内皮抑素基因腺病毒载体的构建及表达

Study on the construction of recombinant adenovirus vector expressing human endostatin and its expressional activity

目的:构建人内皮抑素(Hum an Endostatin,hE)基因的腺病毒载体,并研究其对胃癌细胞株SGC-7901、MKN-45及人脐静脉内皮细胞系ECV304生物学特性的影响。方法:采用L ipofectam ine2000法将含有人内皮抑素基因的质粒pCA13-hE与pBGHE3共同转染293细胞;免疫荧光法及W estern B lot检测hE的表达;用不同感染复数(Mu ltip lic ity of infection,MO I)的重组人内皮抑素基因的腺病毒感染ECV304细胞,观察细胞生长。结果:成功构建了含hE基因的腺病毒载体;经含hE基因腺病毒载体感染的人SGC-7901胃癌细胞株、MKN-45胃癌细胞株均表达hE蛋白;表达的hE蛋白具有一定的生物学活性,可以抑制人静脉内皮细胞系ECV304的生长。结论:获得了有表达功能活性的Endostatin腺病毒载体,表达产物可抑制ECV304细胞的增殖,为肿瘤的抗血管基因治疗提供了必要条件。

Objective： To construct the recombinant adenovirus vector expressing human endostatin and explore its biological activity which treated with the cellines of SGC-7901, MKN-45 and ECV304 by the adenovirus vector. Methods： Recombinant plasmid pcA13-hE containing human endostatin gene was cotransfered with pBGHE3 into 293 packaging cells by lipofectine-mediated transfection, the expressions of human endostatin were detected by immunofluorescence and Western -Blot, the growth behavior of ECV304 cells were obsevered when the cells were infected by different MOI recombinant adenovirus vector containing human edostatin gene. Results： The expression vector of human endostatin was successfully constructed and tranfected into the cellines of human stomach cancer SGC-7901 and MKN-45. The two cellines also expressed the human endostatin protein, and the expressional protein had biological activity and could inhibit the growth of ECV304 cells. Conclusion： The recombinant adenovirus vector of functional human endostatin gene was obtained, the expressional products inhibited the growth of ECV304 cells. The study has significance for gene therapy of tumor.