沉默H-ras基因对人涎腺腺样囊性癌裸鼠移植瘤的抑制作用

Inhibitory effect of silencing H-ras gene by shRNA on growth of xenografted human salivary adenoid cystic carcinoma in nude mice

目的:构建H-ras靶向短发夹RNA(short hairpin RNA,shRNA)质粒,研究其对人涎腺腺样囊性癌裸鼠成瘤的抑制作用及其对移植瘤细胞增殖活性及细胞凋亡率的影响。方法:构建含H-ras靶向特定序列的真核表达质粒pHRAS,稳定表达pHRAS质粒的细胞为实验组,未处理的SACC-M细胞为对照组,建立裸鼠荷瘤模型,计算成瘤率;移植瘤原代培养检测质粒表达及瘤细胞增殖活性;免疫组织化学法检测瘤内H-ras蛋白表达;采用组织学观察、流式细胞术及TUNEL法检测肿瘤细胞凋亡情况。结果:pHRAS质粒转染组成瘤率及肿瘤体积显著低于对照组,原代培养可见大量瘤细胞表达绿色荧光。实验组瘤内H-ras蛋白表达量明显减少,实验组肿瘤细胞凋亡率为(41.55±4.25)%,明显高于对照组的(4.73±1.35)%(P<0.05)。结论:shRNA沉默H-ras基因能抑制SACC-M细胞裸鼠皮下移植瘤的形成,在体内有效下调H-ras蛋白的表达,且对肿瘤细胞具有促凋亡作用。

Objective：Short hairpin RNA （shRNA） plasmid was constructed to investigate the inhibitory effects of silencing H-ras gene by shRNA on growth of xenografted human salivary adenoid cystic carcinoma （SACC） in nude mice,and its influence on proli-feration and apoptosis of SACC-M cells.Methods：The plasmid pHRAS containing the sequence of shRNA targeting H-ras and EGFP gene was constructed. The SACC-M cells transfected with stable pHRAS plasmid were regarded as experimental group. The SACC-M cells without treated by pHRAS plasmid were as control. The xenografted tumor model was established in nude mice and the tumor formation rate was calculated. The plasmid expression and the proliferation of primary xenografted SACC-M cells were detected. H-ras protein expression was measured by immunohistochemistry. Apoptosis of SACC-M cells was assessed by histological examination, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick and end-labeling （TUNEL） assay.Results：The tumor formation rate and tumor size in pHRAS plasmid transfection group was markedly lower than control group （P〈0.05）. After transfection of pHRAS plasmid, green fluorescence was observed in a large amount of primary SACC-M cells. Expression of H-ras protein was significantly decreased in pHRAS plasmid transfection group compared with control. The apoptotic index was much higher in pHRAS group than control group [（41.55±4.25） % vs （4.73±1.35） %, P〈0.05]. Conclusion：Silencing H-ras gene by shRNA pHRAS effectively inhibited the tumorigenesis of subcutaneously xenografted SACC-M cells in nude mice, down-regulated the expression of H-ras protein in vivo, and induced apoptosis of SACC-M cells.