PEI介导的E1A基因对结肠癌细胞放疗增敏效应的研究

The radio-sensitivity effect of E1A gene transfected by PEI on colon carcinoma cell in vitro

以自行合成的聚乙烯亚胺纳米凝胶(Polyethylenimine,PEI)为载体转染E1A基因,观察E1A基因对结肠癌细胞体外生长的抑制作用和对电离辐射的增敏效应,并初步探讨其作用机理。经PEI介导将真核细胞高效表达E1A基因的重组质粒psv-E1A导入人结肠癌细胞系SW480,RT-PCR证实其转染,G418筛选出阳性克隆细胞。通过测绘生长曲线,观察E1A基因对该细胞系生长的抑制作用。流式细胞术检测转染前后SW480细胞的细胞周期分布变化。用噻唑蓝(Methyl thiazolyl tetrazolium,MTT)法测定电离辐射对SW480转染E1A基因前后的效应。用Western-blot方法测定结肠癌细胞SW480转染质粒psv-E1A前后HER-2蛋白表达量的变化。(1)G418筛选出阳性克隆细胞。(2)RT-PCR结果显示PEI能转染质粒psv-E1A并有稳定的表达。(3)流式细胞仪测细胞周期,与SW480细胞相比,SW480-E1A细胞S期呈下降趋势(p<0.001);G2/M期增加显著(p<0.001);G1期细胞无明显变化(p>0.05)。(4)MTT法测量SW480细胞及SW480-E1A细胞不同时间的吸光度值,可见转染E1A基因细胞群体生长缓慢。(5)转染E1A基因后,SW480-E1A细胞对电离辐射的敏感性显著提高(p<0.001)。(6)E1A基因明显降低结肠癌细胞中HER-2蛋白的表达量。PEI能转染质粒psv-E1A,E1A基因能够明显抑制结肠癌细胞的生长,并明显提高其对放射线的敏感性。

As a neotype nonviral vector,（Polyethylenimine,PEI） has been studied in gene transfection experi-ment.This study was investigated the growth inhibition and radio-sensitizing effect of E1A gene transfected by PEI on human colon carcinoma cell in vitro.The PSV-E1A recombinant plasmid,which was designed for high-level ex-pression of E1A gene in a variety of eukaryotic cell lines,was transfected into SW480 cells by PEI.The transfection was confirmed by RT-PCR and G418 was used to get colon carcinoma cells stably expressed E1A gene.The cell growth curve were investigated to observe the growth inhibition induced by E1A gene.The redistributions of cell cy-cle were analyzed by flow cytometry.Cells before and after transfection were treated with irradiation,then the changes of radiation-sensitivity were tested by MTT assay after 24 h meanwhile the expression of HER-2 gene in SW480 cells before and after transfection was detected by western-blot.As results,（1） the colon carcinoma cells ex-pressed E1A gene was confirmed by G418.（2）The result of RT-PCR demonstrated that PEI could transfect plasmid psv-E1A and the cells could stably express E1A gene.（3）Flow cytometry revealed that E1A gene transfected into hu-man colon carcinoma cell could induce S stage suppression（p0.001） and G2/M stage arrest（p0.001）.（4）Compared with the Non-transfected cells,the E1A-transfected cells（SW480-E1A cells） grew slowly observed by MTT assay which was used to get the absorbance of SW480 cell and SW480-E1A cell.（5）The radiation-sensitivity of SW480 cells transfected with E1A gene was up-regulated obviously（p0.001）.（6） The E1A gene obviously down-regulated HER-2 protein expression in colon carcinoma cells.Anyway,PEI can transfect plasmid psv-E1A gene which can sig-nificantly inhibit the growth rate of SW480 cell.Moreover,it also obviously enhanced the cell sensitivity to irradia-tion.