大麻素Ⅰ型受体基因(CNR1)特异性siRNA表达载体的构建及稳定干扰阳性L6细胞克隆的筛选

摘要

The cannabinoid receptor type 1 (CNR1) is a key component of the endocannabinoid system, which has been reported to play a pivotal role in modulating feeding behavior and energy balance. In order to further study on gene function of CNR1, this study was conducted to construct and identify CNR1 gene small interfering (siRNA) expression vectors and screened the stable CNR1- interference positive L6 cell clones. Three pairs of CNR 1-specific double-strand siRNAs were designed and inserted into the pYr-1.1 vector. The CNR1 gene siRNA expression vectors were identified by restriction enzyme digestion and sequencing. After that siRNAs were transfected with L6 cells by LipofectamineTM(Lip)2000. Then, the transfection efficiency was detected by EGFP and FCM. CNR1 gene expression was determined by Real-time PCR and the stable transgenic L6 cell clones were screened by G418. The results revealed that the CNR1 gene siRNA expression vectors have been constructed successfully. The transient transfection efficiencies of L6 cells were 10.45% (P<0.01), 8.57%(P<0.01)and 8.71% (P<0.01) respectively, and the silencing efficacies of the transient transfected L6 cells were 39%(P<0.05), 64%(P<0.01) and 68%(P<0.01), respectively. The optimal selection concentration of G418 for stable transfected L6 cell clones was 800 μg/mL. The silencing efficacies of CNR-1-positive transgenic cell clones were 43%(P<0.05), 18%(P<0.01) and 91%(P<0.01), respectively. The results showed that CNR1-3 expression vector was optimal silencing vector and CNR1-3 stable transgenic cell clones were best silencing cell line. This study successfully provides CNR1 gene silencing method by siRNA and the screening of CNR1-interference positive L6 cell clones renders basic tools for further studying the functions of CNR1 gene.%大麻素Ⅰ型受体(CNR1)是介导内源性大麻素发挥作用的关键分子,在食欲和能量代谢调控中发挥着重要作用.为了更深入研究CNR1的基因功能,本实验旨在构建和筛选有效沉默CNR1基因的干扰表达载体,并筛选出稳定转染干扰质粒的阳性细胞系.设计合成3对CNR1基因的特异性发夹小干扰RNA(siRNA)干扰引物,将其连接入干扰载体pYr-1.1,构建可沉默CNR1基因的siRNA表达载体CNR1-1、CNR1-2和CNR1-3.并采用LipofectamineTM(Lip)2000介导质粒转染L6细胞,绿色荧光蛋白的表达和流式细胞仪监测转染效率,通过实时荧光定量分析siRNA表达载体的干扰效果.并进一步用G418进行了稳定转染细胞筛选.结果显示,CNR1基因的siRNA表达载体构建正确,瞬时转染L6细胞的转染效率分别为10.45％(P＜0.01)、8.57％(P＜0.01)和8.71％(P＜0.01);干扰效率为39％(P＜0.05)、64％(P＜0.01)及68％(P＜0.01).稳定筛选的最佳G418浓度为800 μg/mL,稳定筛选后干扰效率分别为43％(P＜0.05)、78％(P＜0.01)及91％(P＜0.01).干扰效率较高的CNR1-3表达载体和稳定转染CNR1-3的细胞系为构建筛选成功的siRNA表达载体和阳性细胞系.本研究提供了一种有效沉默CNR1基因表达的方法,同时稳定沉默的阳性L6细胞系成功筛选为进一步研究大麻素Ⅰ型受体的基因功能提供了基础资料.