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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 observe the effect of co-transfected antisense DNMT1 and DNMT3b eukaryotic expression vector on the growth of human cholangiocarcinoma cell line QBC-939. Preliminary study of DNMT1, DNMT3b gene in the role of cholangiocarcinoma. Methods The antisense DNMT1 gene and antisense DNMT3b gene eukaryotic expression vector were constructed. The two genes were transfected into human cholangiocarcinoma cell line QBC-939 by lipofectamine. Western blot was used to detect the expression of the corresponding protein before and after transfection. The growth curve of each group was observed by MTT colorimetric assay. The cell growth cycle and apoptosis rate were observed by flow cytometry. The size of tumor was observed under the skin of nude mice. Results (1) Western blot showed that transfection of antisense gene could decrease the expression level of corresponding protein; (2) Co-transfection of antisense DNMT1 and DNMT3b genes and antisense DNMT1 alone could affect the growth curve of QBC-939 (3) Co-transfection of antisense DNMT1 and DNMT3b genes and antisense DNMT1 transfected alone can affect the cell cycle of QBC-939 cells and block the cell cycle in G_1 phase. The rates of death increased from (1.63 ± 0.27)% to (18.47 ± 1.46)% and (6.19 ± 0.78)%, respectively. (4) Combined transfection of antisense DNMT1 and DNMT3b genes And antisense DNMTl alone transfected nude mice subcutaneously tumor volume decreased. The effect of the former was more obvious. (5) In the above effect test, the combination of empty plasmid transfected with antisense DNMT3b gene alone and experiment group had no significant effect on the growth of QBC-939 cells. CONCLUSION: (1) DNMT1 and DNMT3b can down-regulate the expression of DNMT1 and DNMT3b in QBC-939 cells by co-transfection of antisense DNMT1 and DNMT3b eukaryotic expression vectors and can inhibit the growth of cholangiocarcinoma cells in vitro and in vivo and promote apoptosis Occurred, the effect was significantly better than the DN-MT1 transfection alone antisense gene. (2) DNMT1 plays a major role in DNA methylation. DNMT3b plays a synergistic role, both of which may be related to the occurrence of cholangiocarcinoma through methylation pathway.