大鼠结缔组织生长因子基因miRNA表达质粒的构建及其稳定转染大鼠肝星状细胞系的建立

Construction of miRNA expression vector for rat connective tissue growth factor and establishment of stably transfected rat hepatic stellate cell line

目的构建大鼠结缔组织生长因子(Connective tissue growth factor,CTGF)基因miRNA表达质粒,并建立稳定转染大鼠肝星状细胞(Hepatic stellate cell,HSC)系。方法根据大鼠CTGF基因mRNA序列,设计并合成3对寡聚单链DNA X191-1、X191-2和X191-3及1对阴性对照序列DNA X191-4,将4对寡聚单链DNA退火成双链后,分别与载体pcDNA6.2-GW/EmGFP-miR连接,构建CTGF基因miRNA重组表达质粒,分别转染HSC-T6细胞,荧光显微镜观察细胞的转染效率,RT-PCR检测转染细胞中CTGF基因mRNA的转录水平;取干扰效率最高的重组质粒及阴性对照质粒,分别转染HSC-T6细胞,经杀稻瘟菌素持续加压筛选。结果经测序鉴定,重组表达质粒构建正确,插入片段的碱基序列与设计相符;细胞的瞬时转染效率约为50%;3组干扰质粒转染的HSC-T6细胞中,CTGF基因mRNA的转录水平明显低于空白对照组(P<0.01),其中X191-2质粒对CTGF基因转录的干扰效率最高;获得了稳定转染的HSC-T6细胞。结论成功构建了CTGF基因miRNA表达质粒,并获得了稳定转染的肝星状细胞系,为进一步研究肝纤维化的形成机制及其治疗奠定了基础。

Objective To construct a miRNA expression vector for rat connective tissue growth factor（CTGF） and establish a stably transfected rat hepatic stellate cell（HSC） line.Methods According to the mRNA sequence of rat CTGF,three pairs of oligomeric single-stranded DNA,X191-1,X191-2 and X191-3,as well as one pair of negative control oligomeric single-stranded DNA,DNA X191-4,were designed and synthesized.The four oligomeric single-stranded DNAs were annealed into double chains and linked to vector pcDNA6.2-GW / EmGFP-miR respectively.HSC-T6 cells were transfected with the constructed recombinant plasmids,observed for transfection efficiency by fluorescence microscopy,and determined for transcription level of CTGF mRNA by RT-PCR.HSC-T6 cells were transfected with the recombinant plasmid with the highest interference efficiency and negative control plasmid respectively,of which the positive clones were screened by continuous pressure screening.Results Sequencing proved that the recombinant plasmids were constructed correctly,and the base sequences of inserted gene fragments were consistent with those designed.The transient transfection efficiency of HSC-T6 cells was about 50%.The transcription levels of CTGF mRNA in HSC-T6 cells transfected with three interfering plasmids were significantly lower than that in blank control group（P 0.01）.Of the three interfering plasmids,the one containing X191-2 showed the highest interfering efficiency to CTGF mRNA transcription.Stablely transfected HSC-T6 cells were obtained.Conclusion The miRNA expression vector for CTGF was successfully constructed,and stably transfected HSCs were obtained,which laid a foundation of further study on formation mechanism and therapy of liver fibrosis.