内皮抑素和血管抑素融合基因在杆状病毒中的表达及抗肝癌效果评价  

Expression of endostatin and angiostatin fusion gene in baculovirus and evaluation of its anti-hepatoma effect

作　　者：王志昇 李梦婷 姬勇敢 杨萌萌 WANG Zhisheng;LI Mengting;JI Yonggan;YANG Mengmeng(Laboratory Animal Center,School of Pharmacy,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学实验动物中心,银川750004 [2]宁夏医科大学药学院,银川750004

出　　处：《实用医学杂志》2020年第15期2053-2058,共6页The Journal of Practical Medicine

基　　金：国家自然科学基金项目(编号:31460242);宁夏自然科学基金项目(编号:NZ17067)。

摘　　要：目的评价杆状病毒介导的内皮抑素和血管抑素融合蛋白(hEA)的抗肝癌效果。方法以质粒pFastBac DUAL为骨架,将pPh启动子替换为CMV,然后在其下游插入hEA基因,获得重组质粒pBac-ChEA,将pBac-ChEA转化DH10Bac感受态细胞,经蓝白斑筛选获得重组Bacmid,然后经脂质体法转染Sf-9昆虫细胞,获得重组杆状病毒Bac-ChEA。将重组病毒转导HUVEC细胞,通过ELISA、MTT、细胞划痕和血管网络形成检测hEA蛋白的表达以及对HUVEC细胞的增殖、迁移和血管形成的影响;建立裸鼠皮下肝癌肿瘤模型,评价杆状病毒介导的hEA的抗肝癌效果。结果经PCR鉴定成功构建了重组杆状病毒Bac-ChEA;hEA蛋白在Bac-ChEA转导的HUVEC细胞中成功获得了表达并且能够有效抑制HUVEC细胞的增殖、迁移以及血管网络的形成;体内结果表明Bac-ChEA组能有效抑制肝癌肿瘤体积的增长,小鼠寿命(中位生存期)从Bac-CMV组的24 d显著延长至Bac-ChEA组的36 d(P <0.05)。结论本研究成功让hEA蛋白在杆状病毒中获得了表达,并且能够有效地抑制HUVEC细胞的增殖、迁移和血管形成以及小鼠肝癌肿瘤体积的增长和提高小鼠生存率。Objective To evaluate the anti-hepatoma tumor effect of baculovirus-mediated endostatin and angiostatin fusion protein(hEA).Methods Using the plasmid pFastBac DUAL as the backbone,the pPh promoter was replaced with CMV,then the hEA gene was inserted downstream of the CMV promoter to obtain the recombinant plasmid pBac-ChEA.The pBac-ChEA plasmid was transformed into DH10 Bac competent cells,and the recombinant bacmids were obtained by blue-white screening.Then the recombinant bacmids were transfected into Sf-9 insect cells by liposome,resulting in recombinant baculovirus Bac-ChEA.The recombinant virus was transduced to HUVEC cells,and the expression of hEA protein and its effects on proliferation,migration and angiogenesis of HUVEC cells were detected by ELISA,MTT,cell scratch and vascular network formation.The subcutaneous hepatocellular carcinoma tumor model was established in nude mice to evaluate the anti-hepatoma tumor effect of baculovirus-mediated hEA protein.Results The recombinant baculovirus Bac-ChEA was successfully constructed by PCR identification.The hEA protein was successfully expressed in Bac-ChEA transduced HUVEC cells,as well as effectively inhibited HUVEC cell proliferation,migration,and formation of vascular networks.In vivo results demonstrated that Bac-ChEA group could effectively inhibit the growth of tumor volume of liver cancer,and the lifespan(median survival) of mice was significantly extended from 24 d in Bac-CMV group(P <0.05) to 36 d in BacChEA group.Conclusion In this study,the hEA protein was successfully expressed in baculovirus and effectively inhibited the proliferation,migration and angiogenesis of HUVEC cells,as well as the growth of tumor volume of liver cancer in mice,and improved the survival rate of mice.

关 键 词：杆状病毒 内皮抑素 血管抑素 肝癌 基因治疗 

分 类 号：R735.7[医药卫生—肿瘤]