5-Aza-2-deoxyctidine On Cell Cycle, Chromosome And Apoptosis Of Fibroblast

Low efficiency of somatic cell nuclear transfer is the main problem to limit the application of this technology.Now it is generally accepted that the main reason for the failure of nuclear transfer is the apparent incomplete reprogramming of donor nuclear that leads to abnormal expression or the failure expression of genes during development process.Now,DNA Methyl-transferase inhibitor is the common used drug.However, there are some problems for the DNA Methyl-transferase inhibitor,such as:the cytotoxic of nucleoside analogues,the stability and long-term effects of oligonucleotides in vivo. Added this reagent to the culture medium may induce gene mutation,change of cell morphologic,cell apoptosis,as well as the inactivation of X chromosome and the re-activation of imprinting genes,all of which affected the application of the methylation inhibitors.Therefore,accoding to these problems,our experiment designed as follows:1.Established donor cell lines for somatic cell nuclear transfer.In this experiment the method of cow ear tissue adherence was used and then by purification procedure to obtain fibroblasts.Through the frozen preservation,recovery,mapping cell growth curve and analysing cell karyotype,we got dairy fibroblast cell line which had low rate of chromosome mutation,growed well and can steady passage.2.DNA Methyl-transferase inhibitor(5-aza-dc) was used to treat donor cells to seeking for its best concentration in this experiment.With this concentration,we could not only reduce the somatic cell nucleus for the methylation status but also have no adverse effect on the characteristics of cell biology,thereby providing a more efficiency method for somatic cell cloning.In this experiment different concentration of 5-aza-dc was used to treat cow fibroblasts for 72h,by flow cytometry,conventional karyotype analysis and agarose electrophoresis respectively,to test the effect of 5-aza-dc on cell cycle,chromosome karyotype and apoptosis.The results were as follows:The rate of G₀/G₁ phase cells treated with 5-aza-dc(0.005-0.1μmol/L) had no significant difference compared to the untreated group(P＞0.05);For chromosome karyotype,there were no significant aberration of chromosome with lower concentration(0.005μmol/L and 0.01μmol/L) 5-aza-dc(P＞0.05).The chromosome can maintain normal shape.While the 0.03μmol/L and 0.05μmol/L group can significantly induce the aberration of chromosome(P＜0.05).The aberration rate of chromosome was the highest in group that treated with 0.1μmol/L 5-aza-dc.From the agarose gel electrophoresis patterns it can be seen that cells treated by 0.005μmol/L,0.01μmol/L concentration of had no "ladder" phenomenon just as control group.While the towing phenomenon began from group of 0.03μmol/L 5-aza-dc.The proportion of apoptosis treated by the concentration of 0.05μmol/L and 0.1μmol/L groups significantly increased compared with the control group. In a word,these results indicated that there was no effect to chromosome structure and its number when the donor cells treated by 0.005μmol/L and 0.01μmol/L 5-aza-dc for reducing the DNA methylation level.While in a high concentration,the donor cells treated by 5-aza-dc can induce higher aberration of chromosome and apoptosis which can't be used in animal somatic cell cloning production.