腺病毒介导干扰RNA对荷人骨肉瘤裸鼠VEGF表达的影响

目的: 探讨AD-VEGF-siRNA对荷人骨肉瘤裸鼠瘤体组织以及血液中VEGF表达的影响。 方法: 应用VEGF高表达人骨肉瘤细胞系MG-63制备荷骨肉瘤裸鼠模型;成瘤组动物随机分为3组,AD-VEGF-siRNA组15只;AD-EGFP组15只;PBS组15只。另留取3只裸小鼠,未接种肿瘤细胞,未注射药物。成瘤裸鼠瘤体内分别注射上述各组药物,隔天1次,200μl/次,共5次,病毒总量2×10⁹PFU。接种后第11天、14天、17天实验各组分别处死3只裸小鼠,用ELISA法检测各组荷瘤小鼠血液和瘤体组织中VEGF蛋白表达情况。试验结束(第19天)处死剩余裸小鼠,用RT-PCR及免疫组化技术检测裸小鼠瘤体组织VEGF的表达,用ELISA法检测各组荷瘤小鼠血液和瘤体组织中VEGF蛋白的表达。 结果: 1)各组裸小鼠约在接种5天成瘤;2)用AD-VEGF-siRNA处理后,RT-PCR检测发现裸小鼠瘤体组织的VEGFmRNA表达水平明显低于两对照组(P<0.05);3)免疫组化检测发现AD-VEGF-siRNA组VEGF蛋白表达明显减少;4)ELISA检测发现成瘤组小鼠血清VEGF蛋白水平明显高于未作任何处理的健康裸小鼠组(P<0.05),在各时间段AD-VEGF-siRNA组血液及瘤体组织中VEGF蛋白的表达水平明显低于两对照组(P<0.05)。 结论: AD-VEGF-siRNA在体内可以抑制荷骨肉瘤裸小鼠瘤体VEGF基因的表达及血液中VEGF的水平;应用siRNA腺病毒表达载体技术为骨肉瘤抗血管生成的治疗提供了借鉴。

The Effect of Adenovirus-packaged VEGF-siRNA on the Expression of Vascular Endothelial Growth Factor in Osteosarcoma-bearing Nude Mice

Objective: To explore the effect of AD-VEGF-siRNA on the expression of VEGF in neoplasm andblood serum. Methods: A transplantable model of human osteosarcoma was successfully established using subcutaneousinjection of human MG63 osteosarcoma cells with high VEGF expression. The nude mice injected in this manner wererandomly divided into 3 groups: 15 in the AD-VEGF-siRNA group, 15 in the AD-EGFP group, and 15 in the PBS group.Three additional nude mice were raised without injections of tumor cells or drugs. AD-VEGF-siRNA was injected intratu-morally once a day, with a total viral dose of 2× 10⁹ pfu. Three osteosarcoma-bearing mice from each group were sacri-ficed at 11, 14, and 17 days after the implantation of MG63 cells. The remaining mice were sacrificed at the end of theexperiment (19 days after MG63 injection). The expression of VEGF in implanted tumors was detected by RT-PCR andimmunohistochemistry. ELISA was employed to detect Vd:\\PDF\\.pdfEGF protein in implanted tumors and blood serum. Results: (1)Tumors could be seen 5 days after the implantation of MG63 cells. (2) Positive in all groups, the expression level ofVEGF mRNA was lower in the AD-VEGF-siRNA group than in the other two groups(P<0.05). (3) The expression level ofVEGF in the serum of osteosarcoma-bearing mice was higher than that in the 3 healthy mice(P<0.05). (4) The expressionlevel of VEGF protein in blood serum and neoplasm in the AD-VEGF-siRNA group was lower than in the other twogroups (P<0.05). Conclusion: AD-VEGF-siRNA can effectively inhibit VEGF expression in vivo. AD-VEGF-siRNA mayprovide a new therapy to inhibit angiogenesis in osteosarcoma.