论文部分内容阅读
目的 探讨抗凋亡基因survivin在细胞转化中的作用机制。方法 利用RT PCR获得survivin编码区序列 ,双酶切后 ,将其定向克隆至原核和真核表达载体 ;生长曲线观察高表达survivin对细胞增殖的影响 ;软琼脂集落形成实验观察细胞的接触抑制生长能力 ;WesternBlot检测survivin高表达引起的蛋白质分子改变。结果 通过分子克隆 ,构建survivin原核和真核表达载体 ,并可在大肠杆菌和真核细胞中表达。生长曲线表明 ,转染survivin的 2 93细胞的增殖速度略快于对照细胞 ,尤其在细胞生长至高密度时 ;Survivin可以明显提高 2 93细胞的软琼脂集落形成能力 ;Survivin的高表达可以引起癌基因cyclinD1和c myc的蛋白表达水平增高。结论 抗凋亡基因survivin具有促进细胞转化的作用 ,这种作用可能依赖于cyclinD1和c myc的功能
Objective To investigate the mechanism of anti-apoptotic gene survivin in cell transformation. Methods The sequence of the survivin coding region was obtained by RT-PCR and cloned into prokaryotic and eukaryotic expression vector after double enzyme digestion. The growth curve was used to observe the effect of survivin on cell proliferation. Soft agar colony formation assay was used to observe the cell growth inhibition Ability; Western blot detection of high expression of survivin protein molecules caused by changes. Results The prokaryotic and eukaryotic expression vectors of survivin were constructed by molecular cloning and expressed in E. coli and eukaryotic cells. The growth curve showed that the proliferation of 293 cells transfected with survivin was slightly faster than that of the control cells, especially when the cells grew to high density. Survivin could significantly enhance the soft agar colony-forming ability of 293 cells. The high expression of Survivin could cause oncogene cyclinD1 and c myc protein expression increased. Conclusion The anti-apoptotic gene survivin can promote cell transformation, which may depend on the function of cyclinD1 and c-myc