慢病毒携带shRNA对内皮细胞组织因子表达的抑制作用

Inhibition of tissue factor expression in endothelial cells by lentivirus mediated shRNA

目的:构建RNA干扰慢病毒载体,研究慢病毒携带的短发夹干扰RNA(short hairpin RNA,shRNA)对内皮细胞中组织因子(tissue factor,TF)表达的抑制作用。方法:将靶向人TF基因的2条shRNA表达序列克隆到慢病毒载体pENTRTM/U6的黏性末端,测序鉴定后,与目标载体pLenti6/BLOCK-iTTM-DEST vector进行位点特异性重组,再测序;经脂质体导入293FT细胞,包装成慢病毒,收集病毒上清并测定病毒滴度。RT-PCR和ELISA检测干扰后内皮细胞TF的表达。结果:将目的序列成功连接到载体上,并经测序分析证实载体构建成功;在293FT细胞中进行慢病毒包装,收集上清并检测病毒滴度为5×105/TU。RT-PCR和ELISA检测结果证实构建的携带shRNA的慢病毒可显著抑制内皮细胞TF的表达。结论:成功构建了携带人TF基因的RNAi慢病毒载体,慢病毒携带的短发夹干扰RNA能干扰内皮细胞中TF的表达,可为血栓栓塞性疾病的防治提供一种有效的方法。

Objective To construct the recombinant lentivirus RNAi vector, and to determine whether the lentivirus mediated short hairpin RNA （shRNA） can inhibit the tissue factor （TF） ex- pression in endothelial cells. Methods Two short hairpin RNAs targeting to human TF were cloned into pENTRTM/U6 plasmid to obtain an entry clone, and the positive clones were verified by sequencing. A recombination reaction was performed between the pENTR/U6 entry construction and pLenti6/ BLOCKiTYM-DEST vector, and then the positive clones were confirmed by sequencing. The 293FT cell line was transfected by the above recombined plasmid and lentivirus packing materials, the culture supernatant was harvested, and the virus titer was determined. RT-PCR and ELISA were used to observe the inhibition of TF gene expression after the lentivirus transduction in human umbilical vein endothelial cells. Results The shRNA sequences targeting to human TF were cloned into the vectors, and an entry clone and an expression clone were constructed successfully, which were proved by sequence determination. Viral particles were packaged in the 293FF cell line,all virus stocks were collected, and the transfection titer was 5×10^5/ transduced unit. RT-PCR and enzyme linked immunosorbent assay demonstrated that the lentivirus stocks could suppress the TF exgene are successfully constructed, and lentivirus mediated shRNA can inhibit the TF expression in endothelial cells, which may provide a highly effective method for the prevention and treatment of throm-bo-embolic diseases.