Biological Characteristic Analysis Of Ear Fibroblast Cell Lines Of Meishan Pig

Domestic and foreign breeders pay more attention to local pig breed resource of China because of its richness and unique genetic characteristics.However,due to some different reasons,the number of some local pig breeds has dropped sharply or even extincted.So it is necessary to preserve local pig breed resources by biotechnology.Somatic cell cryopreservation has a widely application prospect in the conservation of endangered species and breeding livestock resources because it is simple,effective and more operable,and ear tissue collection does not affect animal health.Meishan pig is a good local native breed in China.It is famous for its reproductive capacity and delicious meat quality.In order to preserve the germplasm resources of Meishan pig breeds,this paper established the ear fibroblast cell line of Meishan by tissue mass culture method and analyzed the biological characteristics of the cell lines.It could properly protect this excellent resource,and provide references for preserve seeds of other animals.This study was to establish ear fibroblast cells from Meishan eartissues with direct tissue mass culture method,and the biological characteristics of these cell lines were studied.The results showed that the ear fibroblast cells of Meishan pig have a good growth situation,and the survival rate of fibroblasts was 95.21% and 90.08% respectively before and after cryopreservation.The growth curves of cryopreserved fibroblasts were "S" type.After bacteria,fungi and mycoplasma detection,all the cells were not contaminated by microorganisms.The percentage of metaphase diploid cells(2n=38)was 92.31% in metaphase,and it accorded with the requirement demands of the fibroblast cell line.The banding patterns of malic dehydrogenase(MDH)and lactic dehydrogenase(LDH)showed that there were no contaminations with other cells.After transfected with green fluorescent protein(EGFP)gene,the positive rates of 5 ear fibroblast cells were form 45.23% to 67.32%,and all the 5 cells after G418 selection could form monoclonal colony.In conclusion,the method for establishment of ear fibroblast cells makes the preservation of genetic resource of Meishan pig more efficiency in cell levels,and it can be used as a reference for the preservation of other animal genetic resources.This study explored the effect of vitamin E on the anti-apoptotic ability in Meishan pig ear fibroblasts during subculture.The cell proliferation was detected with MTT method,the cell mitochondrial membrane potential was detected by using JC-1 staining method,theapoptosis rate of fibroblasts was detected by Annexin V-EGFP/PI,the m RNA expression level of BAX,Caspase-3,BCL-2,SOD-1 were detected by RT-PCR.The results showed that when the cultured cells were treated with vitamin E,the cell relative growth rate and mitochondrial membrane potential were significantly higher than the control group,and the apoptotic rate of fibroblasts decreased significantly.There were no significant differences in the gene expression levels of BAX,and Caspase-3 after VE addition.The gene expression levels of BCL-2,SOD-1 after VE addition increased significantly.The expression of BCL-2 gene of the 100 μmol/L VE treatment group was significantly higher than that of 0 VE and 50μmol/L VE treatment groups(P<0.01 group),and there was no significant difference between 100 and 150 μmol/L VE treatment groups(P>0.05);SOD-1 gene expression level of 100 μmol/L VE treatment group was significantly higher than the other three groups(P<0.01).The results indicated that 100 μmol/L vitamin E addition during cell culture could inprove mitochondrial function,inhibit cell apoptosis,and increase cell growth ability.This study explored the effects of cryopreservation on the methylation status of H19 and IGF2 R genes in porcine somatic cells,DNA methylation status and expression levels of H19,IGF2 R in Meishan pig ear fibroblast cells before and after cryopreservation were detected with bisulfitesequencing and RT-PCR technology.The expression levels of methylation related genes were also detected and analyzed.The results showed that the methylation rates of H19 DMR1 and DMR3(84.48%,69.17%)in cryopreserved cells were significantly higher than those in fresh cells(58.28%,48.33%,P<0.01).H19 DMR2 was demethylation,and the methylation rates of H19 DMR2 was significantly lower than that in fresh cells(26.40% vs 48.00%,P<0.01),and expression level of H19 gene was significantly higher than that in the fresh cells(P<0.01).The IGF2 R DMR2was hypermethylation,and its methylation rates was significantly higher than that in the fresh cells(75.88% vs 42.94%,P<0.01),but there was no significant difference in the expression level of IGF2 R DMR2 between fresh and cryopreserved cells(P>0.05).The expression levels of DNMT3 A and DNMT1 in cryopreserved cells were significantly higher than those in fresh cells(P<0.01),and there was no significant difference in the expression level of DNMT3B(P>0.05)between two groups.The results indicated that the cryopreservation could affect the methylation status of H19 and IGF2 R DMR,thus changing their gene expression levels.