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拟通过RNA干扰技术特异下调人血红素加氧酶-1(human heme oxygenase-1,hHO-1)基因的表达,减少hHO-1的产量从而降低胆红素的产生,探讨在胆红素产生前就阻断其产生,为临床早期防治新生儿高胆红素血症及胆红素中毒性脑病探索一种新的有效手段。针对hHO-1基因设计并化学合成三对小分子干扰RNA(small interfering RNA,siRNA)。采用脂质体转染法将siRNA转染入人肝脏细胞株HL-7702;荧光显微镜检测siRNA转染细胞的效率;转染siRNA1~2天后经RT-PCR和Western印迹方法检测hHO-1表达水平和蛋白质量;并采用HO-1诱导剂血红素诱导或hHO-1表达质粒转染细胞以上调hHO-1表达,检测siRNA干扰后hHO-1产量和酶活性。结果显示:设计的三对siRNA能不同程度的特异下调hHO-1表达,筛选获得抑制效果最佳的siRNA-3。siRNA-3抑制hHO-1呈现浓度与时间依赖性。与非特异对照siRNA及未处理组比较,血红素诱导和hHO-1表达质粒转染均能上调HL-7702细胞内hHO-1表达,提高hHO-1产量,但转染siRNA-3后hHO-1表达明显抑制,同时hHO-1活性随着基因表达下调而下降。实验表明设计合成的siRNA-3抑制效果明显。siRNA-3通过降解hHO-1,减少hHO-1产量,降低酶活性,最终减少胆红素产生,从而使RNA干扰技术成为降低新生儿高胆红素血症和胆红素中毒性脑病发生的一种候选方法。
It is proposed that the expression of hHO-1 gene is specifically down-regulated by RNAi technology to reduce the production of hHO-1 and thus reduce the production of bilirubin, Blocking its production before, in order to prevent early neonatal hyperbilirubinemia and bilirubin toxic encephalopathy to explore a new effective means. Three pairs of small interfering RNA (siRNA) were designed and synthesized for hHO-1 gene. The siRNA was transfected into human hepatic cell line HL-7702 by lipofection method. The efficiency of siRNA transfection was detected by fluorescence microscopy. The expression of hHO-1 was detected by RT-PCR and Western blotting And the amount of protein. The HO-1-induced heme-induced or hHO-1 expression plasmid was used to transfect the cells to up-regulate the hHO-1 expression and detect the hHO-1 production and enzyme activity after siRNA interference. The results showed that the designed three pairs of siRNA could down-regulate the expression of hHO-1 to some extent, and screened the siRNA-3 with the best inhibitory effect. siRNA-3 inhibits hHO-1 in a concentration-and time-dependent manner. Compared with nonspecific control siRNA and untreated group, both heme induction and hHO-1 expression plasmid transfection could up-regulate hHO-1 expression and increase hHO-1 production in HL-7702 cells, but hHO- 1 expression was significantly inhibited, while hHO-1 activity decreased with the gene expression decreased. Experiments show that the design of synthesized siRNA-3 inhibitory effect is obvious. By degrading hHO-1, siRNA-3 reduces hHO-1 production, reduces enzyme activity and ultimately reduces bilirubin production, thereby rendering RNA interference technology a potential for reducing neonatal hyperbilirubinemia and bilirubin toxic encephalopathy A candidate method.