表达血管抑制因子的腺相关病毒载体的构建及其体内外活性

Construction and Activity of Recombinant Adeno-associated Virus Expressing Vasostatin

摘要:

血管抑制因子(Vasostatin, VAS), 为集钙蛋白N-末端180个氨基酸大小的蛋白, 是一种内源性血管生成抑制因子, 对多种肿瘤的生长具有很强的抑制作用。近期有研究显示, VAS可以促进神经内分泌肿瘤的恶化, 提醒我们在开发该抗肿瘤药物时必需非常谨慎。将VAS cDNA插入腺相关病毒-2表达质粒pAAV-2, 采用无辅助病毒参与的三质粒共转染法制备rAAV-VAS病毒。体外分别转染小鼠胰内皮细胞MS1和结肠癌细胞HCT-116, MTT法测定对细胞生长的影响, Western blotting方法检测VAS的表达。采用小鼠皮下移植瘤模型, 验证VAS的表达对肿瘤生长、新生血管密度、以及细胞增殖的作用。结果证明构建的rAAV-VAS病毒载体, 能抑制小鼠胰内皮细胞的生长, 转染HCT-116后能有效表达VAS蛋白, 但HCT-116的体外生长不受影响。瘤体注射rAAV-VAS后, HCT-116移植瘤在小鼠体内的生长速度明显减缓,肿瘤新生血管密度明显降低。结果显示, rAAV-VAS可以抑制HCT-116移植瘤的新生血管形成, 但对其细胞增殖无明显作用。

Abstract:

Vasostatin, a 180-amino acid fragment from the N-terminal domain of calreticulin, is a potent endogenous angiogenesis inhibitor, which can inhibit the growth of many kinds of experimental tumor. But a recent report that vasostatin can enhance the malignant behavior of neuroendocrine tumor reminds us to be cautious to develop it as an anti-tumor medicine. VAS cDNA was cloned into pAAV-2 expression vector; recombinant virus rAAV-VAS was generated by a three plasmids, helper free packaging method. MS1 mouse pancreatic endothelial cell and human colon tumor HCT-116 cell were infected with rAAV-VAS. Transgene expression was analyzed by Western blotting analysis; cell proliferation was determined by MTT assay. The therapeutic potential of rAAV-VAS was evaluated in subcutaneous HCT-116 xenograft mouse model. rAAV-VAS inhibited the proliferation of MS1 but not HCT-116 cell. HCT-116 cell infected with rAAV-VAS secreted VAS protein into the supernatant effectively. The intratumoral delivery of rAAV-VAS inhibited the xenograft growth and microvessel density in tumors significantly. Our results show the effectiveness of rAAV-VAS as an angiogenesis inhibitor in suppressing tumor growth, validating the application of rAAV-VAS gene therapy in treatment against colon cancer.