TCAB1基因RNAi慢病毒载体的构建及在人子宫内膜间质细胞中沉默TCAB1基因的效应

目的构建TCAB1基因RNAi慢病毒载体,为子宫内膜异位症(EMs)的基因靶向治疗提供理论依据。方法化学合成3条靶向TCAB1基因的siRNA,转染HEK-293T细胞,24h后采用荧光素酶报告系统筛选有效siRNA,其对TCAB1mRNA有明显抑制作用。针对已经筛选确定的TCAB1基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经XhoI和HpaI酶切后的LV1载体[含U6启动子和绿色荧光蛋白(GFP)]连接产生LV1-shRNA慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV1-shRNA、pRev、pVsvg、pcgv四质粒共转染包装细胞HEK-293T细胞,包装产生慢病毒,分别于转染后48h、72h收取病毒上清,感染人子宫内膜间质细胞(ESC),进行RT-qPCR检测TCAB1基因相对表达量,检测LV1-shRNA干扰效率。结果 PCR和测序证实,成功构建TCAB1 shRNA的慢病毒载体LV1-shRNA。结论成功构建TCAB1基因的RNAi慢病毒载体,其转染ESC细胞后显著抑制了TCAB1的表达,为子宫内膜异位症的基因靶向治疗提供了实验基础。

Studies on the construct of lentiviral vector for RNAi of the TCAB1 gene and provide a theoretical basis of gene targe- ted therapy for endometriosis.

Objective ： To Studies on the construct of lentiviral vector for RNAi of the TCAB1 gene and provide a theoretical basis of gene targeted therapy for endometriosis. Methods： 3 siRNAs of targeted TCAB1 gene were synthesized by a chemical process, and then transfected HEK- 293T cells. 24h later, effective siRNAs were screened by using luciferase reporter assay system. They exhibi- ted inhibition effects to mRNA of TCAB1 gene. The effective sequence of siRNA targeting TCAB1 gene was confirmed. The comple- mentary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the LV1 vector, which contained U6 promoter and green fluorescent protein （ GFP）, and after digestion with XhoI and HpaI. The resulting lentiviral vector containing TCAB1 shRNA was named LV1 - shRNA, and it was confirmed by PCR and sequencing. HEK -293T cells were cotransfected by lentiviral vector LV1 - shRNA, pRev, pVsvg and pcgv. All virus stocks were produced by calcium phosphate- mediated transfection. The supematants were harvested after transfected 48h and 72h respectively and then infected human ESC. The relative expression of TCAB1 gene and LV1 -shRNA interference efficiency were detected by real -time PCR. Results： The lentivirus LV1 - shRNA vector of TCAB1 shRNA was constructed successfully confirmed by PCR and sequencing. Conclusion： The lentivirus RNAi vector of TCAB1 was constructed successfully. It exhibited inhibition effects to TCAB1 by infecting ESC cell. It provides an experi- mental basis of gene targeted therapy for EMs.