ssc-let-7c对猪甲状腺细胞体外培养增殖研究

Study on the Proliferation of Porcine Thyroid Cell Cultured in vitro by Disposal of SSC-let-7c

Mi R-let-7c是let-7家族的主要成员之一,在甲状腺的生长发育过程中起着重要的作用。本实验通过体外转染mi R-let-7c类似物探索其对猪甲状腺细胞体外培养增殖的影响。观察猪甲状腺细胞在3D条件下的生长状况和猪甲状腺原代及传代细胞的形态变化,并计算细胞生长速度;通过实时荧光定量(q PCR)、MTT技术检测3组体外培养猪甲状腺细胞中mi R-let-7c的表达量和细胞增殖情况;利用在线预测软件mi RWalk2.0对mi R-let-7c的靶基因及其功能进行预测。结果表明:传代2代后的甲状腺细胞生长缓慢,且形态由圆逐渐变长。细胞免疫荧光鉴定结果发现有明显的绿色荧光,表明猪甲状腺细胞功能正常。荧光定量PCR结果显示管家基因Met-t RNA表达稳定,转染mimics组mi R-let-7c表达量明显高于其他2组。利用MTT法的细胞增殖实验中转染mimics组的OD值显著小于其他2组(P<0.05)。利用在线预测软件发现mi R-let-7c的靶基因主要聚集在与胰岛素及癌症等相关的通路。本研究揭示了mi R-let-7c在甲状腺细胞生长过程中的重要作用,为研究mi R-let-7家族在调控猪生长发育过程中的作用提供了有益参考。

MiR-let-7c is one of the main members in miR-let-7 family and plays an important role during the growth and development period. In this experiment, method for transfeeting miR-let-7c mimics in vitro was applied to explore whether there＇s difference in the proliferation of the cultured porcine thyroid cells. Firstly the status of these cells cultured in three-dimensional （3D） system was observed and the morphological change both in the primary and extended cells are also observed by microscope to determine the variation of the cell growth rate. Then the technique of quantitative Real-time PCR and MTF were utilized to detect the expression of miR-let-Tc and the proliferation of thyroid cells. At last, the target genes of miR-let-Tc were predicted by the the online software miRWalk2.0, and with assembling their function pathway. The results showed that after the second passage, the thyroid cells show lower growth rate and mildly change in shapes. The Green fluorescence result indicated the normal function of thyroid cells. The results of the real-time quantitative PCR revealed that the housekeeping gene Met-tRNA expressed steady, and the expression of miR-let-Tc in the miR-let-7 mimics group was significantly higher than that in the other groups, and the OD value in the group of the miR-let-Tc mimics group was markedly less than that of other disposals by the MTY determined method （P〈0.05）. The large amount of target genes were found by using the miRWalk2.0 software, which mainly assemble in ubiquitin mediated proteolysis, pathways in cancer etc. This research revealed that the important effect of let-7c during the progress of thyroid cell＇s development and provides a useful reference for the study of how does the let-7 family regulate the growth of swine.