慢病毒载体介导的结缔组织生长因子基因沉默对下游基因的影响

RNA interference mediated by lent viral vector suppresses the expression of connective tissue growth factor and its effect on downstream gene

目的利用RNA干扰技术,以慢病毒为载体,介导结缔组织生长因子(CTGF)基因沉默,并观察其对下游基因表达的影响。方法用构建的CTGF小干扰RNA(siRNA)慢病毒转移质粒与包装质粒、包膜蛋白质粒共转染293T细胞,包装成介导CTGF基因沉默的慢病毒载体;感染肝星状细胞系HSC-T6,应用Real-time PCR和Western免疫印迹法筛选基因沉默效率最高的慢病毒载体;再根据绿色荧光蛋白(GFP)表达水平检测感染效率;应用Real-time PCR法检测CTGF基因沉默后HSC-T6中金属蛋白酶组织抑制剂-1(TIMP-1)、TIMP-2和I型胶原mRNA表达水平。结果包装的慢病毒载体感染HSC-T6细胞的效率高于50%;感染HSC-T6细胞后,CTGF的表达在mRNA和蛋白水平均受到明显抑制,其中TS1具有最佳的抑制效率;HSC-T6细胞CTGF基因沉默后,其TIMP-1、TIMP-2和I型胶原mRNA水平也显著降低。结论包装的慢病毒载体在HSC-T6中具有较好的感染效率和CTGF基因沉默效果,基因沉默后可进一步抑制HSC-T6细胞TIMP-1、TIMP-2和I型胶原的基因表达,这在抗纤维化的研究中具有积极的意义。

Objective To inhibit the gene expression of connective tissue growth factor （CGTF） by small interfering RNA （siRNA） and observe its influence on downstream gene expression. Methods 293T cells were cotransfected with lent viral transferred plasmid containing siRNA targeted to CTGF,packaging plasmid,and envelope protein plasmid. The lent viral vector was packed to mediate CTGF gene silence and infect hepatic stellate cell line HSC-T6 ceils. The lent viral vector with the highest inhibition efficiency was screened by real- time PCR and Western blot. Efficiency of infection was measured by the expression of green fluorescent protein. mRNA levels of tissue inhibitor of metalloproteinase-1 （TIMP-1）,TIMP-2,and collagen I were detected by realtime PCR. Results Efficiency of infection of the packing lent viral vector was more than 50%. After infecting HSC-T6 cells,the expression of CTGF was suppressed on both mRNA and protein level,with the highest inhibition efficiency by TS1. The expressions of mRNA in TIMP-1,TIMP-2,and collagen I were significantly restrained by inhibition of CTGF （P〈0.05）. Conclusion The packing lent viral vector in HSC-T6 has a better efficiency of infection and CTGF gene silencing effect. Gene silencing can further inhibit the TIMP-1,TIMP-2 and collagen type I gene expression in HSC-T6 ceils. It has an active significance in anti-fibrosis studying.