| During embryonic and individual development,primordial germ cells evolve into sperm and egg cells in adult animal organs through continuous division and development and differentiation.In vitro isolation and culture of rabbit PGCs provides a large number of cells for early embryonic developmental mechanisms,germ cells,cell vaccine production,and immortalized cells.However,there are still many problems in the in vitro culture methods,passage methods and establishment of rabbit PGCs,affecting the application of rabbit PGCs.Experiments in vitro culture of rabbit PGCs,in addition to solving these problems,can also provide references for research on large livestock and experimental animals.Therefore,it is very important to establish a stable and efficient method of proliferation and passage of rabbit PGCs.In this study,16-20 d embryos of rabbits were used as subjects.The effects of gestational age,feeder layer type,feeder layer density,separation methods,passage methods,and cytokines on the isolation and culture of PGCs were compared.Improve the conditions for the isolation and culture of rabbit PGCs in vitro,and increase the number and quality of rabbit PGCs colonies.To provide a reference for the establishment of stable rabbit PGCs in vitro isolation and culture methods.The main results of the test are as follows:1.Isolation of cultured mouse,rabbit embryo fibroblast(MEF,rabbit embryo fibroblast,REF).The 12-15 d fetuses of Kunming white inbred mice and 16-18 d fetuses of Japanese big ear rabbits were selected as material.MEF and REF were isolated to obtain cells with strong growth and proliferation ability,high purity and stable biological traits.The separated MEFs and REFs were divided into 6 groups according to the first exchange time: 12 hours exchange group,24 hours exchange group,36 hours exchange group,48 hours exchange group,60 hours exchange group,72 In the h-fluid group,the primary cells were changed for the first time,passaged once every 2 days,passaged according to the cell density to P3 generation,and the cell density and culture time of 0-3 generations were recorded.The growth curves of MEF and REF in P1,P3,and P8 generations were plotted by cell counting method;the recovery rates of MEF and REF cells after cryopreservation were compared,and P1,P3,and P8 generations frozen for 30 days,60 days,and 90 days were selected.Recovery,and calculate the recovery rate.The results showed that:(1)The total time of P0-P3 cell culture in the first 24 h change group was significantly different from that of other groups(P<0.05).(2)The P3 generation of MEF and REF had fast growth rate,good activity,stable morphological structure,and high purity.The ratio of P1 to P8 was more suitable as a feeder layer cell for subsequent experiments.(3)The P1 and P3 MEFs and REFs that were frozen for 30 days and 60 days(short time)had higher recovery rate,good vigor after resuscitation,and stable biological characteristics.However,due to the high content of P1 hybrid cells,2-3 generations of purification were required after the recovery.In this experiment,P3 MEFs and REFs were cryopreserved for subsequent experiments.The effect of feeder layer and passage method on the isolation and culture of rabbit PGCs.In this study,PGCs from embryonic ploidy were isolated from Japanese big-ear rabbits 16-20 days later.RT-PCR was used to detect the expression of transcription factor Oct-4 and alkaline phosphatase staining.And from the gestational age,separation,passaging methods,feeder layer density and type of rabbit PGCs culture system optimization.The following conclusions were drawn:(1)The morphological structure of the embryonic ridges of rabbit embryos developed well on 18-20 days,and more rabbit PGCs cells could be obtained.(2)The isolated rabbit PGCs were inoculated into 5×104/m L-1,1×105/m L-1,5×105/m L-1 densities of MEF and REF feeder layers,rabbit PGCs at a density of 5× On the 105/m L-1 MEF and REF feeder layer,the formation rate of colonies was high and appeared early,showing a typical mulberry-like,nest-like aggregate growth,maintaining a long undifferentiated time,and a clear boundary with the feeder cells.(3)The growth behavior of the mixed feeder layer at MEF,REF,(MEF,REF ratio of 1:2)was compared by rabbit PGCs.It was proved that the homogenous REF feeder layer was superior to the mixed feeder layer and superior to the heterologous feeder layer.Feeder MEF.(4)The rabbit PGCs were isolated by trypsin digestion and mechanical methods.The number of colonies of primary rabbit PGCs isolated by trypsin digestion method was more than that obtained by mechanical method,and they were successfully transmitted to the P5 generation on the REF feeder layer,which was more suitable for rabbit PGCs.The method of separation.(5)Comparison of trypsin digestion,mechanical method,and feeder layer digestion together.The results showed that mechanical passage was most beneficial to the passage of rabbit PGCs and the formation of colonies.The rabbit PGCs after colonization had good colony morphology and was ideal for the passage of rabbit PGCs.The expression of the transcription factor Oct-4 was detected by RT-PCR,and the result of alkaline phosphatase staining was positive.The effect of cytokines on the isolation and culture of rabbit PGCs.Rabbit PGCs were inoculated into A,B,C,and D culture fluids.Culture A: basic fluid +10% FBS + 5 ng/m L LIF + 2 ng/m L b FGF + 2 ng/m L TGF-?1;Solution B: Base Fluid +10% FBS + 10 ng/m L LIF + 4 ng/m L LIF + 4 ng/m L b FGF + 2 ng/m L TGF-?1;Culture C: Base Fluid +10% FBS + 15 ng/m L LIF+6 ng/m L b FGF+3 ng/m L TGF-?1;culture D: base fluid+10% FBS(control).Adding cytokines to the culture medium can prolong the survival time of rabbit PGCs in vitro.Synergistic effects of cytokines and feeder layer can promote the growth and proliferation of rabbit PGCs,and preserve the undifferentiated state of rabbit PGCs colonies.The results showed that the addition of three cytokines LIF,b FGF,and TGF-?1 in culture medium B had a positive effect on the proliferation of rabbit PGCs and maintenance of their undifferentiated time compared with culture medium D(control group without cytokines);When REF was used as the feeder layer and culture medium B was the medium,rabbit PGCs had the best effect. |
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