【作者】 杜卫华；

【导师】 李宁；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 传统核移植总效率低，且需要熟练的显微操作技术，所需设备昂贵，限制了核移植的广泛应用；因此作为替代，化学去核应运而生。本研究以小鼠为研究对象，探索了第一次减数分裂期卵母细胞的化学去核方法，并以化学去核卵母细胞为核受体进行体细胞核移植；并对去核卵母细胞质中微管组织中心组装能力的恢复和重构胚染色质的凝集与纺锤体的组装进行了分析。 本实验通过免疫荧光染色法确定了小鼠卵母细胞第一次减数分裂的进程，在此基础上分别用脱羧秋水仙碱和放线菌酮处理成熟培养的小鼠卵母细胞，诱导其核物质随极体全部排出。结果表明：体外成熟培养5h（即卵母细胞处于第一次减数分裂前中期），其去核率最高，达85.6％；成熟5h之前各组随成熟培养时间的延长而去核率升高，之后各组则降低；自然裸卵去核率显著低于前者（56％），因此该方法在卵母细胞-卵五细胞复合体的完全去核率与传统的物理去核方法相当，从而达到提高小鼠核移植总效率的目标；同时也为大动物的化学去核研究提供实验基础。 哺乳动物早期胚胎的发育阻滞是胚胎体外培养的普遍现象。针对BALB／C×ICR F₁胚胎的体外发育，研究表明M16能有效克服其体外培养的2—细胞阻滞，使96.2％的1—细胞胚胎发育为4—细胞。另外，本试验通过在M16基础上添加EAA、non-EAA和葡萄糖显著提高了胚胎的桑椹率、囊胚率和孵化率；单独添加葡萄糖的培养液，其桑椹率显著升高，但囊胚率和孵化率的升高并不显著，所以胚胎培养液中氨基酸和葡萄糖能协同促进胚胎发育。试验获得2—细胞胚胎和桑囊胚经过受体移植后均出生仔鼠，表明该胚胎体外培养系统的可行性。 本研究旨在探索一种崭新的、化学试剂诱导去核卵母细胞为核受体的、无透明带的、手工体细胞核移植方法。第一次减数分裂期小鼠卵母细胞经化学去核后，去除透明带，并胎儿成纤维细胞粘合、电融合并SrCl2激活，培养于微孔中。目前此无透明带体细胞核移植胚胎已发育到4—细胞；2—细胞克隆胚胎经输卵管移植到假孕受体后，没有获得怀孕受体。试验还比较了血清饥饿胎儿成纤维细胞、新鲜细胞和冷冻保存细胞对体细胞核移植的影响，发现除融合率外，3组间差异不显著。该方法完全丢弃了传统核移植的显微操作及其繁琐程序，是纯粹的手工克隆（HMC），它的成功将会大大简化核移植程序，提高核移植效率，加快其产业化的步伐。当然目前该方法体系还不成熟，有待于进一步研究和探索。 本试验从化学去核处理卵母细胞移入培养液开始，通过免疫荧光染色对不同时间段细胞中胞质星体的恢复情况进行了跟踪检测。发现，培养0h的去核卵母细胞均没有标记到MTOCs，即在刚刚完成去核的卵母细胞，脱羰秋水仙碱抑制了卵母细胞中微管蛋白单体的聚合作用，无可见MTOCs组装；微管蛋白以单体形式存在，造成免疫荧光标记时胞质深染。培养5h后，53％去核卵母细胞可见标记的MTOCs，但数量明显少于对照组，而又多于培养0 h的去核细胞，说明此时53％的化学去核卵胞质中确有MTOCs的存在，其微管聚合能力正在恢复；MTOCs标记，但其辐射产生的微管纤维短，结构还不够丰满；其余细胞的微管蛋白则丧失重新装配能力。经10h的培养后，42％去核卵母细胞标记出MTOCs，但微管纤丝较短；与培养5h的细胞相比，数中国农业大学博士论文中文摘要量增加，MTOCs明显，因而其中的微管聚合能力又进一步提高。继续加长培养时间的试验表明，M1’0Cs的恢复类似于培养10h，微管组装能力己经最大幅度恢复。将培养10h的化学去核卵母细胞与供体成纤维细胞融合后lh、2h、3h，分别有77.6%、70.6%、58.9%重构胚的染色质发生凝集，其余胚胎的染色体则处于原核期;而只在融合后3h，27.9%重构胚被标记出组装的纺锤体，且其中的同源染色体己经分离。该结果与重构胚的体外发育相符，表明正是这些己恢复微组装功能的化学去核卵母细胞才对其供体核具有重编程的支持能力。更多还原

【Abstract】 Traditionally, Somatic nuclear transfer in mammal is completed by micromanipulation. But micromanipulation is evidently damaging to cytoplast cytoskelton, causing the lost of some important factors involved in programming of donor karyoplast, reducing the volume of cytoplasm. Alternatively, induced enucleation is carried out with agents disturbing the meiosis or inhibiting protein synthesis of oocyte during polar body emites. Consequently all chromatin are extruded as the form of polar body. Here, we used mouse oocytes enucleated by chemical induction as recipients in mouse somatic cloning. Recovery of microtubule organizing centers in enucleated oocytes and condensation of chromosome and organization of spindle in reconstructed embryos were analysed.A non-invasive method of enucleation mouse oocytes has been developed. Firstly, the progression of mouse oocytes in meitosis I was ascertained by immunofluoresence technique. Consequently Cumulus-oocytes complexes were in the stage of germinal vesicle when isolated from the antral follicles. Within 2h isolation from the follicle 100% of oocytes underwent germinal vesicle breakdown and both the nucleolus and the nuclear envelope disappeared. After culturing for 6 h, prometaphase occurred in 90% oocytes. By 8 h of in vitro maturation 33.3% oocytes were at the stage of MI. Anaphase I is most apparent at approximate 12h following isolation with number of 71.4%. At the same time, telophase I occurred in 28.6% of oocytes. After 15h of maturation. 