To investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor γ （PPAR-γ） on the expression of tumor necrosis factor alpha （TNFα） by constructing vectors for RNA interference in...To investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor γ （PPAR-γ） on the expression of tumor necrosis factor alpha （TNFα） by constructing vectors for RNA interference in RAW264.7 cells. Methods： The pSUPER-EGFP vectors were used to transcribe functional small interfering RNA （ siRNA ）. Four pairs of oligonucleotides （ 64 nt ） targeting PPAR-γ gene were inserted into the downstream of the HI promotor, with their veracity confirmed by double digestion and sequencing. Western blotting and immunofluorescence assay were used to examine the silencing effect of PPAR-γ gene in RAW264.7 cells. Meanwhile, the TNFα level was determined by Sandwich ELISA. Results ： Compared with other recombinant pSUPER- EGFP vectors （R-pSUPER. EGFP）, R-pSUPER. EGFP2 induced the best silencing effect on the expression of PPART in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFα expression after the curcumin and lipopolysaccharide （LPS） stimulation. Conclusions： PPARγ-pSUPER-EGFP inducing a silencing effect on the expression of PPART can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.展开更多
为了探讨靶向人端粒酶反转录酶(hTERT)基因的小干扰RNA(s iRNA)表达载体是否具有抑制H eL a细胞端粒酶活性的能力,人工合成2条64个核苷酸(n t)的片段,其中19n t与hTERT基因1789-1807位碱基同源,将其退火、连接到质粒pSU PER中,构建pSU P...为了探讨靶向人端粒酶反转录酶(hTERT)基因的小干扰RNA(s iRNA)表达载体是否具有抑制H eL a细胞端粒酶活性的能力,人工合成2条64个核苷酸(n t)的片段,其中19n t与hTERT基因1789-1807位碱基同源,将其退火、连接到质粒pSU PER中,构建pSU P-hTE。在pSU P-hTE基础上,把该质粒的启动子和64n t插入片段酶切、克隆到质粒pEGFP-C 1中生成pEGFP-hTE,从而获得新霉素抗性筛选质粒。将pEGFP-hTE用脂质体转染H eL a细胞,G 418筛选后获得抗性克隆并将其收获、传代,用不同方法检测hTERT的mRNA和蛋白表达水平、H eL a细胞的端粒酶活性以及细胞的增殖能力。结果显示,pEGFP-hTE转染的H eL a细胞与对照组比较,hTERT的mRNA水平下降及蛋白表达减少、细胞端粒酶活性降低38%,但细胞增殖能力没有明显改变。以上结果表明,pEGFP-hTE能通过RNA干扰(RNA i)途径特异性抑制H eL a细胞hTERT基因的表达,从而有效抑制细胞端粒酶活性,这可能将为肿瘤生物治疗提供一条新的途径。展开更多
基金supported by Open Fund of State Key Laboratory of Trauma,Burns and Combined Injury,Third Military Medical University
文摘To investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor γ （PPAR-γ） on the expression of tumor necrosis factor alpha （TNFα） by constructing vectors for RNA interference in RAW264.7 cells. Methods： The pSUPER-EGFP vectors were used to transcribe functional small interfering RNA （ siRNA ）. Four pairs of oligonucleotides （ 64 nt ） targeting PPAR-γ gene were inserted into the downstream of the HI promotor, with their veracity confirmed by double digestion and sequencing. Western blotting and immunofluorescence assay were used to examine the silencing effect of PPAR-γ gene in RAW264.7 cells. Meanwhile, the TNFα level was determined by Sandwich ELISA. Results ： Compared with other recombinant pSUPER- EGFP vectors （R-pSUPER. EGFP）, R-pSUPER. EGFP2 induced the best silencing effect on the expression of PPART in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFα expression after the curcumin and lipopolysaccharide （LPS） stimulation. Conclusions： PPARγ-pSUPER-EGFP inducing a silencing effect on the expression of PPART can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.
文摘为了探讨靶向人端粒酶反转录酶(hTERT)基因的小干扰RNA(s iRNA)表达载体是否具有抑制H eL a细胞端粒酶活性的能力,人工合成2条64个核苷酸(n t)的片段,其中19n t与hTERT基因1789-1807位碱基同源,将其退火、连接到质粒pSU PER中,构建pSU P-hTE。在pSU P-hTE基础上,把该质粒的启动子和64n t插入片段酶切、克隆到质粒pEGFP-C 1中生成pEGFP-hTE,从而获得新霉素抗性筛选质粒。将pEGFP-hTE用脂质体转染H eL a细胞,G 418筛选后获得抗性克隆并将其收获、传代,用不同方法检测hTERT的mRNA和蛋白表达水平、H eL a细胞的端粒酶活性以及细胞的增殖能力。结果显示,pEGFP-hTE转染的H eL a细胞与对照组比较,hTERT的mRNA水平下降及蛋白表达减少、细胞端粒酶活性降低38%,但细胞增殖能力没有明显改变。以上结果表明,pEGFP-hTE能通过RNA干扰(RNA i)途径特异性抑制H eL a细胞hTERT基因的表达,从而有效抑制细胞端粒酶活性,这可能将为肿瘤生物治疗提供一条新的途径。
