| Embryonic stem cells (ES cells) were isolated from animal inner cell mass (ICM) and cultured in vitro to establish cell clone lines. ES cells are topitent cells and have differentiation ability similar to embryo, they can be cultured and generated in vitro and maintained in undifferentiated states, also have the ability to form chimera. The purpose of this study is to find the best isolation method and the best digesting time of chicken ES cells, ES cells were cultured and passaged to 6th generation. Further studied the identification of chicken ES cells and induced the ES cells to ossification cells, fat cells and nerve cells. The results showed:1. In this study, three different methods were used to isolate stageⅩblastodermal cells embryos. The cells were isolated using spoon, filter paper wreath and hair wreath, the result shows the using the spoon can obtain more whole blastoderms and can save more time.2. In this study, the blastoderm cells were digested for 2 min, 5 min, 8 min and 11 min then the cells were culture and the clones were recorded. The result was the number of clones of digested 11 min was significantly lower than other digested time(P<0.01). But in the 4th generation cultivation, the numbers of clones formed by the cells digested 5 and 8 min were significantly higher than the cells digested 2 and 11 min(P<0.01).3. ES cells were induced into osteoblasts in the media containing different concentrated groups of desamethason, glycerol 2-phosphate disodium salt hydrate and vitamin C. The result showed induced by the media containing 1×10-7 mol/L desamethason, 0.01 mol/L 2-phosphate disodium salt hydrate and 0.05 g/L vitamin C, the different rat was 72%, higher than other groups. ES cells induced 3 days later, the cells grow as fibroblast-like cells, protuberance grew out in 9 days. Granular mineralize node appeared after 15 days'induction. When differentiated for 21 days, the induced cells showed positive for ALP, the Von Kossa's stain and the immunohistochemistry stain respectively.4. ES cells were induced into neuron-like by all-trans retinoic acid (RA). The induced cells were identified by the specific toluidine blue stain and the immunohistochemistry stain. RA and IBMX was used individually and combined. The result showed induced by the medium containing 2×10-6 mol/L RA, the different rate was higher than other group. ES cells induced 48 h later, most single ES cells grew out protuberance, some neuron-like cells with more progress were present. Almost all of the clones lost undifferentiated state, large amount of neuron-like cells appeared with the formation of neuron-like networks. Tree-like but long protuberance appeared in single neuron-like cells after 4 days'induction. Special Nissl body was found by by toluidine blue stain after induced for 7 days. Immunohistochemistry results indicated that differentiated ES cells expressed NSE and NF positive, GFAP negative.5. ES cells were induced into adipocytes in the media containing desamethason, insulin and IBMX. The induced cells were identified by the oil red-O stain and PT-PCR was done to detect the PPAR in adipocytes. The result showed combined with the induced medium containing 1×10-6 mol/L desamethason, 10 mg/L insulin, 5×10-4 mol/L IBMX and the circle medium containing 10 mg/L insulin, 3 days later, the fat drops could be seen in the ES cells, the drops began to assemble in 10 days and then form annular structure. When differentiated for 21 days, the induced cells were red by the oil red-O stain and there was a single trap about 301bp showed PPAR in the induces cells.
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