Ki67启动子调控的肿瘤增殖腺病毒对黑素瘤细胞的杀伤作用

The therapeutic efficiency of conditionally replicative adenovirus regulated by Ki67-promoter targeted at melanoma cells

目的:研究Ki67启动子调控增殖腺病毒Ki67-ZD55对黑素瘤A375细胞的杀伤作用。方法:将PBS、ZD55-EGFP和Ki67-ZD55分别作用于A375细胞。RT-PCR法检测Ki67-promoter mRNA的表达;荧光显微镜观察Ki67-ZD55增殖过程;MTT法检测细胞存活率;Western Blot法检测E1A蛋白及线粒体途径凋亡相关蛋白BAX、Bcl-XL、MCL-1和Caspase-3的表达。结果:RT-PCR检测结果显示Ki67-ZD55能够在A375细胞中表达Ki67-promoter序列;荧光显微镜观察结果显示Ki67-ZD55增殖明显;MTT结果表明Ki67-ZD55组对A375细胞的增殖具有明显抑制作用,抑制效果大于ZD55-EGFP组(P<0.05);Western Blot法检测结果表明E1A、BAX的表达量增加,Bcl-XL、MCL-1的表达量均降低,Procaspase-3发生了明显剪切。结论:Ki67-ZD55能够明显抑制A375细胞增殖,促进A375细胞凋亡。

Objective： To evaluate the therapeutic efficiency of conditionally replicative adenovirus regulated by Ki67 - promoter Ki67 - ZD55 targeted at A375 ceils of human melanoma cell lines. Methods： The A375 cells were treated with PBS, ZD55 - EGFP and Ki67 - ZD55 respectively. The expression of Ki67 - promoter mRNA was detected by RT- PCR. Fluorescence microscopy was used to observe the proliferation of Ki67 - ZD55. The survival rate of A375 cells was calculated by M＇IT assay. Furthermore, the proteins of E1A, BAX, Bcl - XL, MCL- 1 and Caspase - 3 were measured by western blotting. Results： It was confirmed by RT - PCR that the Ki67 - promoter mRNA was expressed only in Ki67 - ZD55 group. Fluorescence microscopy indicated obvious proliferation of Ki67 - ZD55. M3T assay showed that Ki67 - ZD55 could induce more obviously growth inhibition of A375 cells than ZD55 - EGFP （P 〈 0.05）. Western Blotting showed that Ki67- ZD55 can upregulating the proapoptotic proteins E1A and BAX and downregulating the antiapoptotic proteins Bcl - XL and MCL- 1, as well as, activation of Procaspase - 3. Conclusion： This study proved that Ki67 - ZD55 exhibited a remarkably increased cytotoxic and apop- tosis - inducing effects on A375 cells.