The Analysis And Research Of The Human Diploid Cell Strain Genetic Characteristics

Human diploid cell strains (HDCSs) are cell populations which derived from normal human embryos or other tissues. These normal karyotype cells contain two sets of identical chromosome. The number of Chromosomes of these cells remain the same with the original donor and it can be stably inherited in the cell passaging; furthermore, there is no tumourigenicity when these cells are transplanted between different species and these finite cell lines are also free of adventitious agents. For decades, HDCSs have been applied for manufacturing viral vaccines and have been proved to be a safe cell substrate.HDCS has unique advantages in mass production of vaccines. First, it can be stored in liquid nitrogen for a long time and re-established when needed. HDCS remains the characteristics of the original tissue after recovery. Therefore, we can obtain a large number of identical cells in once time. In a word, a successfully established HDCS can be used for many years. Second, HDCS is a safe cell substrate that has no tumourigenicity and known exogenous pollution agents. Finally, HDCS can be detail identified in order to avoid cell cross-contamination and misidentification. For viral vaccine production, human diploid cell is recognized as the best cell substrate now, which has better Immunogenicity and tolerance. Therefore, the establishment of human diploid cell line plays an important role in the control of infectious diseases and the development of vaccinology. However, it is extremely difficult to obtain qualified HDCSs that can satisfy requirements for massive produce of vaccines.In our study, we have developed four new HDCSs by cell culture techniques, namely N0, N1, N2, N3, which all derived from human embryonic lung tissue. NO strain attained 48 passages of cell doublings in vitro, N1 strain attained 43 passages, N2 and N3 strain attained 47 passages. The cell life of these four new HDCSs is similar or identical to WI-38,MRC-5 that currently serve as international standardized cell strains. We also cryopreserved a lot of cells according to cryopreserve procedures, as material foundation for long-term study of the cells.In this study, we preliminarily analyzed the biological characteristics and genetic characteristics of these four new human embryonic lung diploid cell strains after the success of cell cultivation.1. Gene expression of these four HDCSs was tested using Agilent Whole Hum-an Genome Oligo Microarray. Comparisons between different strains, different passages of the same strain and with the control group, KMB-17 were performed. Result shows that differences between comparison groups are significant. There are plenty of genes up regulated or down regulated. Moreover, we classified and gathered the GO (Gene Ontology) of these differentially expressed genes. We show that differentially expressed genes involved in cell division and proliferation, signal transduction, transcription regulator activity, translation regulator activity, cell cycle, cell communication, cell adhesion, cell recognition, cell senescence and apoptosis, cell growth, endomembrane system, cell metabolism, cytokine production, viral reproduction, immune response, etc. KEGG pathway enrichment analysis shows that these genes are mainly related to olfactory transduction, protein digestion and absorption, ECM-receptor interaction, RNA transport, pathways in cancer, Rapl signaling pathway, cytokine-cytokine receptor interactions, PI3K-Akt signaling pathway, hippo signaling pathway, ubiquitin mediated proteolysis and other passways.2. We also assessed the susceptibility of these cells to EV71 and analyzed the gene expression after EV71 infection. NO strain was selected to represent the four HDCSs. Analysis of virus titers shows that NO strain is equal to KMB-17 cells for cultivating these viruses. Result of gene expression shows that differences between comparison groups are significant. There are plenty of genes up regulated or down regulated. Moreover, we classified and gathered the GO (Gene Ontology) of these differentially expressed genes. We show that differentially expressed genes involved in cell division and proliferation, signal transduction, transcription regulator activity, translation regulator activity, cell cycle, cell communication, cell adhesion, cell recognition, cell senescence and apoptosis, cell growth, endomembrane system, cell metabolism, cytokine production, viral reproduction, immune response, etc. KEGG pathway enrichment analysis shows that these genes are mainly related to olfactory transduction, TNF signaling pathway, Transcriptional misregulation in cancer, Raminoacyl-tRNA biosynthesis, microRNAs in cancer, p53 signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, natural killer cell mediated cytotoxicity, Ras signaling pathway, FoxO signaling pathway, TGF-beta signaling pathway and other passways.3. These four HDCSs were authenticated by STR profiling and SNP typing. Result shows that the gender of cell-derived individual is identical to the gender which was recorded in the raw data. These four HDCSs derive from distinct individuals, however, no cell cross-contamination and misidentification was tested.