重组腺病毒Ad-mALR和Ad-hALR的构建及其对棕榈酸(PA)诱导的L02细胞凋亡的影响

Construction of recombinant adenovirus Ad-mALR and Ad-hALR and its influence on the L02 cell apoptosis induced by palmitic acid

目的构建含肝再生增强因子(ALR)基因的重组腺病毒并探讨其抗凋亡功能。方法采用基因重组法构建重组穿梭载体p Ad Track-TO4-m ALR和p Ad Track-TO4-h ALR,同源重组法构建重组腺病毒Ad-GFP-m ALR和Ad-GFP-h ALR,4轮扩增后获得高滴度Ad-GFP-m ALR和Ad-GFP-h ALR。将上述腺病毒感染L02细胞,通过绿色荧光蛋白(GFP)的表达了解其感染率;Western blotting法检测ALR及Bcl-2/Bax蛋白表达;CCK-8法检测细胞增殖活性;流式细胞仪检测ALR对棕榈酸(PA)诱导的L02细胞凋亡的影响。结果重组腺病毒Ad-GFP-m ALR和Ad-GFP-h ALR构建成功,且均能高效感染并稳定表达于L02细胞。与非感染组和感染空载病毒组相比,过表达ALR能促进L02细胞的增殖,并抵抗PA诱导的L02细胞凋亡作用,明显降低Bax,增加Bcl-2蛋白的表达。结论 ALR对L02细胞具有促增殖及抗PA诱导的细胞凋亡的作用。

Objective To construct recombinant adenovirus expressing augmenter of liver regeneration（ALR） gene and investigate its anti-apoptosis effect.Methods Recombinant shuttle vector p Ad Track-TO4-m ALR and p Ad Track-TO4-h ALR were constructed by recombinant DNA technology and recombinant adenovirus Ad-GFP-m ALR and Ad-GFP-h ALR were acquired by homologous recombination.After four cycles of amplification,high titers of the recombinant adenovirus Ad-GFP-m ALR and AdGFP-h ALR were obtained.The expression of green fluorescent protein was detected to evaluate the infection efficiency of Ad-GFPm ALR and Ad-GFP-h ALR,Western blotting was applied to assess the expression of ALR and Bcl-2/Bax protein,and CCK-8 assay was used to evaluate the proliferation activity of L02 cells when L02 cells were infected with either Ad-GFP-m ALR or Ad-GFPh ALR.Flow cytometry was utilized to detect the L02 cells＇ apoptosis rate induced by palmitic acid（PA）.Results p Ad Track-TO4-m ALR,p Ad Track-TO4-h ALR,Ad-GFP-m ALR and Ad-GFP-h ALR were constructed successfully;the recombinant adenovirus AdGFP-m ALR and Ad-GFP-h ALR could infected L02 cells efficiently and expressed steadily in L02 cells.The L02 cells infected with either Ad-GFP-m ALR or Ad-GFP-h ALR had significantly increased proliferation activity compared with those uninfected and those infected with Ad-GFP.L02 cells with the infection of either Ad-GFP-m ALR or Ad-GFP-h ALR had significantly lower apoptosis rate,enhanced the Bcl-2 expression and decreased the Bax expression when L02 cells were treated with palmitic acid compared with those uninfected and those infected with Ad-GFP.Conclusion Recombinant adenovirus Ad-GFP-m ALR and Ad-GFP-h ALR here have been constructed successfully and the over expression of either m ALR or h ALR has the ability to promote the proliferation activity of L02 cells and the effect against L02 cell apoptosis induced by palmitic acid.