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目的 探讨核酶 (RZ)在细胞水平调节雄激素受体 (AR)基因表达的作用。方法 设计并合成针对AR的锤头状核酶序列 ,分子克隆技术构建活性核酶和无活性核酶表达载体 ,脂质体介导下转染雄激素受体表达阳性细胞株T47D。转染第 1～ 3天 ,应用免疫组织化学和逆转录 聚合酶链反应 (RT PCR)技术检测细胞内AR基因表达水平。结果 AR活性核酶表达载体转染后 ,T47D细胞ARmRNA水平降低 2 2 .4%～ 50 .7% (P <0 .0 5) ,AR蛋白阳性细胞减少 7.1 0 %～1 1 .90 % (P <0 .0 5)。而无活性核酶对细胞AR基因表达差异无显著性 (P >0 .0 5)。结论 成功构建AR锤头状核酶表达载体 ,能特异性切割ARmRNA ,阻断雄激素受体基因表达
Objective To investigate the role of ribozyme (RZ) in the regulation of androgen receptor (AR) gene expression at the cellular level. Methods The hammerhead ribozyme sequence targeting AR was designed and synthesized. The active ribozyme and inactive ribozyme expression vector were constructed by molecular cloning technique. The positive expression of androgen receptor positive cell line T47D was transfected by liposome. The expression of AR gene in cells was detected by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) 1 to 3 days after transfection. Results After transfection with AR-ribozyme, AR mRNA level in T47D cells decreased by 22.4% -50.7% (P <0.05), AR protein-positive cells decreased by 7.1 0% -1.1.90% (P < P <0. 05). No activity of ribozyme on the AR gene expression was no significant difference (P> 0.05). Conclusion AR hammerhead ribozyme expression vector was successfully constructed, which can specifically cut AR mRNA and block the expression of androgen receptor gene