重组腺病毒mIκBα基因的构建及体外抑制血管生成的作用

Construction of Recombinant Adenovirus Encoding mIκBα and the Suppression of Angiogenesis in Vitro

目的应用同源重组法构建mIκBα基因的腺病毒真核表达载体,探讨其体外抑制血管形成的作用。方法通过Lipofectamine介导构建重组腺病毒pAdmIκBα,有限稀释法测定病毒滴度;以MOI为10、20、30、50感染HCC9204细胞,RT-PCR方法检测细胞NFκ-B(p65)的表达,ELISA法检测细胞上清液p65的含量;鸡胚绒毛尿囊膜检测(CAM)了解pAdmIκBα对血管形成的抑制。结果经PCR检测提示重组体腺病毒含有pAdmIκBα基因;pAdmIκBα的滴度是1.252×109pfu/ml;将pAdmIκBα转染HCC9204细胞,当MOI=10时,p65 mRNA表达和上清液p65蛋白含量开始下降,在MOI=50时降低到最低水平;pAdmIκBα对p65表达的抑制呈剂量依赖性;pAdmIκBα对血管新生有很强的抑制作用,而且随着剂量的增加作用越发明显。结论成功构建出含有mIκBα基因的具有感染能力的高滴度的重组腺病毒,并且该重组腺病毒具有体外抑制血管生成的作用。

Objective To construct the recombinant adenovirus vector expressing the human mutant IКBa（mIКBa） gene by homogenous recombination, and investigate whether mIКBa expressed by infected HCC9204 cells can inhibit angiogenesis. Methods mIКBa cDNA from plasmid PcDNA3- mIКBa cDNA was subcloned into pAdtrack-CMV vector, pAdtrack-mIКBa and the adenovirus plsmid were cotransferred into BJ5183 cells to produce adenovirus vector encoding mIКBa. The adenovirus vector was then transferred into HEK293 ceils by liposome to get recombinant adenovirus particals pAdmIКBa, and the pAdmIКBa was purified by CsC1 density purification, and the titer of the adenovirus particles was measured with limited dilution method; HCC9204 cells with MOI 10, 20, 30, 50 were infected, respectively. The expression of p65 was investigated by RT-PCR, and the quatity of p65 was detected in superreminents through ELISA. The ability of mIКBato inhibit angiogenesis was by CAM assay. Results PCR indicated that the recombinant adenovirus contained mIКBa gene, and the title was 1. 252 ×109pfu/ml. The expression of p65 protein and p65 mRNA began to desease when MOI was 10, and the lowest when MOI was 50 , they were all does-dependant, pAd mIКBa had a strong effect on suppress angiogenesis in vitro. Conclusion Infective recombinant adenovirus encoding mIКBa can be obtain by homogenous recombination, which can suppress angiogenesis in vitro.