摘要:目的 观察小干扰RNA通过沉默Rhc糖蛋白调节血氨的效果,为肝性脑病的靶向药物治疗提供依据.方法 培养原代小鼠脑微血管内皮细胞,用阳离子脂质体将小干扰RNA序列Rhcg-Mus-455、Rhcg-Mus-607、Rhcg-Mus-883转染进小鼠脑微血管内皮细胞中,提取并转染细胞蛋白与RNA,行蛋白质免疫印迹实验与琼脂糖凝胶电泳实验,观察细胞内氨转运蛋白及其mRNA的表达情况,以此观察小干扰RNA对细胞Rhcg基因的沉默效果与沉默效率,筛选出能够高效抑制小鼠脑微血管内皮细胞中氨转运体基因的小干扰RNA序列.结果 蛋白质免疫印迹实验中,3个小干扰RNA序列Rhcg-Mus-455、RhcgMus-607、Rhcg-Mus-883的Rhcg蛋白水平两两比较,差异无统计学意义(P>0.05);但Rhcg-Mus-455、Rhcg-Mus-607、RhcgMus-883的Rhcg蛋白水平均低于生理盐水与阴性对照,差异有统计学意义(P<0.05).琼脂糖凝胶电泳实验中,3个小干扰RNA序列Rhcg-Mus-455、Rhcg-Mus-607、Rhcg-Mus-883的Rhcg mRNA水平两两比较,差异无统计学意义(P>0.05);但RhcgMus-455的Rhcgm RNA水平低于生理盐水与阴性对照,差异有统计学意义(P<0.05).结论 3个小干扰RNA序列对小鼠脑微血管内皮细胞中的氨转运体均有沉默表达作用,其中,小干扰RNA序列Rhcg-Mus-455具有更高的沉默效率.因此,通过基因沉默技术,在体外找到能够特异性结合氨转运蛋白的小干扰RNA,可抑制血氨转运,进而改善肝性脑病.%Objective To investigate the effect of small interfering RNA on regulating blood ammonia by silencing Rhcglycoprotein, and to provide a basis for targeted drug treatment of hepatic encephalopathy.Methods The original generation of small mouse brain microvascular endothelial cells was cultivated, using cationic liposome small interfering RNA sequence Rhcg-Mus-455, Rhcg-Mus-607and Rhcg-Mus-883 transfected into small mouse brain microvascular endothelial cells, the protein and RNA cells were extraction and transfected, protein western blot experiment and agarose gel electrophoresis experiment were conducted to observe the expression of intracellular ammonia transporter protein and its mRNA, so as to observe the silencing effect and silencing efficiency of small interfering RNA on Rhcg gene of cells, and to screen out the small interfering RNA sequence that can effectively inhibit the ammonia transporter gene in rat brain microvascular endothelial cells.Results The Rhcg protein levels of Rhcg-Mus-455, Rhcg-Mus-607 and Rhcg-Mus-883 of 3 small interfering RNA sequences were compared in the Western blot experiment, and the differences were not statistically significant (P>0.05). The Rhcg protein levels of Rhcg-Mus-455, Rhcg-Mus-607 and Rhcg-Mus-883 were all lower than those of normal saline and negative control, and the difference was statistically significant (P<0.05). Rhcg mRNA levels of Rhcg-Mus-455, RhcgMus-607 and Rhcg-Mus-883 were compared in the agarose gel electrophoresis experiment, and the differences were not statistically significant (P>0.05). The Rhcg mRNA level of Rhcg-Mus-455 was lower than that of normal saline and negative control (P<0.05). Conclusion All of the 3 small interfering RNA sequences have the effect of silencing the expression of ammonia transporters in the mouse cerebrovascular endothelial cells, the small interfering RNA sequence 455 may have obvious silencing efficiency.Therefore, small interfering RNA that can specifically bind ammonia transporters can be found in vitro through gene silencing technology to inhibit blood ammonia transport and thereby improve hepatic encephalopathy.