NDRG1干扰载体的构建及稳定过表达NDRG1人脑微血管内皮细胞的建立

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BamH Ⅰ和Bbs Ⅰ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BamH Ⅰ和Hind Ⅲ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。

Construction of NDRG1 interference vector and establishment of human brain microvascular endothelial cells stably over-expressing NDRG1

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 over-expressing NDRG1. Methods The pGPU6-GFP plasmid was double digested by BamH 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 BamH 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 NDRG1 protein in the hCMECs was increased compared with control group. Conclusion The pGPU6-GFP-NDRG1 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.