【摘要】 目的：构建N-myc下游调节基因1 (NDRG1)的shRNA干扰载体和过表达载体，转染人脑微血管内皮细胞（hCMECs）后验证干扰和过表达效果并获得稳定过表达NDRG1的细胞。方法：将pGPU6-GFP质粒采用Bam HⅠ和BbsⅠ双酶切后，分别与设计的8条shRNA连接构建pGPU6-GFP-NDRG1 shRNA干扰载体，经测序鉴定后采用Lipofectamine 2000转染hCMECs，采用荧光显微镜观察载体转染效率，将PCR扩增的NDRG1编码区序列与pMD19-T连接，经测序正确后将重组质粒与pcDNA3.1空质粒采用限制性内切酶Bam HⅠ和HindⅢ双酶切，连接后构建pcDNA3.1-NDRG1过表达载体，采用Lipofectamine 2000将测序正确的过表达载体转染hCMECs，采用实时荧光定量PCR (RT-qPCR)法检测干扰和过表达的hCMECs中NDRG1 mRNA表达水平，Western blotting法检测hCMECs中NDRG1蛋白表达水平，采用新霉素筛选出阳性克隆hCMECs。结果：pGPU6-GFP载体经双酶切后，电泳结果显示完全线性化，测序结果显示shRNA均成功连接至pGPU6-GFP载体；荧光显微镜观察载体转染效率约为70%;RT-qPCR法检测，有3个shRNA干扰载体的干扰效果良好，转染后hCMECs中NDRG1 mRNA表达水平分别约为对照组的26%、21%和13%;Western blotting法检测到对应的特异性蛋白条带，重组质粒pcDNA3.1-NDRG1经双酶切后，电泳结果可见大小为1 185和5 428 bp的2条DNA条带，测序结果显示NDRG1基因成功插入，成功制备阳性克隆hCMECs。RT-qPCR法检测，阳性克隆hCMEC中NDRG1 mRNA表达水平较对照组升高，约为对照组的25.96倍；Western blotting法检测，hCMEC中NDRG1蛋白表达水平较对照组升高。结论：成功构建pGPU6-GFP-NDRG1 shRNA干扰载体并验证干扰效果；成功构建pcDNA3.1-NDRG1过表达载体和稳定过表达NDRG1的hCMECs。更多还原

【Abstract】 Objective:To construct the shRNA interference vector and over-expression vector of N-myc downstream regulatory gene 1(NDRG1)and transfect the human cerebral microvascular endothelial cells(hCMECs),and to verify the interference and over-expression effects and to obtain the cells stably overexpressing NDRG1. Methods:The pGPU6-GFP plasmid was double digested by Bam H Ⅰ and Bbs Ⅰ,and then ligated with eight designed shRNAs to construct the pGPU6-GFP-NDRG1 shRNA interference vectors respectively,and the hCMECs were transfected by Lipofectamine 2000 after sequencing and identification,and the transfection efficiency of the vectors was observed under fluorescence microscope;the sequence of NDRG1 coding region amplified by PCR was ligated with the pMD19-T,after correct sequencing,the recombinant plasmid and the empty plasmid pcDNA3. 1 were digested with restriction endonucleases Bam H Ⅰ and Hind Ⅲ,and the pcDNA3. 1-NDRG1 over-expression vector was constructed after ligation;the hCMECs were transfected with the correctly sequenced over-expression vector by using Lipofectamine 2000;the expression levels of NDRG1 mRNA in the hCMECs were detected by real-time fluorescence quantitative PCR(RT-qPCR) method,and the expression levels of NDRG1 protein in the hCMECs were detected by Western blotting method;the positive clone hCMECs were screened by neomycin. Results:After the pGPU6-GFP vector was doubly digested,the electrophoresis results showed that it was completely linear,and the sequencing results showed that the shRNAs were all successfully connected to the pGPU6-GFP vector;the transfection efficiency of vector was about 70 % observed under fluorescence microscope;the RT-qPCR results showed that there were three shRNA interference vectors with good interference effects, and the expression levels of NDRG1 mRNA in the hCMECs after transfection were about 26%,21%,and 13% of those in control group,respectively;the corresponding specific protein bands were obtained by Western blotting method,the recombinant plasmid pcDNA3. 1-NDRG1 was identified by enzymatic digestion,and two DNA bands of 1 185 and 5 428 bp were identified by electrophoresis,and the sequencing results showed that the NDRG1 gene was successfully inserted into the pcDNA3. 1 vector,and the positive clone hCMECs were successfully constructed. The RT-qPCR results showed that the expression level of NDRG1 in the positive clone hCMECs was about 25. 96 times higher than that in control group;the Western blotting results showed that the expression level of NDRG1protein in the hCMECs was increased compared with control group. Conclusion:The pGPU6-GFPNDRG1 shRNA interference vector is successfully constructed and the interference effect is verified;the pcDNA3. 1-NDRG1 over-expression vector and the hCMECs stably over-expressing NDRG1 are successfully constructed.更多还原