Engineering Research Center of Key Technology and Industrialization of Cell-based Vaccine, Ministry of Education, Lanzhou, 730030, China; Gansu Tech Innovation Center of Animal Cell, Biomedical Research Center, Northwest Minzu University Lanzhou 730030, China; Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Biomedical Research Center, Northwest Minzu University, Lanzhou, 730030, China.
Engineering Research Center of Key Technology and Industrialization of Cell-based Vaccine, Ministry of Education, Lanzhou, 730030, China.
Biologicals. 2023 Aug;83:101697. doi: 10.1016/j.biologicals.2023.101697. Epub 2023 Aug 14.
MDCK is currently the main cell line used for influenza vaccine production in culture. Previous studies have reported that MDCK cells possess tumorigenic ability in nude mice. Although complete cell lysis can be ensured during vaccine production, host cell DNA released after cell lysis may still pose a risk for tumorigenesis. Greater caution is needed in the production of human vaccines; therefore, the use of gene editing to establish cells incapable of forming tumors may significantly improve the safety of influenza vaccines. Knowledge regarding the genes and molecular mechanisms that affect the tumorigenic ability of MDCK cells is crucial; however, our understanding remains superficial. Through monoclonal cell screening, we previously obtained a cell line, CL23, that possesses significantly reduced cell proliferation, migration, and invasion abilities, and tumor-bearing experiments in nude mice showed the absence of tumorigenic cells. With a view to exploring tumorigenesis-related genes in MDCK cells, DIA proteomics was used to compare the differences in protein expression between wild-type (M60) and non-tumorigenic (CL23) cells. Differentially expressed proteins were verified at the mRNA level by RT-qPCR, and a number of genes involved in cell tumorigenesis were preliminarily screened. Immunoblotting further confirmed that related protein expression was significantly reduced in non-tumorigenic cells. Inhibition of CDC20 expression by RNAi significantly reduced the proliferation and migration of MDCK cells and increased the proliferation of the influenza virus; therefore, CDC20 was preliminarily determined to be an effective target gene for the inhibition of cell tumorigenicity. These results contribute to a more comprehensive understanding of the mechanism underlying cell tumorigenesis and provide a basis for the establishment of target gene screening in genetically engineered non-tumorigenic MDCK cell lines.
MDCK 目前是流感疫苗生产中主要使用的细胞系。先前的研究报告称，MDCK 细胞在裸鼠中具有致瘤能力。尽管在疫苗生产过程中可以确保完全细胞裂解，但细胞裂解后释放的宿主细胞 DNA 仍可能存在致瘤风险。在生产人用疫苗时需要更加谨慎；因此，使用基因编辑技术建立不能形成肿瘤的细胞可能会显著提高流感疫苗的安全性。了解影响 MDCK 细胞致瘤能力的基因和分子机制至关重要；然而，我们的理解仍然肤浅。通过单克隆细胞筛选，我们之前获得了一个细胞系 CL23，其具有明显降低的细胞增殖、迁移和侵袭能力，裸鼠肿瘤实验表明不存在致瘤细胞。为了探索 MDCK 细胞中的肿瘤相关基因，我们使用 DIA 蛋白质组学比较了野生型（M60）和非致瘤型（CL23）细胞之间的蛋白质表达差异。通过 RT-qPCR 验证了差异表达蛋白在 mRNA 水平上的差异，并初步筛选了一些与细胞致瘤相关的基因。免疫印迹进一步证实，非致瘤细胞中相关蛋白表达显著降低。通过 RNAi 抑制 CDC20 表达显著降低了 MDCK 细胞的增殖和迁移，并增加了流感病毒的增殖；因此，CDC20 被初步确定为抑制细胞致瘤性的有效靶基因。这些结果有助于更全面地了解细胞致瘤的机制，并为建立基因工程非致瘤 MDCK 细胞系中的靶基因筛选提供了依据。
