小鼠髓样细胞触发受体-2基因小发卡状RNA慢病毒载体构建  

Construction of shRNA lentiviral vector targeting mouse triggering receptor expressed on myeloid cells 2 gene

作　　者：冉江霞 石胜良[1] 王成志 RAN Jiangxia;SHI Shengliang;WANG Chengzhi(The Second Affiliated Hospital of Guangxi Medical University,Nanning 530007,China)

机构地区：[1]广西医科大学第二附属医院,南宁530007 [2]广西医科大学第三附属医院

出　　处：《山东医药》2018年第43期39-42,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81460183)

摘　　要：目的构建小鼠髓样细胞触发受体-2(TREM2)基因小发卡状RNA(sh RNA)慢病毒表达载体,并检测其功能。方法针对小鼠TREM2基因设计合成3对sh RNA序列,将其分别与线性化GV248载体连接,转化至感受态大肠埃希菌DH5α,构建TREM2基因sh RNA重组慢病毒载体(TREM2-shRNA-1、TREM2-shRNA-2、TREM2-shRNA-3)。用重组载体转染293T细胞,荧光显微镜下观察绿色荧光(GFP)后,进行滴度测定。取对数生长期BV2小胶质细胞,分为TREM2-shRNA-1感染组、TREM2-shRNA-2感染组、TREM2-shRNA-3感染组、阴性对照组及空白对照组。TREM2-shRNA-1感染组、TREM2-shRNA-2感染组、TREM2-shRNA-3感染组、阴性对照组分别感染TREM2-sh RNA-1、TREM2-shRNA-2、TREM2-shRNA-3、sh RNA空质粒,空白组不做任何处理。感染120 h取各组细胞,Q-PCR法检测各组TREM2 m RNA,计算细胞沉默效率。结果 TREM2-shRNA-1、TREM2-shRNA-2、TREM2-shRNA-3转染293T后镜下均观察到GFP,包装浓缩的慢病毒滴度分别为(4×108)、(4×108)、(5×108) TU/m L。与空白对照组和阴性对照组比较,TREM2-shRNA-1感染组、TREM2-shRNA-2感染组、TREM2-shRNA-3感染组TRME2 m RNA相对表达量均降低(P均<0. 05);与TREM2-shRNA-1感染组、TREM2-shRNA-3感染组比较,TREM2-shRNA-2感染组TRME2 m RNA相对表达量降低(P均<0. 05)。TREM2-shRNA-1感染组、TREM2-shRNA-2感染组、TREM2-shRNA-3感染组、阴性对照组的沉默效率分别为49. 4%±3. 98%、79. 7%±2. 01、65. 3%±2. 41%和39. 2%±6. 98%,TREM2-shRNA2组沉默效率最高。结论成功构建TREM2-shRNA-1、TREM2-shRNA-2、TREM2-shRNA-3。TREM2-sh RNA-1、TREM2-shRNA-2、TREM2-shRNA-3均可沉默小鼠BV2小胶质细胞中TREM2基因表达,以TREM2-shRNA-2沉默效果最好。Objective To construct shRNA lentiviral vector targeting triggering receptor expressed on myeloid cells 2(TREM2)gene,and to detect its silencing efficiency.Methods Three pairs of shRNA of mouse TREM2 gene were designed,synthetized,and ligated separately to the linearized GV248 vector.Then these recombinant lentiviral vectors were transformed into competent E.coli DH5αto construct shRNA lentiviral vector targeting mouse TREM2 gene(TREM2-shRNA-1,TREM2-shRNA-2,and TREM2-shRNA-3).After 293T cells were transfected with the recombinant vector,and green fluorescence(GFP)was observed under a fluorescence microscope,the virus titer was detected.These recombinant lentiviral vectors were used to infect mouse BV2 microglia in the logarithmic growth phase and were divided into TREM2-shRNA-1 infection group,TREM2-shRNA-2 infection group,TREM2-shRNA-3 infection group,negative control group,and blank control group.After 120 h,the silence efficiency were detected by Q-PCR.Results GFP was observed after the TREM2-shRNA-1,TREM2-shRNA-2,and TREM2-shRNA-3 transfected after 293T cells,and the titer of the concentrated virus was 4×10 8,4×10 8 and 5×10 8 TU/mL,respectively.Compared with the blank control group and the negative control group,the relative expression of TRME2 mRNA in the TREM2-shRNA-1 infection group,TREM2-shRNA-2 infection group,and TREM2-shRNA-3 infection group decreased(P<0.05).Compared with the TREM2-shRNA-1 infection group and the TREM2-shRNA-3 infection group,the relative expression of TRME2 mRNA in the TREM2-shRNA-2 infection group decreased(P<0.05).The silencing efficiency of the TREM2-shRNA-1 infection group,the TREM2-shRNA-2 infection group,the TREM2-shRNA-3 infection group,and the negative control group were 49.4%±3.98%,79.7%±2.01,65.3%±2.41%,and 39.2%±6.98%,respectively,of which the TREM2-shRNA2 infection group had the highest silencing efficiency.Conclusions TREM2-shRNA-1,TREM2-shRNA-2,and TREM2-shRNA-3 are constructed successfully.TREM2-shRNA-1,TREM2-shRNA-2,and TREM2-shRNA-3 may silence TREM2 gene e

关 键 词：小发卡状RNA慢病毒载体 髓样细胞触发受体-2 迟发性阿尔茨海默病 阿尔茨海默病 

分 类 号：R749[医药卫生—神经病学与精神病学]