Vasostatin基因重组腺病毒载体的构建及其对胰腺癌生长的抑制作用

Construction of replication-deficient recombinant adenovirus carrying vasostatin gene and its inhibitory effect on pancreatic cancer

目的 以复制缺陷型腺病毒为载体 ,构建vasostatin基因重组腺病毒 ,并观察其对胰腺癌裸鼠移植瘤生长的抑制作用。方法 以人肌肉cDNA文库为模板应用PCR的方法扩增出vasostatin的DNA片断 ,定向插入腺病毒穿梭质粒 pshuttle CMV经酶切、测序鉴定正确后 ,电穿孔法将重组穿梭质粒转化E .coliBJ5 183 AdEasy 1感受态细菌 ,行细菌内同源重组。将抗性筛选和酶切鉴定正确的重组质粒 pAd CMV vasostatin转染 2 93细胞进行包装。病毒扩增纯化后 ,病毒感染人胰腺癌细胞 ,RT PCR和Western印迹法检测vasostatin的表达状况。复制人胰腺癌裸鼠移植瘤模型 ,瘤内注射 10 7pfupAd CMV vasostatin、10 8pfupAd CMV LacZ及PBS ,观察移植瘤的生长曲线及瘤重。 结果 PCR产物电泳后可见长度为 5 6 0bp左右的目的条带 ;酶切及测序鉴定表明穿梭质粒 pshuttle CMV中插入了vasostatin的DNA片断 ;卡那霉素抗性筛选及PacI酶切鉴定证实腺病毒重组质粒 pAd CMV vasostatin构建成功 ;转染 2 93细胞 7天后观察到细胞病理学效应出现。PCR鉴定表明重组腺病毒中含有vasostatin片断。RT PCR和Western印迹法证实vasostatinmRNA及蛋白的表达。治疗后 3周 pAd CMV vasostatin组移植瘤的体积及瘤重均显著小于 pAd CMV LacZ组及PBS组。

Objective To construct the replication-deficient recombinant adenovirus carrying vasostatin gene and study its inhibitory effect on pancreatic cancer. Methods A cDNA clone encoding vasostatin was isolated from human skeletal muscle cDNA library by polymerase chain reaction. The shuttle plasmid, pshuttle-CMV-vasostatin, in which the amplified fragment was inserted into the downstream of the cytomegalovirus promoter, was established by ligation. After identification by digestion and sequencing, linearize the shuttle plasmid with Pme I, and then transform the linearized shuttle plasmid into E.coli BJ5183-AdEasy-1 by electroporation. The plasmid was named as pAd-CMV-vasostatin after homologous recombination and identification by kanamycin-selection and restriction digestion. The recombinant plasmid was transfected into cell 293. The recombinant adenovirus was detected by examining the presence of cytopathic effect and identified by polymerase chain reaction. Both RT-PCR and western blotting were used to identify the expression of vasostatin. The xenografted nude mice with pancreatic cancer were established to observe the inhibitory effect of vasostatin on tumor growth. The volume of xenografts was measured every three days during the 3-week treatment. The weight of xenografts was measured at the end of the 21st day. Results The PCR product was about 560 bp confirmed by agarose gel electrophoresis. The successful cloning of vasostatin into pshuttle-CMV was confirmed by restriction endonuclease analysis, and the gene of interest was identified as vasostatin by nucleotides sequencing technique. The recombinants were selected for kanamycin resistance and the recombination was confirmed by the presence of 4.5kb fragment in the restriction endonuclease analysis. The cytopathic effect present after 7 days in cell 293 transfected with linearized pAd-CMV-vasostatin. Both the cytopathic effect of cell 293 and the 560 bp PCR producet indicated the presence of the recombinant adenovirus containing vasostatin gene. The expression of vasostatin was identified by RT-PCR and western blotting. The volume and weight of xenografts in pAd-CMV-vasostatin group were significantly smaller than those in pAd-CMV-LacZ group and PBS group. Conclusion The recombinant adenovirus containing vasostatin gene was constructed successfully, and could significantly inhibit the growth of xenografted pancreatic cancer in nude mice.