摘要
目的:建立稳定表达候选抑癌基因DMBT1的食管癌细胞系EC9706,并分析转染前后其增殖特点、转移侵袭能力的变化.方法:脂质体介导法将DMBT1的全长表达质粒转染食管癌细胞系EC9706,G418筛选阳性克隆细胞,Western blot及RT-PCR检测DMBT1蛋白及mRNA水平变化,MTT法绘制细胞生存曲线及无血清条件下测细胞生存抑制率,Transwell实验检测细胞趋化迁移能力变化.结果:通过稳定转染后,DMBT1蛋白水平及mRNA水平均比对照组提高3.2倍以上,与对照组比较,差异有显著意义(P<0.01);实验组细胞增殖速度、倍增时间、对数生长期等均比对照组细胞更为缓慢,在无血清培基中增殖抑制率随时间延长不断增高,实验组迁移细胞数为206±25,对照组为367±42,2组比较差异有显著意义(P<0.01).结论:在体外成功建立稳定表达DMBT1的细胞系,DMBT1过表达在体外显著抑制食管癌细胞系EC9706的生长增殖及趋化迁移.
AIM： To establish the cell line EC9706 of esophagus carcinoma with stable expression of a new candidate tumor suppressor gene DMBT1 and to analyze cell invasiveness and cell proliferation compared to the control group so as to provide any insights into the function of the newly-discovered tumor suppressor gene. METHODS： The full-length DMBT1 expression plasmid pTRexDest30_DMBT18kb.2 was transfected into esophagus carcinoma cell line EC9706 with Lipofectamine 2000 which was subsequently screened with G418. DMBT1 transcript and protein were determined by semiquantitative RT-PCR and Western blot; then cell growth and proliferation was evaluated with MTT; finally transwell assay was applied to evaluate the ability of its migration before and after the transfection. RESULTS： After transfection, the levels of DMBT1 protein and mRNA were 3.2 times higher than control group. There was significant difference between transfection group and the control group. The growth curves mapped with MTT indicated that the pTRexDest30_DMBT1 group transfected with showed slower speed of proliferation versus the vector control and the untreated group. In addition, cell survival in serum-free medium was markedly inhibited compared to the vector control. Transwell assay further confirmed conspicuous discrepancy in migration （206 ± 25 vs 367 ± 42, P 〈 0.01）, the experimental group versus the control group. CONCLUSION： The biological model that stably expresses DMBT1 was successfully established; DMBT1 over-expression significantly inhibits the proliferation and invasive process of esopha- gus carcinoma cell line in vitro.
出处
《世界华人消化杂志》
CAS
北大核心
2009年第17期1759-1763,共5页
World Chinese Journal of Digestology
关键词
脑恶性肿瘤缺失基因1
稳定转染
食管癌
Deleted in malignant brain tumors 1
Stable transfection
Esophagus carcinoma
