CKI p15INK4B高表达对人黑色素瘤细胞cyclinDl和c-myc表达的影响及生长特性的改变

通过 DNA体外重组和转染技术 ,将已构建好的含有 p1 5基因的质粒 p XJ- 41 - p1 5转染 p1 5缺失的人黑色素瘤细胞 A 375,经 G 41 8筛选出阳性单克隆 ,并经 PCR、Western等检测 ,证明建立了 p1 5稳定高表达的细胞模型 .生长曲线和 FCM实验表明 ,与对照组细胞相比 ,实验组细胞增殖能力减弱 ,G1期细胞增加 ,S期细胞减少 .同时 ,细胞血清依赖性有所恢复 ,在软琼脂的集落形成能力下降 ,显示出细胞部分恶性表型逆转 .进一步免疫印迹实验表明 ,癌基因 c- myc的蛋白表达水平明显下降 .Cyclin D1的表达受到抑制 ,而 CDK4的表达则基本不变 .以上结果显示出 ,p1 5基因高表达能够抑制人黑色素瘤细胞的增殖及部分恶性表型 ,负调细胞周期进程 ,而 c- Myc蛋白和 G1期引擎分子 Cyclin D1表达的被抑制可能是 p1 5负调细胞增殖的分子机理之一

Effect of Overexpression of CKI p15INK4B on the Expressions of CyclinD1 and c-myc and the Change of Growth Characteristic

Using DNA in vitro recombination and gene transfection,the plasmid pXJ 41 p15 which included the p15 INK4B gene was introduced into human melanoma cell A375.G418 was used to select the G418 resistant clone.With the analysis of PCR and Western blot,the human melanoma cell model which overexpressed the p15 gene was shown to have established.Growth curve and FCM showed that the proliferation of melanoma cell overexpressing was inhibited in contrast of control. In addition,p15 overexpression altered some of the malignant morphology.For example,it recovered the serum dependence of the cells,and decreased the capacity of growth in soft agar.Furthermore,Western blot showed that the expression of c myc was strongly declined.Investigation of engine molecules of G 1 phase showed that the level of CyclinD1 was inhibited,but the expression of CDK4 almost had no change.In summary,overexpression of p15 can inhibit the proliferation and some of the malignant morphology of human melanoma cell and negatively regulate the procession of the cell cycle.The inhibition of the expressions of c Myc and CyclinD1 may be one of the molecular mechanisms of p15 on negative regulation of cell proliferation.