摘要
目的构建MAD2(有丝分裂阻滞缺陷蛋白2)特异性RNA干扰真核表达载体,体外观察抑制MAD2基因表达对胃癌细胞生长和细胞周期的影响。方法构建MAD2siRNA真核表达载体,脂质体法将pSilencer3.1空载体和pSilencer3.1/MAD2-siRNA1和pSilencer3.1/MAD2-siRNA2真核表达载体分别导入人胃癌细胞系SGC7901。稳定转染后Westernblot和RT-PCR检测胃癌细胞内MAD2蛋白及mRNA水平的表达情况,挑选出抑制效果最好的单个克隆。MTT法检测各实验组细胞生长情况,流式细胞术检测纺锤体抑制剂长春新碱作用后,胃癌细胞MAD2-siRNA转染组和空载体组细胞周期的分布。结果成功构建MAD2siRNA真核表达载体,转染胃癌细胞SGC7901可使其MAD2蛋白及mRNA表达显著下调。MAD2表达降低的胃癌细胞生长速度明显增快(P<0.01),经长春新碱作用后不能被阻滞于有丝分裂期。结论小干扰RNA技术可以有效地特异性抑制胃癌细胞纺锤体检查点关键蛋白MAD2的表达。MAD2的抑制可使胃癌细胞SGC7901生长加速,增殖能力增强,同时减弱纺锤体抑制剂长春新碱阻滞细胞周期的作用。
AIM： To construct the RNA interference eukaryotic expression vector specific for human MAD2 gene and to observe its effect on the growth of gastric cancer cell line SGC7901. METHODS： The expression vectors of pSilencer 3.1/MAD2-siRNA1 and pSilencer 3.1/MAD2-siRNA2 were constructed by gene recombination and then were stably transfected into the gastric carcinoma cell line SGC7901 by liposome mediation. The expression of MAD2 in the levels of protein and mRNA was detected by Western blot and RT-PCR, and the monoclone with the highest inhibition efficiency was selected. The growth of the transfected cells was assessed by MTT. And the cells treated with 1.0 mg/L vincristine （VCR） for 24 hrs were analyzed by FCM for cell cycle. RESULTS： Sequence-specific siRNAs targeting MAD2 significantly down regulated the expression of MAD2 in SGC7901 cells. In MAD2-siRNA transfected cells, the rate of cell growth increased markedly and cell cycle couldn＇t be arrested in M phase induced by VCR, compared with the cells transfected with the mock vector. CONCLUSION： Down regulation of MAD2 expression of SGC7901 bv sequence-specific siRNA could accelerate the cell growth and impair the mitosis arrest of SGC7901 induced by VCR.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第3期290-292,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30200337
313024002)
