【作者】 李凤珍；

【导师】 易建明；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 核移植效率低是限制体细胞核移植技术应用的主要原因,目前普遍认为,供体核的不完全表观重编程导致在发育过程中有重要作用的基因异常表达或没有表达是动物核移植失败的主要原因。DNA甲基化酶抑制剂是目前应用较普遍的一种药物。然而DNA甲基化酶抑制剂存在一些问题:如核苷类似物的细胞毒性、寡核苷酸在体内或细胞内作用的稳定性与长期疗效性等。在培养液中添加这种试剂可以诱发细胞形态变化、细胞中的基因突变、细胞凋亡以及失活X染色体和印记等位基因的重新活化等,这些都影响了甲基化抑制剂的应用。故本实验针对以上问题设计如下试验:1.建立体细胞核移植所需要的供体细胞细胞系,本实验采用荷斯坦奶牛耳组织块贴壁法获得原代细胞,经过传代培养得到纯化的成纤维细胞。通过细胞冷冻保存与复苏、细胞生长曲线的绘制及细胞染色体的核型分析等得到了生长旺盛、染色体变异率低、可以稳定传代的荷斯坦奶牛成纤维细胞系。2.采用DNA甲基化酶（5-aza-dc）抑制剂处理供体成纤维细胞,尝试建立一种既不影响体细胞生物学特性,又可降低供体细胞细胞核的甲基化状态方法,从而提供一种提高体细胞核移植效率的方法。实验中用不同浓度的甲基化酶抑制剂（5-aza-dc）处理奶牛成纤维细胞72 h,分别采用流式细胞仪检测细胞周期方法、常规染色体核型分析方法及琼脂糖凝胶电泳测细胞凋亡法,检测5-aza-dc对细胞周期、染色体及细胞凋亡的影响。结果显示:经甲基化酶抑制剂5-aza-dc（0.005-0.1μmol/L）处理的细胞在G₀/G₁期的百分比与没有经5-aza-dc处理的对照组相比差异不显著（P>0.05）;低浓度的甲基化酶抑制剂（0.005μmol/L和0.01μmol/L）处理的细胞,染色体没有发生明显的畸变（P>0.05）,染色体能够保持正常的形态,高浓度（0.03μmol/L与0.05μmol/L）处理导致染色体畸变率显著增加（P<0.05）,而高浓度0.1μmol/L的5-aza-dc处理后染色体畸形率增加更为显著（P<0.01）;从琼脂糖凝胶电泳图谱上可见经0.005μmol/L、0.01μmol/L浓度组的5-aza-dc处理的细胞与对照组一样都没有出现明显的DNA拖带现象,而从0.03μmol/L组就开始有拖带现象,0.05μmol/L和0.1μmol/L浓度组与对照组相比细胞发生凋亡的比例显著增加。总之,此实验的结果说明在对供体细胞做甲基化处理时用0.005μmol/L和0.01μmol/L的5-aza-dc处理不会影响细胞的染色体结构和数目,并且细胞凋亡率很低,同时不会影响细胞周期,但是高浓度处理则会导致染色体畸变率与细胞凋亡显著增加,理论上不能用于体细胞克隆动物的生产。更多还原

【Abstract】 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.更多还原