摘要
目的探讨哺乳动物雷帕霉素靶蛋白（mTOR）基因在人结肠癌组织中的表达及其对HT-29细胞增殖与凋亡的影响。方法免疫组织化学方法检测磷酸化mTOR（p-mTOR），在郑州大学第一附属医院普通外科2009年10月至2010年6月50例结肠癌组织和50例正常结肠组织中的表达情况。体外合成mTOR小干扰RNA（siRNA），转染人结肠癌HT-29细胞为实验组，同时设空白对照组（不转染）及无义对照组（转染无义siRNA），Western印迹法检测各组HT-29细胞的mTOR蛋白表达；四甲基偶氮唑蓝法检测细胞增殖；原位末端标记法检测细胞凋亡。结果结肠癌组织中p-mTOR的表达高于对照组[60％（30例）比14％（7例），P〈0．05]，mTOR的表达和淋巴结转移、肿瘤的分化程度相关（r=0．311、0．427，均P〈0．05）。实验组HT-29细胞mTOR的表达和细胞增殖均低于空白对照组和无义对照组（0．39±0．25比1．18±0．05、1．46±0．09，0．275±0．033比0．460±0．028、0．450±0．037，均P〈0．05）；细胞凋亡指数则均高于空白对照组和无义对照组（12．33±1．53比0．33±0．31、1．67±0．58，均P〈0．05）。结论在结肠癌组织中存在mTOR高表达，mTORsiRNA对HT-29细胞mTOR基因的表达有抑制作用，能抑制HT-29细胞的增殖并促进其凋亡。
Objective To explore the expression of mammalian target of rapamycin （roTOR） in colon cancer and the inhibitory effect of roTOR siRNA on the proliferation and apoptosis of human colon cancer HT-29 cells. Methods Immunohistochemistry was employed to detect the expression of phosphorylated mTOR （p-roTOR） in colon cancer tissues （n = 50） and normal colon tissues （n = 50） from First Affiliated Hospital of Zhengzhou University （October 2009 to June 2010 ）. The correlations were examined between the expression of p-roTOR and such clinical pathological data as colon cancer staging and lymph node metastasis. The oligonucleotide templates of roTOR siRNA were designed and employed to transfect HT-29. The nonsense control groups were established accordingly by siRNA with an insignificant order. And the blank group had no transfection. The protein level of mTOR was measured by Western blotting. The method of MTF was used to detect the cell proliferation. And the method of terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） was employed to detect the apoptosis. Results The expression of p-roTOR was higher in the colon cancer group than the control group [ 60% （30 cases） vs 14% （7 cases）, P 〈 0.05 ]. And the expression level was correlated with the degree of lymph node metastasis and tumor differentiation （ r = 0. 311, 0. 427, both P 〈 0. 05 ）. The expression of roTOR protein in the transfection group was lower than the blank and the nonsense control groups （0. 39 ± 0. 25 vs 1.18 ±0.05, 1.46 ± 0.09, both P 〈 0. 05 ）. The proliferation was lower than those of the blank and the nonsense control groups （0. 275 ± 0. 033 vs 0. 460 ± 0. 028, 0. 450± 0. 037, both P 〈 0. 05 ）. The apoptotic indices were higher than those of the blank and control groups （ 12. 33 ±. 1.53 vs 0. 33 ± 0. 31, 1.67± 0. 58, both P 〈 0. 05 ）. Conclusion The expression of mTOR was higher in colon cancer tissues than that in normal colon tissues. The RNA interference of roTOR in HT-29 cell line can effectively knock down the expression of roTOR so as to significantly inhibit cell proliferation and promote cell apoptosis.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第41期2899-2902,共4页
National Medical Journal of China
