转基因诱导永生化鸡细胞系的建立

Establishment of Transgenic Induced Immortal Chicken Cell Line

传统上从鸡胚中分离病毒用于疫苗的生产常常不能满足需求,研究从体外培养的细胞中分离病毒的途径成为当前疫苗生产领域的迫切需要。本研究利用体细胞诱导重编程技术建立永生化细胞系,为应用于疫苗制备等研究和生产奠定基础。通过慢病毒载体转染转录调控因子(NANOG, LIN28和C-MYC)重编程鸡成纤维细胞,并稳定培养至100代,获得永生化的细胞,而后逐步去除细胞因子、血清等添加物,优化细胞系培养体系,并对细胞驯化实现悬浮生长,以获得单位体积最大密度的细胞。研究结果表明:通过转基因将NANOG,LIN28和C-MYC 3个因子整合入细胞中表达,细胞系对碱性磷酸酶染色呈阳性反应,端粒逆转录酶(cTERT)基因在细胞系中显著上调,并稳定培养至100代,使得这些细胞具有自我更新特性和永生化的潜能。确定了培养基中的血清替代物KSR浓度为20%,并撤除了培养基中bFGF等生长因子,为细胞大规模培养应用提供了可能。永生细胞实现悬浮生长,细胞生长倍增时间为21.71 h,最大密度为1.3×106 cells/m L。因此,我们通过重编程技术建立了一株稳定的永生化细胞系,并且使它能在低浓度KSR中悬浮培养,这为疫苗制备等研究和生产提供科学依据。

Traditionally, viruses isolated from chicken embryos have not been sufficient for vaccine production. It has become an urgent need in the field of vaccine production to study ways to isolate virus from cultured cells in vitro. This research aimed to establish an induced immortal cell line with inducible reprogramming technology from somatic cells and establish the foundation for research and production of vaccine application. Chicken embryonic fibroblast cell lines(CEF) were transfected with transcription factors LIN28, NANOG and C-MYC with lentiviral vectors and cultured stably to 100 thgeneration and then the induced immortal cell line was established. After that, the cytokines and serum were gradually removed and the culture system was optimized.The derived cells were harvested in the maximum density and cultured in suspension in serum-reduced medium,and the results as follow: Transcription factors NANOG, LIN28 and C-MYC were incorporated in cells and the telomerase reverse transcriptase(cTERT) gene was significantly up regulated in cells, then 100 generations were passaged, which indicating the immortality and self-renewal ability in these cells. The derived cells were subjected to attritions in culture medium with 20% KSR and in the absence of bFGF, which provided the possibility for large scale culture application. Finally, the derived cells were trained to adapt to suspension culture,it provided a possibility for the application of large-scale cell culture. The stable cell line in suspension culture could grow at doubling of 21.7 hours and maximum density at 1.3×106 cells/mL. Therefore, we generated an immortal cell line by using cellular reprogramming technology and these cells were proliferative in low KSR and suspension culture system, which laid a foundation for vaccine preparation and research.