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

Study on silencing effect of lentivirus-mediated RNA interference on galectin-1 in mice pancreatic stellate cells

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<0.01)。结论:构建的galectin-1 RNAi慢病毒载体能显著降低mPSC中galectin-1的表达量,galectin-1沉默后mPSC增殖能力下降。

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（P0.05）.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.