| 慢病毒介导的RNA干扰对小鼠胰腺星状细胞中半乳糖凝集素-1基因沉默效应的研究 |
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| 引用本文： | 袁中旭,汤东,陆子鹏,卫积书,张静静,蒋奎荣,苗毅. 慢病毒介导的RNA干扰对小鼠胰腺星状细胞中半乳糖凝集素-1基因沉默效应的研究[J]. 外科理论与实践, 2011, 16(5): 455-460. DOI: 10.16139/j.1007-9610.a2743 |
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| 作者姓名： | 袁中旭 汤东 陆子鹏 卫积书 张静静 蒋奎荣 苗毅 |
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| 作者单位： | 南京医科大学第一附属医院胰腺外科江苏省临床医学研究院肿瘤生物研究所; |
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| 基金项目： | 国家自然科学基金(30972911,30972912) |
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| 摘 要： | 目的:评价慢病毒介导的RNA干扰技术对小鼠胰腺星状细胞(mPSC)中半乳糖凝集素-1(galectin-1)基因的沉默效应,并观察其对mPSC增殖的影响。方法:设计并合成3条galectin-1基因特异性RNAi靶序列,构建到慢病毒PLKO.1-PURO载体中。从慢病毒包装转染的293T细胞,获得病毒上清感染的mPSC;采用嘌呤霉素(2μg/μL)筛选出慢病毒感染成功的mPSC。实时荧光定量RT-PCR和Western印迹法检测mPSC中galectin-1 mRNA和蛋白的表达,MTT法检测mPSC的增值能力。结果:PCR扩增和测序表明成功构建galectin-1慢病毒干扰载体,可以有效沉默mPSC中galectin-1基因的表达。构建的RNAi慢病毒载体感染mPSC后galectin-1 mRNA表达量同对照组比较,分别为(19.4±4.8)%、(6.2±4.3)%、(27.6±5.7)%,galectin-1蛋白表达量同对照组比较分别为(25.9±3.6)%、(10.8±4.1)%、(30.2±3.2)%。稳定的galectin-1沉默mPSC在第4天细胞增殖能力下降(22.9±2.7)%(P
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| 关 键 词： | 胰腺星状细胞 半乳糖凝集素-1 慢病毒 RNA干扰 |
Study on silencing effect of lentivirus-mediated RNA interference on galectin-1 in mice pancreatic stellate cells |
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| YUAN Zhong-xu,TANG Dong,LU Zi-perg,WEI Ji-shu,ZHANG Jing-jing,JIANG Kui-rong,MIAO Yi. Study on silencing effect of lentivirus-mediated RNA interference on galectin-1 in mice pancreatic stellate cells[J]. Journal of Surgery Concepts & Practice, 2011, 16(5): 455-460. DOI: 10.16139/j.1007-9610.a2743 |
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| Authors: | YUAN Zhong-xu TANG Dong LU Zi-perg WEI Ji-shu ZHANG Jing-jing JIANG Kui-rong MIAO Yi |
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| Affiliation: | YUAN Zhong-xu,TANG Dong,LU Zi-peng,WEI Ji-shu,ZHANG Jing-jing,JIANG Kui-rong,MIAO Yi.Department of Pancreatic Surgery,the First Affiliated Hospital of Nanjing Medical University,Jiangsu Province Academy of Clinical Medicine Institute of Tumor Biology,Nanjing Jiangsu 210029,China. |
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| Abstract: | Objective To explore the silencing effect of the constructed mice galectin-1 RNA interference(RNAi) lentivirus vector on pancreatic stellate cells of mice(mPSC).Methods Three effective sequences of RNAi targeting galectin-1 were confirmed.Both sense and antisense Oligo DNA of the targeting sequence were designed,synthesized and cloned into the PLKO.1-PURO vector.After co-transfection of the T293 cells,the lentivirus were produced.mPSC was infected with the lentivirus and screened by puromycin(2 μg/μL),the expression of galectin-1 mRNA and protein in mPSC was detected by real-time quantitative polymerase chain reaction and Western blotting.The proliferation of mPSC was detected by MTT.Results A recombinant lentiviral vector expressing shRNA against galectin-1 gene was obtained and confirmed by DNA sequencing.The mRNA and protein expression in mPSC were decreased remarkably after infected with the lentiviral vector.The mRNA expression of galectin-1 was(19.4±4.8)%,(6.2±4.3)%,(27.6±5.7)%,and the protein expression of galectin-1 was(25.9±3.6)%,(10.8±4.1)%,(30.2±3.2)%,as compared with the control group,respectively.And the mPSC proliferation was reduced by 22.9%at the 4th day after shRNA virus infection and puromycin screening(P<0.05).Conclusions The lentiviral shRNA expression vector targeting mice galectin-1 gene can decrease the galectin-1 expression markedly in mPSC with stable galectin-1 gene knock-down effect and inhibit the proliferation of mPSC. |
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| Keywords: | Pancreatic stellate cells Galectin-1 Lentivirus vector RNA interference |
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