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目的探讨抑癌基因p53对人乳腺癌细胞MCF-7生长以及周期相关基因cyclinD1、cyclinE、CDK2和CDK4表达的影响。方法将正义的p53真核表达载体导入MCF-7细胞,获得稳定表达正义p53的细胞模型,并经PCR和Western blot鉴定。分析了细胞的各种生长特性包括生长曲线、血清依赖、单细胞克隆形成、软琼脂成集落等;利用TdR双阻断法同步化细胞,流式细胞术分析了细胞周期;Western blot检测了其他周期相关基因的表达。结果p53的高表达可以使细胞的生长速度减慢、血清依赖性增加、单细胞克隆成集落能力降低、非锚定依赖性生长能力减弱,S期的进程减缓,一定程度上抑制cyclinD1和CDK2的表达,cyclinE和CDK4的表达未见明显变化。结论抑癌基因p53的表达能够抑制人乳腺癌细胞MCF-7的生长,并可能通过阻抑cyclinD1和CDK2的表达而阻抑细胞周期S期进程,实现其调节功能。
Objective To investigate the effects of tumor suppressor gene p53 on the growth of human breast cancer cell line MCF-7 and the expression of cyclinD1, cyclinE, CDK2 and CDK4. Methods The recombinant p53 eukaryotic expression vector was transfected into MCF-7 cells, and a cell model stably expressing p53 was obtained and identified by PCR and Western blot. Various growth characteristics of the cells were analyzed including growth curve, serum dependent, single cell colony formation, colonies of soft agar. The cell cycle was analyzed by TdR double-block method and cell cycle was analyzed by flow cytometry. Cycle related gene expression. Results The high expression of p53 could slow down the growth of the cells, increase the serum dependency, decrease the colonization ability of single cell clone, weaken the ability of non-anchorage dependent growth, slow down the progression of S phase and inhibit the expression of cyclinD1 and CDK2 to a certain extent Expression, cyclinE and CDK4 expression did not change significantly. Conclusion The expression of tumor suppressor gene p53 can inhibit the growth of human breast cancer cell line MCF-7, and may inhibit the cell cycle S phase by inhibiting the expression of cyclinD1 and CDK2 to regulate its function.