Establishment Of Reindeer Fibroblast Cell Line And Analysis Of Their Biological Characteristics

Reindeer,as a vulnerable animal in the world,is only distributed in the Lugenhe area in the northwest of the Daxing’an Mountains in China.In this experiment,the ear tissues of female and male reindeer from the hinterland of Daxing’an Mountains in Hulunbuir League of Inner Mongolia were used as experimental materials,and female and male reindeer fibroblast cell lines were successfully established and subcultured.Four groups of reindeer fibroblasts of different generations were analyzed for morphology,survival rate before and after cryopreservation,adhesion rate,growth curve,chromosome karyotype and G-band.For investigation of cell proliferation,different generations were extracted RNA from female and male reindeer fibroblasts,real-time quantitative PCR technology was used to detect intracellular the expression levels of stem cell-related genes Oct4,Sox2,Nanog-like,cell growth and division-related genes MYC,ERRFI1,ANAPC2,BCCIP,CUL1,CKS1B,CCNF,CDKNIA,MCM2,PCNA,RPA3,RAD51 and also the expression level of cell telomerase activity gene TERT.The results show that the fibroblasts from the ear tissue of the reindeer are spindle-shaped and flat with a centered nucleus.The average subculture time is 3 to 4days and can be stably passaged for more than 60 times.The attachment rate of cell starts at 6h and reaches 85%at 12h,which shows that the reindeer fibroblasts established in this experiment was stable.The survival rate of reindeer fibroblasts after cryopreservation was between 65-85%in the four cell lines,which was significantly lower than that before cryopreservation(P<0.05).In the growth curve,it was found that the four cell lines of reindeer fibroblasts grew rapidly from D2 to D5,and there was a growth turning point from D5 to D6.The growth curves of all fibroblasts showed a typical"S-shape",and the maximum number of proliferating cells was 37.1×10~4.The karyotype test results showed that 84～88%were 2n=70,which including 34 pairs of autosomes and 1 pair of sex chromosomes XY.The G-band of chromosomes analysis results also showed band stability and reindeer species specificity.The ATP6,COX2,ND4 and D-loop control region genes of the mitochondrial DNA of reindeer fibroblasts were sequenced,and the gene sequences of 17 species were used to construct a phylogenetic tree with reindeer.Except for the D-loop control region,other gene sequences were shown by reindeer system evolutionary characteristics earlier than other deer species.In order to investigate the reasons for the high passage activity of reindeer fibroblasts,the results of AP staining,stem cell and cell proliferate key genes and telomerase activity detection,showed that the fibroblast division activity of reindeer is generally higher than that of Sika deer,but there are gender differences with some species.In summary,this study successfully established reindeer fibroblast cell lines and stably subcultured for more than 60 generations,confirming that the standard karyotype of reindeer was 2n=68+XY.By analyzing the biological characteristics of reindeer fibroblasts,the rapid proliferation of reindeer cells was revealed its special cell characteristion,which provided important research materials for the reindeer cell genetic resource bank,the conservation and genetic studies of reindeer.