糖皮质激素受体α基因RNA干扰的生物学效应研究

Inhibition of glucocorticoid receptor α using hairpin siRNA expression vector in vitro

目的 采用RNA干扰 (RNAinterference ,RNAi)技术 ,通过构建人糖皮质激素受体α(glucocorticoidreceptorα ,GRα)基因的特异性siRNA(smallinterfereRNA)真核表达载体 ,体外观察对GRα基因的沉默作用及生物学效应。方法 采用基因克隆技术 ,将合成的发卡样特异性GRα干扰寡核苷酸序列插入真核表达载体 (pPUR/U6) ,构建GRαsiRNA的表达载体 ,转染体外ECV 3 0 4细胞 ,48h后观测胞内GRα蛋白水平 (Westernblot)及对LPS刺激后细胞因子产生的影响作用。结果 ①成功构建发卡样GRαsiRNA真核表达载体 (pPUR/U6 GRαsiRNA) ;②pPUR/U6 GRαsiRNA转染 (脂质体法 )ECV 3 0 4细胞 ,48h后明显下调胞内GRα蛋白水平 ;③转染pPUR/U6 GRαsiRNA的细胞 ,在LPS刺激后 ,其TNF α、IL 1β的表达释放显著高于空载体对照组。结论 该RNA干扰真核表达载体能明显干扰GRα蛋白的表达、释放 ,结果促进了LPS刺激后炎性细胞因子的表达释放。进一步说明严重创伤所引起的糖皮质激素受体下调是机体应激紊乱的重要原因。

Objective To investigate the silence effect and biological effects of glucocorticoid receptor α (GRα) on LPS-induced cytokine expression by designing a hairpin GRα siRNA expression vector (pPUR/U6-GRα siRNA). Methods The pPUR/U6-GRα siRNA expression vector was constructed by gene recombination, and then transferred to the cultured ECV-304 cells. At 48 h after culture, the whole cell protein was extracted and detected by Western blotting using rabbit-anti-human GRα polyclonal antibody. The cells were stimulated with LPS at 48 h after pPUR/U6- GRα siRNA transference. The TNF-α and IL-1β productions in the supernatants were detected by ELISA. Results The pPUR/U6-GRα siRNA expression vector was successfully constructed. Western blot analysis demonstrated that siRNA against GRα reduced the expression of this protein at 48 h after pPUR/U6- GRα siRNA transference, but the productions of TNF-α and IL-1β in the supernatants were enhanced when the cells were stimulated with LPS. Conclusion The pPUR/U6-GRα siRNA expression vector can reduce the expression of GRα protein and enhance the productions of TNF-α and IL-1β after LPS stimulation, which would be harmful to tissue repair after trauma.