98% of oocytes released the first polar body and thus the first meiosis of mouse oocytes ended. Secondly, Strong chromosome to chromosome binding was induced by culturing pro-MI oocytes in demecolcine supplemented medium. Subsequent expulsion of entirely chromosome complex during polar body extrusion was facilitated by exposing the demecolcine treated oocytes to a combination of cytoheximide and demecolcine. This simple two-step chemical enucleation procedure yields fully enucleated mouse oocytes in 85.6% of cumulus-oocytes complexes and 56% of natural oocytes, significantly different (p < 0.05). Since the enucleation rate of cumulus-oodles complexes is equivalent to that by micromanipulation, induced enucleation will greatly improve the whole efficiency in mouse somatic nulear transfer.The preimplantation embryos of several mammalian species undergo blocks in development in vitro. The stage when the block occurs has been correlated to the time the embryonic genome takes over control of development from the maternal genome, which occurs in mouse at the 2-cell stage. The embryos of BALB/CxICR Fl mouse show 2-cell block in vitro and the effects of several culture media on overcoming the 2-cell block in embryos development were studied. M16(Sigma) , in which 96.2% of 1-cell embryos developed to 4-cell stage, were shown to be effective in overcoming the 2-cell block. The modified M16 added with essential amino acid (1%), non-essential amino acid (1%) and glucose significantly improved the developmental rate such as morula rate> blastocyst rate and inhatching rate after the 4-cell stage embryos were removed into the medium. We conclude that the amino acid and glucose may promote the lately development of mouse embryos. Birth of mouse offspring after transferring of embryos in vitro cultured further demonstrated the availability of theculture system of mouse embryos.Since the success of Dolly, the first cloned sheep with the adult somatic cells as karyoplast donor, new approaches have been developed for nuclear transfer technology. Here we describe a handmade cloning method which combines the chemical induced enuleation and zona-free technology in embryo culture. Enuleated oocytes were derived by exposing the oocytes to demecolcine and cytoheximide supplemented mdium sequently and its chromosome was depleted to the first polar body. Then the zona and polar body of oocytes treated with drug were removed by transferring into the M2 containing 0.5% protease. The mouse fetal fibroblast cells were glued to the oocytes memb更多还原