短发夹状RNA沉默H-ras基因对人涎腺腺样囊性癌细胞增殖的影响  

Effect of shRNA-mediated silence of H-ras gene onproliferation of human SACC-M cells

作　　者：于利洁[1] 王洁[1] 董福生[2] 石宏[1] 李荷香[1] 顾洪涛[1] 

机构地区：[1]河北医科大学口腔医学院口腔病理研究室,石家庄050017 [2]河北医科大学口腔医学院 口腔颌面外科,石家庄050017

出　　处：《中华口腔医学杂志》2008年第2期113-117,共5页Chinese Journal of Stomatology

基　　金：河北省自然科学基金（C2006000796）

摘　　要：目的探讨H—ras基因对涎腺腺样囊性癌（salivary adenoid cystic carcinoma，SACC）生物学特性的影响以及作为SACC基因治疗靶点的可行性。方法构建H—ras靶向H-ras—shRNA质粒及阴性对照HK-shRNA质粒，经脂质体介导转染SACC—M细胞，G418筛选建立稳定转染细胞株，将细胞分为H—ras-shRNA转染组、HK—shRNA转染组及未转染细胞组。绘制细胞生长曲线检测细胞体外增殖能力；反转录聚合酶链法分析各组细胞中H—ras基因mRNA表达变化；荧光蛋白定量分析H—ras蛋白表达水平；流式细胞术测定细胞周期及细胞凋亡情况；裸鼠成瘤实验检测细胞在体内的成瘤能力，分析H—ras沉默对细胞增殖及凋亡的影响。结果成功构建重组质粒并导入SACC—M细胞，建立稳定表达质粒的细胞株。H—ras—shRNA质粒能有效降低SACC—M细胞中H—ras表达，H—ras基因抑制率为61．80％，H—ras蛋白表达抑制率为62．76％。细胞增殖活性明显受抑，G0G1期比例增加；流式细胞术结果显示实验组细胞凋亡率为30．82％，显著高于阴性对照组及空白对照组的4．29％和4．16％。H—ras沉默细胞的裸鼠体内成瘤能力降低。结论H—ras靶向shRNA干扰质粒能持续有效的抑制SACC—M细胞中H—ras基因及蛋白水平的表达，降低细胞体内外的增殖活性，并能有效诱导细胞凋亡。Objective To examine the effects of H-ras gene silence on cell cycle, proliferation and apoptosis of salivary adenoid cystic carcinoma -M （SACC-M）cell lines. Methods The plasmid H-ras- shRNA, containing the sequence of shRNA targeting H-ras, and HK-shRNA （ without interfering effect） were constructed and transfected into SACC-M cells. The cell line with shRNA plamid stable expression was isolated by G418. The expression levels of H-ras were detected by RT-PCR and protein immunofluorescent assay; cell cycle and cell apoptosis were analyzed by flow cytometry （FCM）. The proliferation of cell was also determined by subcutaneous tumor formation in nude mice. Results After transfection of H-ras-shRNA plasmid, the mRNA expression of H-ras in SACC-M cells was down-regulated by 61.80% and protein expression of H-ras was inhibited by 62.76% ; the cell proliferation was inhibited obviously; the G0G1 phase cells were increased. The cell apoptosis rate of H-ras-shRNA group was significantly higher than that of HK- shRNA group（P 〈0. 05）. The volume of subcutaneous tumor in nude mice was significantly smaller in H-ras-shRNA group than in control group. Conclusions The recombinant plamid HRAS-shRNA could efficiently down-regulate the expression of H-ras gone and protein, induce apoptosis of SACC-M cells and simultaneously inhibit proliferation of these cells in vitro and in vivo.

关 键 词：基因 ras 癌 腺样囊性 质粒 RNA干扰 

分 类 号：R686[医药卫生—骨科学]