含MMP-9信号肽、人MMP-2PEX片段腺病毒载体的构建及其生物学活性

Construction of a recombinant adenovirus containing MMP-9 signal peptide and PEX domain of human MMP-2 and study of its activity

目的:构建含MMP-9信号肽、人MMP-2PEX片段融合蛋白的重组腺病毒(ET-M9-PEX),通过基因治疗策略建立患者体内药物生物反应器,以期利用抗血管因子的体内表达实现肿瘤的有效治疗。方法:利用PCR、基因重组等技术,构建含M9-PEX融合蛋白的腺病毒载体,经L293细胞包装、扩增后得到有感染力的ET-M9-PEX重组腺病毒。用Westernblotting和免疫荧光检测其感染细胞后PEX的表达和分泌,通过生长曲线观察其感染内皮细胞(endothelialcell,EC)的培养上清对EC增殖的影响,利用鸡胚绒毛尿囊膜(chickenchorioallantoicmembrane,CAM)实验检测其对血管发生和种植瘤生长的影响。结果:ET-M9-PEX构建成功,感染细胞后有PEX的表达和分泌。体外实验表明,ET-M9-PEX感染细胞的培养上清能抑制EC增殖。体内实验证实,ET-M9-PEX感染后的PG细胞接种于CAM,与E-T相比,对瘤重的抑制率为57.57%(P<0.01),对一级血管的抑制率为33.52%(P<0.01);而E-T和PBS组之间瘤重和一级血管数均无显著性差异。结论:构建的ET-M9-PEX重组腺病毒能显著抑制EC细胞增殖,抑制CAM种植瘤生长和血管发生,ET-M9-PEX有望成为肿瘤基因治疗的有效药物。

Objective： To construct a recombinant adenovirus （abbreviated as ET-M9-PEX） containing MMP-9 signal peptide and noncatalytic carboxyl-terminal hemopexin domain of human MMP-2, and to use the constructed adenovirus as a drug bioreactor in vivo to enhance the expression of an anti-angiogenesis factor for treatment of tumor by a gene therapy strategy. Methods ： Adenovirus vector containing M9-PEX gene was constructed by PCR and gene recombination, and was packaged and amplified in L293 cells to obtain ET-M9-PEX recombinant adenovirus with infective ability. The expression and secretion of PEX in ET-M9-PEX-infected cells were detected by Western-blotting and immunofluorescent staining. The inhibitory effect of ET-M9-PEX-conditioned medium on EC cells proliferation was detected by growth curve and its inhibitory effects on angiogenesis and tumor growth were determined by chicken chorioallantoic membrane （CAM） assay in vivo. Results：ET-M9-PEX was successfully constructed and the expression and secretion of PEX in ET-M9-PEX-infected cells were verified. The ET-M9-PEX conditioned medium significantly inhibited the proliferating rate of EC cells. The tumor weights from ET-M9-PEX-infected PG cells in CAM and grade I vessel number were reduced by 57.57% （P〈0.01 ） and 33.52% （P〈0.01 ） , respectively, compared with E-T. However, neither the tumor weight nor the vessel number was significantly different between E-T and PBS groups. Conclusion： ET-M9-PEX constructed in the present study shows remarkable inhibitory effects on EC proliferation, tumor growth and angiogenesis on CAM, suggesting that ET-M9-PEX may be a promising candidate for tumor gene therapy.