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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 evaluate the silencing effects of lentivirus-mediated RNA interference on galectin-1 gene in mouse pancreatic stellate cells (mPSC) and to observe its effect on the proliferation of mPSCs. Methods: Three galectin-1 gene-specific RNAi target sequences were designed and synthesized and constructed into lentivirus PLKO.1-PURO vector. 293T cells transfected with lentivirus were harvested to obtain mPSC infected with virus supernatant. Purified mPSCs with lentivirus infection were screened with puromycin (2μg / μL). The expression of galectin-1 mRNA and protein in mPSC was detected by real-time fluorescent quantitative RT-PCR and Western blotting. The added value of mPSC was detected by MTT assay. Results: PCR amplification and sequencing showed that galectin-1 lentiviral vector was successfully constructed and could effectively silence the expression of galectin-1 gene in mPSC. The mRNA expression of galectin-1 in mPSC infected with RNAi lentiviral vector was (19.4 ± 4.8)%, (6.2 ± 4.3)% and (27.6 ± 5.7)%, respectively. The expression of galectin-1 protein was The control group were (25.9 ± 3.6)%, (10.8 ± 4.1)% and (30.2 ± 3.2)%, respectively. Stable galectin-1 silencing mPSC decreased cell proliferation ability by day 4 (22.9 ± 2.7)% (P <0.01). CONCLUSION: The constructed galectin-1 RNAi lentiviral vector significantly reduces the expression of galectin-1 in mPSC, and the proliferation of mPSC is decreased after galectin-1 silencing.