| Pluripotent stem cell is significant for animal husbandry and medical research. As a kind of traditional laboratory animal, rabbit has been applied in the fields of transgenic animal research and human disease studies. But the pluripotent stem cells-embryonic stem cell(ESC) and induced pluripotent stem cell(i PSC) of rabbit are still in their primary state, which require further explorations of methods and techniques in derivation and identification. In this study, we isolated, cultured and characterized Japanese white rabbit ES-like cells, and tried to induce rabbit embryo fibroblast(REF) cell to i PSC at the same time. The results were as follows:1. Rabbit ES-like cells were derived from blastocysts. 60 rabbits were superovulated with FSH and h CG, 38 embryos were obtained from one rabbit averagely, and the blastocyst rate was 79.96%. The components of rabbit ES-like cells basic medium were 76% Knockout DMEM+20%KSR+1%Glutamax+1%NEAA+1%β-mercaptoethanol+1%penicillin-streptomycin+10 ng/m L b FGF+1000 IU/m L h LIF. Different concentrations of melatonin(10-6, 10-7, 10-8, 10-9, 0 M) were supplemented to the basic medium to study the melatonin’s effects on rabbit blastocysts, inner cell mass(ICM) and ESC. The results indicated that melatonin had no significant effect on blastocysts adherence, which were about 50% in the five groups. However, melatonin showed positive effects on the proliferation and passage of ICM and ES-like cells, in which, 10-7 M melatonin exhibited the most significant effects. 10-7 M melatonin could improve ICM proliferation rate significantly(P<0.05), which increased from 19.97% to 34.57%. Rabbit ES-like cells were isolated after the machinery segmentation of ICM, which showed flat morphologies and sharp edges. The supplement of 10-7 M melatonin in medium could improve rabbit ESC passages from 4 to 6, and improved passage rate significantly(P<0.05) in P4 and P6.2. We identified P5 rabbit ES-like cells, the results suggested that rabbit ES-like cells were pluripotent. P5 rabbit ES-like cells showed alkaline phosphatase activity; Pluripotent marker genes Oct4,Sox2,Klf4, c-Myc, Nanog, Line28 a were detected by RT-PCR; Results of q PCR showed that the relative expression level of Oct4,Sox2,Nanog in ES-like cells were apparently higher than that in REF cells; Immunochemical analyses indicated that pluripotent proteins OCT4, SOX2, NANOG and surface marker proteins SSEA-1, SSEA-4 and TRA-1-60, TRA-1-81 were recognized by antibodies against these proteins, and SSEA-4, TRA-1-60 were faintly expressed. Rabbit ES-like cells had normal karyotypes which were 2n = 44; After removing feeders and pluripotent-maintaining factors, P5 rabbit ES-like cells could spontaneously differentiate into lipocyte-like, neuron-like, fibroblast-like cells and beating cardiomyocytes in vitro; After 8 days hanging-drop, embryo body(EB) were formed. After 14 and 21 days differentiation, tissue-specific genes Bmp4, Afp, Desmin, Sox17, Pax6 and proteins VIMENTIN, β-TUBULIN, AFP, ALB, DESMIN could be detected, and the expression quantity increased with the time, which indicated that rabbit ES-like cells could differentiate to neuron, fibroblast, hepatocyte and muscle cells. We injected P3 ES-like cells to groin hematoma of a NOD-SCID mouse, after 8 weeks, a teratoma with three germ-layers including neuron, muscle, gland tissues was obtained.3. Explore the method of inducing REF cells to rabbit i PSC. Primary REF cells were infected by human reprogramming transfactors(Oct4, Sox2, Klf4, c-Myc) using retroviral vectors. Cell clones were collected and passaged after 12-18 d of infection, the clone formation efficiency was 0.03%. However, the exogenous genes were not silenced after passaging to P10, and AP activity were negative. The results suggested that we failed to induce REF to rabbit i PSC. |
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