Study On The Culture And Biological Characteristics Of Rabbit Primordial Germ Cells(PGCs) In Vitro

Under specific culture conditions in vitro,PGCs can be reprogrammed into germ stem cells(EGCs).EGCs have similar biological characteristics with ES,which provide suitable seed cells for the research of regenerative medicine.On the other hand,PGCs can differentiate into mature germ cells in vitro by adding specific inducers in culture medium,which represents a research field and plays an important role in reproductive biology.In this study,fetal rabbits were used as the research objects.The effects of fetal age,serum and serum substitutes,different concentrations and kinds of small molecular compounds on PGCs isolation and culture were compared.The biological characteristics of PGCs were identified,and the conditions of PGCs isolation and culture in vitro were improved to pave the way for the next step.1.The rabbit embryonic fibroblast(REF)was isolated and cultured from E14-16 fetal rabbits of Japanese big eared white rabbits.After subculture,the REF with sufficient quantity,high purity and good activity was obtained.After cell inoculation,the first fluid change was performed 24 hours later,and then the cells were subcultured 2-3 days later according to cell density.The density and culture time of each generation were recorded and the cell growth curve was drawn.According to the principle of slow freezing and fast dissolving,REF was cryopreserved and resuscitated.The results are as follows:(1)P3-P5 REF has less heterogenous cells,more quantity and better activity,while P1,P7 and P10 have lower purity and activity.The former is more suitable for making feeder layer.(2)After different time(30 days,90 days,180 days)cryopreservation,according to the cryopreservation method of this experiment,the cell viability has little effect,especially after the recovery of P3-P5 REF,the cell viability is better and the purity is higher,so it can be used for PGCs culture.2.To explore the biological characteristics of rabbit PGCs in vitro and the effects of fetal age,small molecular compounds,FBS and KSR on the separation and culture of rabbit PGCs.In ths study,PGCs were isolated from the reproductive crista of E10.5-15.5 Japanese white rabbits and identified by morphological observation,AKP staining,RT-PCR detection of transcription factor Oct-4.The results showed positive.When PGCs were inoculated on the feeder layer made of 5×105 REF,different concentrations and kinds of small molecular compounds,as well as serum and serum substitutes were added into the culture medium,respectively.the following conclusions were drawn:(1)PGCs can be reprogrammed as EGCs,and EGCs can be differentiated into neuroid,epithelioid and embryoid cells.(2)Compared with E12.5,E13.5,E14.5 and E15.5,PGCs isolated from the reproductive crista of E10.5 and E11.5 fetal rabbits had stronger proliferation ability and higher cell activity in vitro.(3)When 3um RA,20 um FK,and 100 nm 4OHT were added to the culture medium,PGCs had more colonies,better morphological characteristics and better cell activity.(4)Compared with 15％serum added in the culture medium,when 15%KSR was added,the number of PGCs colonies was more and the morphological characteristics were better.