| Somatic cell cloning technique is of great study value at improvement livestock breed, drug production, protecting endangered species, and in which the donor nuclear and recipient cytoplasm synchronization is a key factor, it was widely used in serum starvation method inducing donor cells in G0/G1phase. Mice as the most widely used and study model of somatic cell cloning has incomparable advantages in other species, but its’ rate of embryo development by cloned is low, is still a problem needed to resolve. There is research shows that this may be related to the serum starvation induced a large number of DNA damage lead to the apoptosis rates go up. Therefore, this test is based on the growth state of donor cells, attempts to study effects of antioxidant how to inhibition mice fetal fibroblast apoptosis and the the subsequent reconstructed embryos development in mice nuclear transfer.Objective:(1) To determine the activity of given stress stimulation on different passages fibroblasts of Kunming mice;(3) To explored the effects of different concentration of VC,VE and MT on donor cells’activity.(4) To explored the effects of different concentration of VC,VE and MT on donor cell’s subsequent reconstructed embryos development, thereby obtain the optimum concentration of VC,VE and MT.Methods:(1) Prepared and established fibroblasts lines,Treated different passages fibroblasts with frozen thaw for measure the cell viability by MTT and drawn the growth curve to compared with normal fibroblasts of same passages, and select the optimal algebra of mice fetal fibroblasts as donor cells;(2) Treated the lth generation of MEF cells with VC (0μM,100μM,150Mm and200μM). VE (0μM,25u.M,50μM,100μM), MT (0M,10-8M,10-9M,10-10M) respectively, use the MTT method to measure the cell viability of each group and compared.(3)use the treated MEF by VC,VE and MT as the donor cell to get reconstructed embryo, Data were analyzed with SPSS11.0.Results:(1) There was no difference in morphological characteristics of fibroblasts between each passages;(2) In the normal passaged group, There was no difference in viability between each passages; The1and2passages frozen-thaw group were significantly higher than3and4passages cells, but there was no difference between former; The results indicated that the resistance to outside stimuli was inversely proportional to sub-cultured frequencies; thereby select the lth passage MEF as the donor cell;(3) cells of lth passage were detected in2d cultured in10%FBS treatments with antioxidant increased viability compared with control, and there was no significantly differences (P<0.05),while there was no difference between experimental groups and control group in0.5%FBS(P>0.05)(4) In VC study, On the fusion rate, results showed that the three experimental groups were all significantly higher than the control group (P<0.05); on the cleavage rate,150μM group were higher than200μM and100μM group(P>0.05),but they were all higher than control group (P<0.05);On the blastocyst rate, experimental groups were all significantly higher than the control group (P<0.05);it indicated that the optium concentration of VC to treated the donor cell was150μM;(5) In VE study, On the fusion rate, results showed that the three experimental groups were all significantly higher than the control group (P<0.05); on the cleavage rate,100μM group were higher than50μM and150μM group(P>0.05),but they were all higher than control group (P<0.05); On the blastocyst rate, experimental groups were all significantly higher than the control group (P<0.05); it indicated that the optium concentration of VC to treated the donor cell was100μM;(6) In MT study, On the fusion rate experimental groups were all significantly higher than the control group (P<0.05);On the cleavage rate, results showed that10-9M group was higher than10-8M(P>0.05),but was significantly higher than 10-10M and control group(P<0.05); On the blastocyst rate,10-9M was higher than10-10M and10-8M group(P>0.05),but they were all higher than control group (P<0.05). it indicated that the optium concentration of MT to treated the donor cell was10-9M. |
PDF Full Text Request