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大肠癌是常见的恶性肿瘤,其发病率和病死率正逐年递增。过氧化物酶体增殖因子活化受体δ(peroxisome pro1iferator-activated receptorδ,PPARδ)是结肠腺瘤性息肉基因通路的下游靶标,很可能参与了大肠癌发病机制。然而,目前对该基因在大肠癌中的功能尚未阐明。本实验采用慢病毒三质粒包装系统构建特异靶向人PPARδ基因的RNA干扰(RNAinterference,RNAi)慢病毒载体,用以建立PPARδ基因稳定沉默的结肠癌细胞株。测序证实,成功构建编码表达PPARδ短发夹结构RNA(sh-PPARδ)的慢病毒载体pLVshPPARδ以及对照载体pLVControl。病毒载体转导结肠癌细胞株KM12C后,荧光显微镜及流式细胞术(fluorescence-activated cell sorting,FACS)证实细胞转染效率>90%,RT-PCR测定实验组KM12C细胞PPARδ的mRNA表达比未处理细胞下降70%,对照组与未处理细胞无显著差异。Western blot显示实验组PPARδ蛋白表达量比未处理细胞明显下降,对照组与未处理细胞无显著差异。结果显示成功构建特异靶向人PPARδ基因的RNAi慢病毒载体,并成功建立PPARδ表达稳定干扰的人结肠癌细胞株KM12C,为PPARδ在大肠癌中的功能研究提供了新的细胞模型。
Colorectal cancer is a common malignancy, and its morbidity and mortality are increasing year by year. Peroxisome proliferator-activated receptor δ (PPARδ) is a downstream target of adenomatous polyp pathways and is likely to be involved in the pathogenesis of colorectal cancer. However, the function of this gene in colorectal cancer has not been elucidated yet. In this study, a lentivirus three plasmid packaging system was constructed to construct a RNA interference (RNAi) lentivirus vector targeting human PPARδ gene to establish a stable and silenced colon cancer cell line with PPARδ gene. Sequencing confirmed that the lentiviral vector pLVshPPARδ encoding the PPARδ short hairpin RNA (sh-PPARδ) and the control vector pLVControl were successfully constructed. After transfecting the colon cancer cell line KM12C with a viral vector, the transfection efficiency was confirmed to be> 90% by fluorescence microscopy and fluorescence-activated cell sorting (FACS). The mRNA expression of PPARδ in experimental group was lower than that of KM12C Treatment of cells decreased by 70%, no significant difference between the control group and untreated cells. Western blot showed that the expression of PPARδ in experimental group was significantly lower than that in untreated cells, while no significant difference was found between control group and untreated cells. The results showed that the RNAi lentivirus vector targeting human PPARδ gene was successfully constructed and the human colon cancer cell line KM12C with stable expression of PPARδ was successfully established, which provided a new cell model for the study of the function of PPARδ in colorectal cancer.