【摘要】 目的:研究小干扰RNA(small interfering RNA,siRNA)方法抑制蛋白酶体激活剂REGγ的基因表达对人甲状腺髓样癌TT细胞株凋亡的影响。方法:培养人甲状腺髓样癌TT细胞株,构建真核表达载体pREGγ-shRNA及阴性对照质粒pNeg-shRNA,在脂质体Lipofectamine 2000的介导下转染人甲状腺髓样癌TT细胞株,采用RT-PCR和WesternBlot法检测pREGγ-shRNA转染和pNeg-shRNA转染的TT细胞之间REGγmRNA及REGγ蛋白质表达之间的差别;然后采用TUNEL法检测REGγshRNA转染和阴性质粒转染的TT细胞的凋亡率之间的差别,以及West-ernBlot法检测pREGγ-shRNA转染和pNeg-shRNA转染的TT细胞凋亡因子Caspase-3表达之间的差别。结果:真核表达载体pREGγ-shRNA转染TT细胞的REGγmRNA和REGγ蛋白表达水平明显低于pNeg-shRNA转染的TT细胞(P<0.05),pREGγ-shRNA转染的TT细胞的凋亡水平明显高于pNeg-shRNA转染的TT细胞(P<0.05)。结论:应用siRNA技术能有效的抑制人甲状腺髓样癌细胞REGγ基因的表达,抑制蛋白酶体激活剂REGγ基因的表达能明显地促进TT细胞的凋亡,提示REGγ可阻止细胞凋亡。REGγ在人甲状腺髓样癌细胞凋亡中的地位,为肿瘤的生物学治疗提供了新的思路。更多还原

【Abstract】 Objective: To study the effects of specific inhibition to proteasome inhibitor REGγ gene expression by small interfering RNAs (siRNA) on the apoptosis of human medullary thyroid carcinoma TT cell line. Methods: An eukaryotic expression vector carrying human pREG γ-shRNA and a negative control plasmid pNeg-shRNA were constructed. The vector carrying pREGγ-shRNA or the negative control plasmid was transfected respectively into human medullary thyroid carcinoma TT cells by Lipofectamine 2000. REGγ expres- sion in TT cells transfected with pREGγ-shRNA and TT cells transfected with pNeg-shRNA was detected by RT- PCR and Western blot. TUNEL was employed to detect the apoptosis rate and Western blot was used to detect caspase-3 expression in TT cells transfected with pREG γ-shRNA and TT cells transfected with pNeg-shRNA. Results: The expression of REGγmRNA and REGγ protein in TT cells transfected with pREG γ-shRNA was lower than that in TT cells transfected with pNeg-shRNA (P<0.05). The apoptosis rate was high- er in TT cells transfected with pREGγ-shRNA than in those transfected with pNeg-shRNA (P<0.05). Conclusion: siRNA targeting REGγ mRNA can specifically suppress REGγ gene expression and can significantly promote apoptosis of TT cells, suggesting that REGγ can prevent apoptosis and is helpful for developing new methods of cancer biotherapy.更多还原