慢病毒载体构建小鼠CMKLR1基因缺陷性血管平滑肌细胞系

Construction of mouse CMKLR1 gene knock-down vascular smooth muscle cell lines mediated by lentivirus vector

背景:趋化素在动脉粥样硬化时的表达水平上调,趋化素及其受体CMKLR1可能参与了动脉粥样硬化的病理过程。目的:建立CMKLR1基因缺陷性小鼠血管平滑肌细胞株。方法:以小鼠CMKLR1基因mR NA序列作为干扰靶点,设计3组靶向CMKLR1基因的shR NA序列,构建慢病毒载体,筛选出干扰最佳的shR NA,在293T细胞对慢病毒进行包装和滴度测定。体外培养小鼠血管平滑肌细胞,转染慢病毒形成CMKLR1基因缺陷性血管平滑肌细胞株,用real-time PCR检测感染慢病毒后细胞中CMKLR1 mR NA水平。结果与结论:基因测序结果表明慢病毒载体构建成功,慢病毒的滴度约为8.7×106 TU/m L,p LVX-sh RNA3对CMKLR1基因的抑制效果最显著(P<0.001)。将p LVX-shR NA3转染至血管平滑肌细胞中形成基因缺陷细胞株,用real time PCR血管平滑肌细胞中CMKLR1 mR NA水平,该细胞株中CMKLR1 mR NA水平显著下降(P<0.001),表明慢病毒介导的si RNA可有效沉默小鼠血管平滑肌细胞中CMKLR1基因的表达。

BACKGROUND：Levels of chemerin are elevated in arteriosclerosis, indicating chemerin and its receptor （CMKLR1） may participate in the pathological process of arteriosclerosis. OBJECTIVE： To establish the stable CMKLR1 gene knock-down mouse vascular smooth muscle cel lines. METHODS： Three shRNA sequences targeting the coding region of mouse CMKLR1 mRNA were synthesized and employed to construct lentivirus recombinant vectors. The best pLVX-shRNA was picked up to package recombinant lentivirus in 293T cels, which were infected in cultured mouse vascular smooth muscle cels. The CMKLR1 mRNA level of vascular smooth muscle cels was verified by real-time PCR. RESULTS AND CONCLUSION： The lentiviral vectors were successfuly constructed, which was confirmed by DNA sequencing. The titer of lentivirus reached 8.7×10^6 TU/mL in the packaging cels, and pLVX-shRNA3 showed the most significant interfering effects on CMKLR1 gene （P 〈 0.001）. The pLVX-shRNA3 was chosen to establish the stable CMKLR1 gene knock-down vascular smooth muscle cel lines, in which the expression of CMKLR1 mRNA was also significantly inhibited shown on real-time PCR （P 〈 0.001）. We finaly confirmed that the expression of CMKLR1 gene can be effectively silenced by lentivirus-mediated siRNA in mouse mouse vascular smooth muscle cels.