摘要
目的:探讨抑癌基因Pdcd4的表达对羟基喜树碱细胞毒活性的影响及其机制.方法:采用脂质体转染法将带有全长Pdcd4cDNA的质粒转入BGC-823细胞,MTT法测定羟基喜树碱对Pdcd4cDNA转染BGC-823细胞的生长抑制作用,流式细胞术检测羟基喜树碱作用前后细胞周期和凋亡率的变化,Western blot蛋白印迹法检测Pdcd4蛋白表达.结果:建立了稳定转染Pdcd4cDNA的BGC-823细胞系;羟基喜树碱作用转染细胞24,72h后,药物对Pdcd4cDNA转染组细胞的半数抑制浓度均低于两对照组(pCDNA3.1与pCDNA-Pdcd4-D418A)(74.48μmol/L vs87.67,102.30μmol/L;14.30μmol/L vs40.59,29.54μmol/L,均P<0.05);流式细胞仪测定结果表明,80μmol/L羟基喜树碱作用转染细胞24h,处于细胞周期G0/G1期的细胞比例下降,S期细胞比例升高,出现细胞周期S期阻滞,72h,细胞凋亡率显著升高(pCDNA3.1:45.40%vs5.65%;pCDNA-Pdcd4-D418A:36.21%vs3.07%;pCDNA-Pdcd4:46.17%vs4.25%,P<0.05);Western blot结果表明80μmol/L羟基喜树碱作用转染细胞72h,Pdcd4蛋白表达水平升高.结论:提高人胃腺癌细胞BGC-823中Pdcd4蛋白的表达能增强其对羟基喜树碱的敏感性;羟基喜树碱能引起BGC-823细胞S期阻滞,并能诱导细胞凋亡;羟基喜树碱本身也可上调BGC-823中Pdcd4的表达.
AIM： To investigate the effect of Pdcd4 tumor suppressor gene expression on the cytotoxic activity of hydroxycamptothecine （HCPT） to the human gastric cancer cell line BGC-823 and its mechanism,METHODS： Lipid-mediated transfection was used to obtain Pdcd4 high-expressed BGC-823 cell line and the control cell lines. MTT method was used to determine the growth inhibition of HCPT to the transfected BGC-823 cells. Flow cytometry was used to analyze the effects of HCPT to the cell cycle and apoptosis rate of the transfected BGC-823 cells. Western blot was used to analyze the transfection results and the expression of Pdcd4 protein in the transfected BGC-823 cells treated with HCPT.RESULTS： A stable Pdcd4 high-expressed BGC-823 cell line was established. MTT results showed that after the transfection treated with HCPT for 24 and 72 hours, the IC50 of the Pdcd4 high-expressed group was lower than that of the control groups （pCDNA3.1 and pCDNA-Pdcd4- D418A） （74.48 μmol/L vs 87.67, 102.30 μmol/L; 14.30 μmol/L vs 40.59, 29.54 μmol/L, all P 〈 0.05）. After the transfected BGC-823 cells were treated with 80 μmol/L HCPT for 72 hours, the cell pro- portion in G0/G1 phase declined, while the cell proportion in S phase increased and the apoptosis cell rate increased significantly （pCDNA3.1： 45.40% vs 5.65%; pCDNA-Pdcd4-D418A： 36.21% vs 3.07%; pCDNA-Pdcd4： 46.17% vs 4.25%, all P 〈 0.05）. Western blot results showed that after transfected BGC-823 cells were treated with 80 μmol/L HCPT for 72 hours, the expression of Pdcd4 protein was increased.CONCLUSION： High expression of Pdcd4 increases the sensitivity of BGC-823 cells to HCPT. HCPT induces BGC-823 cells S-stage arrest and apoptosis. HCPT up-regulates the expression of Pdcd4 protein in BGC-823 cells.
出处
《世界华人消化杂志》
CAS
北大核心
2009年第7期647-651,共5页
World Chinese Journal of Digestology
