摘要
为了探讨人野生型p53(wt-p53)基因增强大肠癌细胞化疗敏感性的分子生物学机制,将携带wt-p53基因的质粒分别转染两种p53基因突变的人大肠癌细胞系HT-29及SW620,分析细胞中p53及细胞周期蛋白D1(cyclinD1)蛋白的表达水平;将化疗药物5-氟尿嘧啶(5-fluorouracil,5-FU)以不同浓度、不同时间分别作用于HT-29及SW620细胞,另外将已转染wt-p53基因的大肠癌细胞用5-FU进行诱导,Western印迹分析上述干预条件下细胞中p53蛋白及细胞周期蛋白D1表达水平的变化;流式细胞术检测wt-p53基因联合5-FU组及对照组中细胞凋亡的改变情况.结果表明,wt-p53基因能增加癌细胞中细胞周期蛋白D1的表达,与wt-p53基因呈剂量依赖性关系;5-FU则降低其蛋白表达,与5-FU呈时间和剂量依赖性关系,而5-FU所致的细胞周期蛋白D1表达水平的降低在细胞预先转染了wt-p53基因时会被抑制;wt-p53基因与5-FU联合使用能提高大肠癌细胞凋亡率.结果提示,wt-p53基因可提高大肠癌细胞中细胞周期蛋白D1的表达水平,并抑制5-FU所致的细胞周期蛋白D1降解,从而提高大肠癌细胞对化疗药物5-FU的敏感性.
The molecular mechanisms of p53 gene enhancing chemosensitivity of the human colorectal cancer cells were currently studied. The plasmid with wild-type p53 (wt-p53) gene were transfected into mutant p53- expressing HT-29 and SW620 cell lines. After administration of 5-fluorouracil (5-FU) at the various concentrations and time points to HT-29 and SW620 cell lines, Western blotting was used to detect the level of p53 and cyclin D1 in Wt-p53-infected HT-29 and SW620 cells. The apoptosis of cells was analyzed by the flowcytometry. The results showed that transfection of wt-p53 gene could result in an increment of cyclin D1 protein in HT-29 and SW620 cells in a dose-dependent manner. Whereas, 5-FU could markedly decrease the level of cyclin D1 protein in a dose- and time-dependent manner. 5-FU-induced down-regulation of cyclin D1 expression could be apparently blocked by wt-p53 gene transfection. Wt-p53 transfection associated with the treatment of 5-FU could significantly increase the percentage of apoptotic cells. All results indicated that introduction of the wt-p53 gene into human colorectal cancer cells might prevent the degradation of cyclin D1 protein by 5-FU and, furthermore, significantly result in the increase of cyclin D1 expression. As a result, the sensitivity of human colorectal cancer cells to chemotherapeutic agents 5-FU was hereby increased.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第8期638-643,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
江西省科技攻关计划项目资助(No.S-1-11-a)~~