腺病毒介导的反义VEGF与可溶性VEGF受体联合对实验性MM45T.Li小鼠肿瘤生长及转移的影响

Inhibition of Soluble VEGF Receptor Gene and Antisense VascularEndothelial Growth Factor Nucleotide on Growth,Metastasis, and Mortality Rate in ExperimentalMurine Hepatoma Mediated by Adenovirus

目的:构建并观察含反义VEGF、sflt1、sflt1联合反义VEGF及lacZ的重组腺病毒对MM45T.Li小鼠肿瘤生长及转移的影响。方法:通过RTPCR从胚胎小鼠中克隆可溶性VEGF受体基因sflt1,并比较sflt1与3′末端缺失可溶性VEGF受体基因3′Δflt1在COS7细胞中的表达效率;将含反义VEGF的重组腺病毒和含lacZ报告基因的重组腺病毒分别感染MM45T.Li细胞株,观察反义VEGF体外抑制VEGF分泌的作用;采用CAM实验模型,观察含目的基因的重组腺病毒对鸡胚血管形成的影响;分别将含不同目的基因的重组腺病毒进行瘤内直接注射,每周体外测量肿块大小,7周后处死小鼠,观察有无转移,并对肿瘤组织冰冻切片后进行VEGF、可溶性VEGF受体(sflt1)表达及新生血管形成的免疫组织化学分析;余下的小鼠共观察13周,记录小鼠死亡时间,计算生存函数。结果:含反义VEGF的重组腺病毒感染MM45T.Li细胞后,能明显减少VEGF的分泌P<0.01;鸡胚注射Adsflt1、AdantisenseVEGF及Adsflt1/antisenseVEGF后,其血管的分布较注射AdlacZ组明显减少,甚至造成死胚;经sflt1、反义VEGF治疗的荷瘤小鼠肿瘤生长速度较对照组明显减慢(P<0.01),肿瘤体积明显变小(P<0.01),肿瘤转移率减少(P<0.05),13周生存率明显延长(P<0.01);而且反义VEGF与sflt1联合具有正叠加效应。

Objectives: To determine the inhibition of soluble VEGF receptor gene (sflt 1) and antisense VEGF nucleotide on growth, metastasis, and morality rate of the mice with experimental murine hepatoma. Methods: sflt 1 gene was cloned from the 12th day mouse embryos, the expression efficiency of sflt 1 was compared with that of 3′ termini deleted flt 1 (3′Δflt 1) in COS 7 cells. Suppression of antisense VEGF on VEGF secretion in MM45T.Li cells was performed through testing the VEGF concentration of cultured supernatant by ELISA 72 h after infection with Ad antisense VEGF. Inhibition of Ad antisense VEGF and Ad sflt 1 on neovasculrization in vitro was assayed using CAM model. Experimental murine hepatoma model was produced by inoculating the MM45T.Li cells in forelimb axilla of the syngenic Balb/c mouse, tumor bearing mice were divided into 4 groups, each group was treated separately by recombinant adenoviruses containing different foreign gene; the sizes of tumors were measured every week; The metastatic tumor was investigated seven weeks after treatment; Immunohistochemistry of VEGF, sFlt 1 and CD31 were performed on cryostat section of tumor; the remaining mice would be investigated for a total of thirteen weeks, the death time of the mice would be accounted, the survival time was calculated by SAS software. Results: Expression of sflt 1 was stronger than that of 3′Δflt 1 in COS 7 cells; secretion of VEGF can be decreased after MM45T.Li cells infected with Ad antisense VEGF(P< 0.01). Blood vessels of chicken embryo treated with Ad sflt 1 or Ad antisense VEGF grew more slowly than that of the control chicken embryo, some treated chicken embryos were even died. Tumor size, growth rate of tumor, and metastatic rate in the mice treated with Ad sflt 1 or Ad antisense VEGF were significantly slower than that of the control. Survival rate of the mice treated with Ad sflt 1 or Ad antisense was prolonged significantly, furthermore, there was enhanced effect in combining therapy of antisense VEGF with sFl 1. Conclusion: Both Ad antisense VEGF and Ad sflt 1, can effectively inhibit neovascularization, Ad antisense VEGF combined with sflt 1 would increase the inhibition effect. Moreover, antisense VEGF and sFlt 1 strongly inhibited both tumor metastasis and neovascularization, and the survival time of the treated mice was prolonged. Therefore, inhibiting vascularization may be an effective way in tumor gene therapy.