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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 recombinant adenovirus (ET-M9-PEX) containing MMP-9 signal peptide and human MMP-2PEX fragment fusion protein and to establish the in vivo drug bioreactor by gene therapy strategy in order to make use of in vivo expression of anti-vascular factors Effective treatment of cancer. Methods: Recombinant adenovirus vector containing M9-PEX fusion protein was constructed by PCR and gene recombination. The recombinant adenovirus vector was packaged by L293 cells and amplified. The recombinant adenovirus containing ET-M9-PEX was obtained. The expression and secretion of PEX in infected cells were detected by Western blotting and immunofluorescence. The growth curves of endothelial progenitor cells (ECs) infected with endothelial cells were observed by the growth curve. The effects of culture supernatant on the proliferation of EC were observed by using chicken chorioallantoic membrane (CAM) ) Experiments were performed to examine the effect on angiogenesis and tumor growth. Results: The ET-M9-PEX was successfully constructed and the expression and secretion of PEX after infection of cells. In vitro experiments showed that the culture supernatant of ET-M9-PEX infected cells can inhibit the proliferation of EC. In vivo experiments showed that PG cells inoculated with ET-M9-PEX were inoculated into CAM cells in a dose-dependent manner. Compared with ET, the inhibitory rates of tumor cells were 57.57% (P <0.01) and 33.52% (P <0.01). There was no significant difference in tumor weight and primary vessel between ET and PBS group. CONCLUSION: The constructed ET-M9-PEX recombinant adenovirus can significantly inhibit the proliferation of EC cells and inhibit the growth and angiogenesis of CAM implants. ET-M9-PEX is expected to become an effective drug for tumor gene therapy.