected into human hepatoma Hep G2 cells. Methods： Human hepatoma Hep G2 cells were inoculated into the armpit of the forelimb of nude mice to establish subcutaneous xenograft model. The experiment was divided into three groups： tumor group, NC group and r AAV-sh RNA-CDK2 group. All groups are through the tail vein injection. The tumor long diameter（a）, short diameter（b） and tumor volume were measured and calculated with a vernier caliper after every five days. According to the mean tumor volume of nude mice in each group, the tumor growth curves were plotted. After last administrations for 24 hours,mice were killed and the tumor inhibition rate was calculated; the effect of r AAV-sh RNA-CDK2 on CDK2 expression levels of m RNA and protein were studied by Real-time PCR and Western blot. Results： r AAV-sh RNA-CDK2 can effectively inhibit the proliferation of Hep G2 hepatoma cells. Its inhibition rate was 72.18%. r AAV-sh RNA-CDK2 make the m RNA and protein expression of CDK2 in liver cancer tissues reduced significantly. Conclusion： Recombinant adeno-associated virus r AAV-sh RNA-CDK2 realized the purpose of targeted therapy of liver cancer in vivo, and determined the quantitative dosing intravenous approach.