|
This thesis affect rabbit embryonic stem cells (embryonic stem Cell, ES) cells and embryonic germ cells (embryonic germ cell, EG cells) in culture, clone, separation and passaged effect factors were studied. Kunming white mouse fibroblast (MEF) feeder layer, low glucose DMEM 10% the NBS 10% FCS 10ng/mlLIF 10ng/mlSCF 0.1 mM β-mercaptoethanol 100IU/ml the penicillin 80IU/ml streptomycin for medium, from 72 ~ 120h embryos isolated rabbit class ES cell lines, clones passaged for four generations, the first generation of which 43 ES cell colony (39.8%), the second generation of eight ES cell colonies (7.4%), the third generation two ES cell colonies (2.7%), the fourth generation of an ES cell colony (0.9%). ES cells cultured in vitro suspension to form embryoid bodies, the monolayer rabbits ES cells cultured on feeder layers of aging, can differentiate into a variety of cell types, such as cardiac muscle and nerve cells. In addition, this experiment also investigated the embryo age, growth factors, feeder layer on the rabbit ES cells isolated and cloned. The results showed that the The 72h rabbit embryos adherence rate and the inner cell mass proliferation rates were 68.1% and 50.0%, the 96h rabbit embryo adherence rate and the inner cell mass proliferation rates were 71.4% and 53.6%, 120h rabbit embryos adherence rate The inner cell mass proliferation rates were 77.4% and 45.2%, respectively. Joint use of growth factors SCF and LIF can significantly improve embryonic adherence rate (71.4%: 34.8%) and ICM proliferation rate (53.6%: 8.7%), the control group did not get the ES cell clones. MEF as feeder layer adherent rate of embryos, the inner cell mass of the proliferation rate of ES clone is superior as a feeder layer in REF. Meanwhile, the growth behavior of rabbit embryonic mouse embryo culture system, and open the feeder layer (1) mouse trophoblast cells directly spread in a petri dish. The rabbit trophoblast and open the feeder layer forces, the the most rabbit embryo spread in the feeder layer. (2) mice ICM lumps growth. The rabbit embryo ICM was polymorphism, aggregation growth, migration growth and monolayer. 4 ~ 5d passaged (3) mice ICM cultured rabbit ICM training 3 ~ 4d passage. 14 ~ 18d rabbit fetal genital ridge and surrounding tissue to its homologous fibroblast co-culture, low glucose DMEM 10% the NBS 10% FCS 10ng/mlLIF 10ng/mlSCF 0.1mMβ-mercapto ethanol 100IU/ml the penicillin 80IU/ml streptomycin for medium, isolated rabbit primordial germ cells (PGC), cloning and multiple passages. EG colony obtained from PGC, 14d fetal primary observed the class EG colony spread to four generations after missing. The 16d fetus class EG only passing second generation, the the 18d fetus does not get EG colony. EG cells have many characteristics of stem cells, were the typical lumpy gathered growth, the AKP staining positive growth in the aging medium feeder layer to form embryoid bodies, epithelial cells, nerve cells, and fibroblasts.
|