摘要
目的:探讨野生型X P D基因对人胆管癌QBC939细胞的生物学影响.方法:用碱裂解法提取空载质粒pEGFP-N2和重组质粒pEGFP-N2-XPD,提取出的质粒以KPNⅠ、BGIⅡ和SPHⅠ酶切鉴定.实验分4组,重组质粒pEGFP-N2-XPD组、空载质粒pEGFP-N2组、脂质体组,并用具有相同遗传背景和代数的QBC939细胞作为空白对照.用脂质体转染法瞬时转染四组细胞.荧光显微镜下观察转染后绿色荧光蛋白报告基因表达情况.提取各组细胞总RNA,合成cDNA,用聚合酶链反应(PCR)检测4组细胞中XPD、p53、cyclin D1、c-myc表达情况.并用四甲基偶氮唑盐(MTT)和流式细胞仪检测细胞增殖及其细胞周期的变化.结果:pEGFP-N2-XPD细胞与pEGFP-N2、脂质体组和空白对照组相比,XPD mRNA表达量明显增加(0.778±0.018vs0.561±0.039,0.544±0.035,0.542±0.034,均P<0.01).pEGFP-N2-XPD细胞中p53mRNA相对表达量与pEGFP-N2、脂质体组和空白对照组比较具有统计学意义(0.421±0.019vs0.256±0.014,0.267±0.015,0.274±0.018,均P<0.01).pEGFP-N2-XPD细胞与其他组相比,cyclin D1mRNA相对表达量明显降低(0.339±0.041vs0.560±0.039,0.558±0.050,0.560±0.041,均P<0.01).pEGFP-N2-XPD细胞与其他组相比,c-myc mRNA相对表达量明显降低(0.355±0.045vs0.570±0.075,0.560±0.041,0.537±0.050,均P<0.01).流式细胞仪检测pEGFP-N2-XPD组细胞周期G1期为81.65%,S期为11.83%,其他组Gl期分别为65.54%、56.61%、63.26%;S期分别为24.10%、29.52%、27.28%,结果具有统计学意义(P<0.05).MTT检测示pEGFP-N2-XPD细胞生长率为0.249±0.02,与其他组相比,细胞增殖力明显减弱(P<0.01).结论:野生型XPD基因可以抑制胆管癌细胞的生长,XPD基因可抑制c-myc、cyclin D1基因的表达,增加p53基因表达.
AIM:To investigate the impact of tranfection of the wild-type xeroderma pigmentosum group D (XPD) gene on the biological behavior of human cholangiocarcinoma cell line QBC939. METHODS:Empty plasmid pEGFP-N2 and recombinant plasmid pECFP-N2-XPD were digested with KPN I,BGI II and SPH I for plasmid identification. Cells were divided into four groups:pEGFP-N2-XPD group,pEGFP-N2 group,Lipofectamine (Lip) group,and blank control group. Cells were transfected with Lipofectamine. The expression of green fluorescent protein (GFP) was observed under a fluorescence microscope. The mRNA expression of wild-type XPD,p53,cyclin D1 and c-myc was detected by reverse transcription-polymerase chain reaction (RT-PCR). Flow cytometry (FCM) was employed for examining the cell cycle of transfected QBC939 cells. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. RESULTS:The relative expression level of XPD mRNA in the pEGFP-N2-XPD group was sig-nificantly higher than those in the pEGFP-N2 group,Lip group and blank control group (0.778 ± 0.018 vs 0.561 ± 0.039,0.544 ± 0.035 and 0.542 ± 0.034,respectively; all P 0.01). The relative expression level of p53 mRNA in the pEGFP-N2-XPD group was also significantly higher than those in the pEGFP-N2 group,Lip group and blank control group (0.421 ± 0.019 vs 0.256 ± 0.014,0.267 ± 0.015 and 0.274 ± 0.018,respectively; all P 0.01). The relative expression level of cyclin D1 mRNA in the pEGFP-N2-XPD group was signif i-cantly lower than those in the pEGFP-N2 group,Lip group and blank control group (0.339 ± 0.041 vs 0.560 ± 0.039,0.558 ± 0.050 and 0.560 ± 0.041,respectively; all P 0.01). The relative expression level of c-myc mRNA in the pEGFP-N2-XPD group was also signifi cantly lower than those in the pEGFP-N2 group,Lip group and blank control group (0.355 ± 0.045 vs 0.570 ± 0.075,0.560 ± 0.041 and 0.537 ± 0.050,respectively; all P 0.01). FCM results showed that the percentage of cells in G1 phase was signifi cantly higher (81.65% vs 65.54%,56.61% and 63.26%,respectively; all P 0.05) and that in S1 phase was signifi cantly lower (11.83% vs 24.10%,29.52% and 27.28%; all P 0.05) in the pEGFP-N2-XPD group than in the pEGFP-N2 group,Lip group and blank control group. MTT assay revealed that the growth rate of cells in the pEGFP-N2-XPD group was significantly lower than those in the other three groups (all P 0.01). CONCLUSION:Transfection of the wild-type XPD gene can inhibit the proliferation of human QBC939 cells in vitro,down-regulate the expression of cyclin D1 and c-myc mRNAs,and upregulate the expression of p53 mRNA.
出处
《世界华人消化杂志》
CAS
北大核心
2010年第15期1531-1536,共6页
World Chinese Journal of Digestology