Cell Biological Research Of Two Strains Newly Established Human Embryonic Lung Diploid Cell Strain

objective:vaccination is one of the most positive and effective measures that can resist the virus infective diseases of humans and animals. With the increase of virus diseases and improvement of acquaintance of humans to virus diseases, the quantity demanded of vaccine increases constantly. Each research and development institution devotes to the research and production of new vaccines, while guaranteeing the continual increase of varieties and quantities of vaccines, the quality has been attached even moreimportance., and the security and effectiveness of vaccine is the base of production quality of vaccine. The control of vaccine production quality includes raw material, intermediate product, final product and whole production process control.The cell substrate as the raw material to produce vaccine is the key to guarantee the quality of vaccine, the cells applied to vaccine production are primary cell, continuous cell and human diploid cell, and human diploid cell has the advantages of identifying adequately and standardization compared with primary cell and continuous cell, which can establish cell seed bank system, and the cell bank can be used to produce in many years, which is conducive to quality control, it is also identified as the safe and stable cell substrate to produce human vaccine as it is non-carcinogenic and not polluted by exogenous factor, and used to produce human vaccine more and more. At present, a large number of vaccines produced by human diploid cell have appeared on the market in China, such as hepatitis vaccine, poliomyelitis vaccine, measles attenuated Live Vaccine, varicella vaccine and others.Certainly, human diploid cell resource is scarce, and its passage generation is limited, the new human diploid cell shall be established constantly to supply the requirement in order to adapt the demand of vaccine research and development and production. Consequently, more and more vaccine research and production institutions devote to research and develop diploid cell so as to culture stable diploid cell which can be used to vaccine production to guarantee the source of raw material of vaccine production. This task is to research characteristics of two strains newly isolated human embryonic lung diploid cell on the basis of KMB-17 cell strain, and aims at establishing a new and good diploid cell strains fit for vaccine research and production.Method:research the characteristics two cell strains (N2 cell strain, N3 cell strain) are separated from human embryo lung respectively, including observing the form and growing status of cells in the process of cell passage, survival status and form of cells after frozen storage and resuscitation, drawing growth curve of cells in different passages in the process of cell passage and realizing form and activity change in the process of cell passage by analyzing cell cycle; identify fibroblast by the method of HE coloration and immunofluorescence; inspect the stability of cell in the process of passage by G-banded chromosomes karyotype analysis; meanwhile, we also analyze the oncogenicity of cells by nude mouse oncogenicity assay and soft agar clone formation assay, evaluate the security of cells preliminary.Results:from the morphologic change and activity analysis of cells in the process of passage, we can see that the growth activity of cells reduces gradually and obviously reduces in high generation with the increase of cell passage generation, the form of cells only changes in high generation; by combining the methods of HE coloration and immunofluorescence, the cell is fusiform, vimentin is identified as positive, and accords with the essential characteristic of fibroblast; the results from analyzing chromosome by G-banding technique show that with the increase of passage number, the hereditary stability of the two strains of cells remains stable, which conforms to the analytical standard of human diploid cell of Chinese Pharmacopoeia; it shows that the two stains of cells have non oncogenicity by analyzing oncogenicity of cells.Conclusion:this task does identification and verification for N2, N3 cell line according to related provisions of human diploid cell strain in zooblast substrates preparation and verification regulation of Chinese Pharmacopoeia, the above experimental method researches prove that N2, N3 are typically human diploid fibroblasts with good cell activity, and without tumorigenicity, inheritable character in passage process and after cryopreservation is stable, which can be considered as a vaccine candidate cell matrix.