Analysis Of Biological Characteristic Changes Of Fibroblast Cells Of Cervus Nippon Following The Space-mutagenic Treatment

This study is first time to investigate the biological characteristic changes of growth curve,karyotype and cell growth related gene expression of Cervus Nippon fibroblast cells,following the space-mutagenic treatment by using the culture method of self-developed Polyvinylidene fluoride(PVDF).Fibroblast cells were obtained from earskin tissue and adhered to the culture,then subcultured by trypsin digestion and cryopreserved it for study.The number of viable cells were counted in 24-well plates and the cell growth curves were plotted,and also the fitting curve of cell growth curves were analyzed.Conventional chromosomal specimens were prepared to detect the karyotype and G-banding of fibroblast cells.Real-time fluorescence quantitative PCR further was used to detect the expression level of fibroblast cells growth related genes,which compared the space-mutagenic treted cell group with that in control.The results show that the new technology of developed PVDF membrane for cell culture could effectively maintain the survival of Cervus Nippon fibroblast cells in the space and the cells can successfully proliferated after recovery.However,the expression level of cell growth related genes is different after the space-mutagenic treatment with control group.Both of male and female fibroblast cells were still maintained normal morphology after the space-mutagenic tretment,and the growth curve of fibroblast cells showed "S" model which was similar to control group,but the logarithmic growth phase of female and male in space-mutagenic treted group were prolonged to 3-4 days compared with 1-2 days of control group.The degree of fitting of growth curve was similar between two groups,however,fibroblast cells inflexion time was later and fibroblast cells volume was lower in space-mutagenic treted group.Karyotype and G-band analysis showed that the chromosome number of fibroblast cells in space-mutagenic fibroblast cells were still remained 2n = 66 with normal chromosome morpholog(female: 2n=66,XX;male: 2n=66,XY).Chromosome type was still 2n = 31 T + 2M,where 1 and 2 are centripetal centromeres,and other are centromeric centromeres.The results of Real-time fluorescence quantitative PCR showed that 4 expression level of 10 cell growth related genes in male and female was significantly increased after the space-mutagenic treatment(genes: CDKN1 A,BCCIP,ANAPC2,CUL1,P<0.05)).This study established a new technology of somatic cell culture for domestic animals in the space condition,and also analyzed the effect of the space-mutagenic treatment on biological characteristics and gene expression changes in Cervus Nippon fibroblast cells,which provided some basic and important information and technique for future studies of animal genetics.