大鼠miR-126慢病毒表达载体的构建及其在肝星状细胞中的表达

Construction of rno-miR-126 Lentiviral Expression Vector and its Expression in HSCs

目的:肝星状细胞(hepatic stellate cell,HSC)激活并发生表型改变是肝纤维化形成的关键,本实验期待通过构建慢病毒rno-miR-126,并体外转染HSC,为研究miR-126在肝纤维化中的作用奠定实验基础。方法:PCR扩增miR-126的前体,构建miR-126的重组表达载体pCDH-CMV-MCS-EF1-copGFP-miR-126,脂质体法转染包装细胞293TN细胞,包装产生慢病毒,以293TN细胞绿色荧光蛋白(green fluorescent protein,GFP)的表达水平测定病毒滴度,构建重组慢病毒(Lentivirus,LV)载体(Lv-miR-126),体外转染活化的HSC-T6,荧光显微镜观察荧光阳性细胞百分率,real-time PCR检测miR-126转入水平。结果:经PCR扩增检测阳性菌落和测序证实,成功构建携带大鼠miR-126基因重组慢病毒载体。倒置荧光显微镜下观察可见包装细胞293TN呈绿色荧光,并测得滴度108>ifu/ml。荧光显微镜下HSC的荧光阳性率在95%以上。Real-time PCR检测证实miR-126转染后获得了较高的表达。结论:成功构建大鼠慢病毒载体Lv-miR-126,并可在体外高效稳定表达,为本研究后续对miR-126靶点验证和功能研究奠定实验基础。

Objective： The activation and transformation of hepatic stellate cells （HSC）is the key link in the development of hepatic fibrosis, we expected to construct a lentiviral expression vector for mo-miR-126 and transfect to HSC, thus laying a scientific basis for the study of miR-126 in hepatic fibrosis. Methods： The pre-miR-126 was amplified by PCR and then constructed the recombinant plasmid pCDH-CMV-MCS-EFl-copGFP-miR-126. 293TN cells were cotransfected with lentiviral vector pCDH-CMV-MCS-EFl-copGFP- miR-126 and Lentivirus Package plasmid mix. Virus titer was measured according to the expression level of GFP. The lentiviral vector encoding miR-126 was constructed and transfected, Furthermore, the expression level of miR-126 was measured by RT-PCR, Results： The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing, The 293TN showed green fluores- cence was observed under fluorescence microscope and virus titer was 108〉ifu/m. the positive rate of HSC is more than 95%, the high ex- pression of miR-126 after transfection was confirmed by Real-time PCR. Conclusion： The recombinant lentiviral expression vector was successfully constructed and efficiently expressed in vitro, laying a good foundation for target verification and its function for miR-126.