CAJ | 학술논문

目的：构建靶向人 BRG1基因的短发夹 RNA（shRNA）慢病毒载体并验证其沉默效率。方法：设计3个针对人 BRG1基因的特异性 shRNA 序列（shBRG1），构建于 pLKO．1慢病毒载体中，并与 psPAX2和 pMD2．G 质粒共同转染293T 细胞以包装成慢病毒颗粒。将包装好的慢病毒感染人主动脉平滑肌细胞（HASMC），应用实时荧光定量 PCR 和 western blot 检测 BRG1 mRNA 和蛋白表达水平，判断其沉默效率。结果：3种 shBRG1干扰序列均成功插入慢病毒载体中且测序正确，3种慢病毒均可有效降低 BRG1 mRNA 表达水平（P 均＜0．01）。其中 shBRG1-3的沉默效率最高，其感染HASMC 后 BRG1蛋白表达水平较对照组显著下降（P ＜0．01）。结论：靶向人 BRG1基因的 shRNA 慢病毒表达载体构建成功，shBRG1-3慢病毒能够有效降低 BRG1 mRNA和蛋白表达水平。
목적：구건파향인 BRG1기인적단발협 RNA（shRNA）만병독재체병험증기침묵효솔。방법：설계3개침대인 BRG1기인적특이성 shRNA 서렬（shBRG1），구건우 pLKO．1만병독재체중，병여 psPAX2화 pMD2．G 질립공동전염293T 세포이포장성만병독과립。장포장호적만병독감염인주동맥평활기세포（HASMC），응용실시형광정량 PCR 화 western blot 검측 BRG1 mRNA 화단백표체수평，판단기침묵효솔。결과：3충 shBRG1간우서렬균성공삽입만병독재체중차측서정학，3충만병독균가유효강저 BRG1 mRNA 표체수평（P 균＜0．01）。기중 shBRG1-3적침묵효솔최고，기감염HASMC 후 BRG1단백표체수평교대조조현저하강（P ＜0．01）。결론：파향인 BRG1기인적 shRNA 만병독표체재체구건성공，shBRG1-3만병독능구유효강저 BRG1 mRNA화단백표체수평。
Objective:To construct short hairpin RNA (shRNA)lentivirus targeting human BRG1 gene and detect its efficiency of gene silence in human aortic smooth muscle cells (HASMC). Methods:Three specific shRNA sequences targeting human BRG1 gene (shBRG1 )were cloned into pLKO.1 lentiviral vector respectively,and pLKO.1-shBRG1 was transfected into 293T cells with psPAX2 and pMD2.G.Lentiviral particles produced by 293T cells were then used to infect HASMC.Silence efficiency of BRG1 mRNA and protein was determined by qRT-PCR and western blot after infection. Results:It was confirmed by DNA sequencing that shBRG1 sequences were correctly inserted into the pLKO.1 lentiviral vector.All three shBRG1 lentivirus effectively downregulated the expression of BRG1 mRNA in HASMC,and shBRG1-3 had the highest silencing efficiency (P < 0.01 ).BRG1 protein expression was significantly downregulated after infection of shBRG1-3 in HASMC compared with negative control (P <0.01 ). Conclusion:Human BRG1 gene RNA interference lentivirus have been successfully constructed. The expression levels of BRG1 mRNA and protein can be significantly downregulated in HASMC when infected with shBRG1-3 lentivirus.