摘要
目的观察联合转染反义DNMT1、DNMT3b基因真核表达载体对人胆管癌细胞QBC-939生长的影响。初步探讨DNMT1、DNMT3b基因在胆管癌发生中的作用。方法分别构建反义DNMT1基因和反义DNMT3b基因真核表达载体,脂质体介导法将两者联合转入人胆管癌细胞QBC-939,Western blot检测转染前后相应蛋白的表达变化,噻唑蓝(MTT)比色法观察各组细胞生长曲线,流式细胞术观察细胞生长周期及凋亡率的变化,裸鼠皮下种植细胞观察成瘤大小。结果(1)Western blot检测证实转染反义基因能使相应蛋白表达水平降低；(2)联合转染反义DNMT1、DNMT3b基因和单独转染反义DNMT1能影响QBC-939的生长曲线,使细胞增殖减慢,其中以前者为甚；(3)联合转染反义DNMT1、DNMT3b基因和单独转染反义DNMT1能影响QBC-939的细胞周期,使之阻滞于G_1期,使细胞凋亡率从(1．63±0．27)%分别增加到(18．47±1．46)%和(6．19±0．78)％；(4)联合转染反义DNMTl、DNMT3b基因和单独转染反义DNMTl能使裸鼠皮下种植成瘤体积减小。生长延缓,前者效果更为明显；(5)在上述效应检测中,联合转染空质粒和单独转染反义DNMT3b基因实验组对QBC-939细胞的生长无明显影响。结论(1)通过联合转染反义DNMT1和DNMT3b基因真核表达载体,可同时下调DNMT1和DNMT3b在QBC-939细胞中的表达,并能在体内外抑制胆管癌细胞的生长,促进凋亡的发生,其效果明显优于单独转染DN—MT1反义基因。(2)在DNA甲基化的过程中,DNMT1起着主要的作用,DNMT3b扮演着协同的角色,两者可能通过甲基化途径与胆管癌的发生有关。
Objective To study the effect of co-transfection of antisense DNMT1 gene and antisense DNMT3b gene eukaryotic expression vector on the growth of human biliary tract cancer cell line QBC-939, and to explore the role of DNMT1 and DNMT3b in the cholangiocarcinoma tumorigenesis. Methods The constructed antisense DNMT1 and DNMT3b gene eukaryotic expression vectors were both transfected into QBC-939 cells using the lipofectamine transfection reagents. The expression level of DNMTI and DNMT3b protein was detected by Western blot assay. The growth curves of transfected cells and un-transfected cells were observed by MTT method respectively. Flow cytometry was used to detect the change of the cell cycle and the apoptosls rate. Transfected cells were injected into the subcutaneous tissue of nude mouse and the volume of tumor in different groups was measured 4 weeks later. Resuits （1） The protein levels of DNMT1 and DNMT3b were decreased significantly after transfeetion; （2） Both co-transfection of DNMT1 and DNMT3b genes and single transfection of antisense DNMT1 gene affected the cell growth curve and slowed down the cell proliferation. The former delayed the cell growth more than the later; （3） Co-transfection of DNMT1 and DNMT3b genes and single transfection of antisense DNMT1 gene arrested the cell cycle in Gl phase and increased the apoptosis rate from （1.63 ± 0.27） % to （18.47 ± 1.46） % and （6.19 ± 0.78） % respectively; （4） The tumor in the subcutaneous tissue of nude mouse in co-transfection group was the smallest, and that in antisense DNMT1 gene transfection group was the second among the all groups; （5） Co-transfection of the plasmid pCMV and pEGFP-C1 and the single transfection of the antisense DNMT3b gene did not inhibit the growth of QBC-939. Conclusion Co-transfection of antisense DNMT1 and DNMT3b gene eukaryotic expression vectors can down-regulate the expression level of DNMT1 and DNMT3b, and can inhibit the growth of human cholangiocarcinoma cell line QBC-939 and induce cell apoptosis. Co-transfection has more effect on the cell growth than single transfection of the antisense DNMT1 gene. Our study also suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support the function in QBC-939 cells. These genes may be related to the tumorigenesis in cholangiocarcinoma via DNA methylation pathway.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第8期912-915,i0001,共5页
Chinese Journal of Experimental Surgery
基金
国家高技术研究发展计划(863计划)资助项目(2002AA214061)
