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目的制备环氧化酶-2(COX-2)的RNA干扰(RNAi)重组慢病毒(lentivirus),观察COX-2表达沉默对人食管鳞癌(ESCC)细胞增殖的影响。方法将目的基因短发卡RNA(sh RNA)载体与包装载体共转染293T细胞,收集上清液后浓缩纯化、滴度测定、感染ESCC细胞;应用荧光显微镜观察包装效率及感染效率,实时荧光定量PCR及Western blot评价COX-2基因沉默效应,MTS法检测细胞的增殖。结果成功制备了沉默COX-2的重组慢病毒,该病毒感染ESCC细胞后,COX-2 m RNA和蛋白的表达量分别降低了80%和50%左右,细胞的增殖明显减慢。结论慢病毒介导的sh RNA干扰能有效沉默ESCC细胞COX-2基因,COX-2表达沉默抑制ESCC细胞增殖。
Objective To prepare the RNA interference (RNAi) recombinant lentivirus of cyclooxygenase-2 (COX-2) and observe the effect of COX-2 silencing on the proliferation of human esophageal squamous cell carcinoma (ESCC) cells. METHODS: 293T cells were co-transfected with the short hairpin RNA (shRNA) vector of the target gene and the packaging vector. The supernatant was collected and purified. The titer and ESCC cells were infected. The packaging efficiency and infection efficiency were observed by fluorescence microscopy. The silencing effect of COX-2 gene was evaluated by PCR and Western blot. The proliferation of COX-2 gene was detected by MTS assay. Results The recombinant lentivirus silenced by COX-2 was successfully prepared. The expression of COX-2 mRNA and protein in ESCC cells was reduced by about 80% and 50%, respectively, and the cell proliferation was significantly slowed down. Conclusion Lentivirus-mediated sh RNA interference can effectively silence the COX-2 gene in ESCC cells. The silencing of COX-2 inhibits the proliferation of ESCC cells.