【作者】 孙伟；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 在体细胞核移植研究中，受体卵母细胞去核和克隆胚胎的构建是两个影响核移植效率的重要环节。传统的去核方法由于其机械损伤大、耗时、设备和技术要求高、去除的胞质量多、去核效率低，所以严重的影响了核移植的整体效率。最近，化学诱导去核具有成批处理、操作简单、物理损伤小、胞质丢失少等特性，因此适合于大规模的卵母细胞去核和克隆动物的生产；本试验利用Demecolcine（下文简称Deme）进行牛MII卵母细胞诱导去核条件的优化和筛选，然后以输卵管上皮全细胞为核供体材料，将供体全细胞胞质注射和Deme化学诱导去核相结合，初步建立一种简化的牛体细胞核移植方法，探讨在大家畜体细胞核移植中的可行性。最终目的是简化体细胞核移植步骤，提高整体效率。主要试验结果如下：1．脱羧秋水仙素诱导牛MII卵母细胞去核效果研究利用不同浓度Deme和不同处理时间对牛MII卵母细胞进行诱导去核结果表明，在对照组中，不同处理时间对卵母细胞去核效率无显著影响（P＞0.05）；在0.4ug/mL处理浓度下，各处理组去核效率均显著高于0min处理组（P＜0.05）；处理30min组与60min、90min处理组间差异显著（P＜0.05），但后两组之间无显著差异（P＞0.05）；在0.5ug／mL和0.6ug／mL浓度条件下，0min、30min、60min、90min处理组也获得相似的结果，三个处理组的去核效率都显著高于对照组（P＜0.05）；而0.4ug／mL处理组与0.6ug／mL处理组之间差异显著（P＜0.05），而0.5ug／mL处理组与另外两个处理组间都无显著差异（P＞0.05）。低剂量和短时间处理负作用小，所以，0.4ug／mL处理60min组的去核率最理想；采用两种不同的处理方法M₁和M₂法，去核率分别为84.97％和78.93％。虽然前者高于后者，但是两者之间无显著差异（P＞0.05），M₂法的优势是对卵母细胞的损伤小，利用率高：利用0.4ug/mL Deme+0.05MSucrose（蔗糖）和0.4ug／mL Deme+5ug/mL CB（细胞松弛素B）配伍，诱导处理1h后去核，去核率分别为70.63％、61.80％，实验组间差异不显著（P＞0.05），但5ug／mL CB组与对照组之间差异显著（P＜0.05）；经Deme诱导后78个凸起和细胞膜相连的卵母细胞，置于Deme-free培养液后在5个时间点0h、2h、4h、6h和8h，观察凸起回复的卵数分别为0、15、33、52和62（回复率分别为0％、19.23％、42.30％、66.67％和79.49％）。这意味着诱导后的卵母细胞应该在2h内进行机械辅助去核，以防染色质回复。2．供体输卵管上皮细胞分离纯化及其相关特性研究利用实验设计的五步纯化法，对上皮细胞进行纯化，经广谱角蛋白（PCK）免疫组化鉴定（SABC法），上皮细胞纯度高达95％以上；通过对生长特性的观察和生长曲线的测定，细胞长势良好，说明培养条件合适；第4代上皮细胞分别添加了0ng／mL（对照组）、25ng／mL、50ng／mL和75ng/mL的EGF，加入EGF后第5d，对照组S期细胞所占的比例为36.70％，G₂-M期细胞为11.50％，而25ng／mL、50ng／mL和75ng／mL实验组S期细胞分别为30.30％、26.90％和29.40％，G₂-M期细胞所占的比例分别为10.80％、11.90％和10.90％，和对照组相比，各期细胞分布发生明显变化，S期的比例呈下降趋势。从凋亡情况来看，对照组高达3.40％，而50ng／mL组最低，仅为0.86％，这表明50ng／mLEGF有利于上皮的生长。3．化学诱导去核联合全细胞胞质注射进行牛重构胚胎构建，比较其发育情况。Deme诱导后经机械辅助去核，利用全细胞胞质注射共构建重构胚胎40枚，卵裂率为62.50％（25／40），囊胚率10.00％（4／40）；诱导后选择凸起明显的卵母细胞，直接进行全细胞胞质注射，共构建胚胎71枚，卵裂率为52.11％（37／71），囊胚率8.45％（6／71），两组之间卵裂率和囊胚率均差异不显著（P＞0.05）。但是在发育过程中两组均出现不同程度的碎裂，卵裂球大小不均，有小球样结构，发育受阻等现象。上述结果表明，化学诱导去核联合全细胞胞质注射进行牛重构胚胎的构建是可行的，相关的机制还有待进一步的研究。更多还原

【Abstract】 Among the various kinds of factors affecting somatic cell nuclear transfer, theenucleation of recipient oocytes and the reconstruction of cloned embryos were thought tobe important. The traditional enucleation method seriously decreased the whole efficiencyof nuclear transfer as a result of invasive damage, time-consuming, high requirement ofequipment and operating techniques as well as removal of a big amout of cytoplasm.Recently, chemical induced enucleation has been successfully developed for thepreparation of cytoplast and production of clone animals, Because this approach makesless physical damage, cytoplast loss and can be used in batch processing, compared withtraditional ones, it is desirable for large-scale enucleation of recipient cells. The objectiveof this study was to investigate the optimal condition of MII bovine oocytes chemicallyinduced by Demecolcine （abbreviated as Deme）. We established a new simplified methodof bovine somatic cell nuclear transfer through combiding donor whole-cellintracytoplasmic injection with Deme-induced enucleation to explore the possibility ofthis means in big livestock somatic cell nuclear transfer. The ultimate objective was tosimplify the procedure of somatic cell nuclear transfer and increase the whole efficiency.The main results were as follows:1. The effect of Deme induced enucleation of bovine MII oocytesThe bovine MII oocytes treated in Deme of different concentration for different timewere confirmed in this study. The results indicated that the time of treatment had nosignificant effect on the rate of enucleation in the control group（P＞0.05）; In 0.4ug/mLgroup, the enucleation rate of induced MII bovine oocytes treated for 30min, 60min,90min was significantly higher than that of oocytes treated for 0min（P＞0.05）, The rateof enucleation of bovine oocytes treated for 30min differed from that of oocytes treatedfor 60min or 90min（P＜0.05）, while there was no obvious between the groups treated for60min and 90min（P＞0.05）. when the oocytes were treated in 0.5ug/mL or 0.6ug/mL for0min, 30min, 60min and 90min seperately, the results were similar to that of 0.4ug/mlgroup. Based on our experimental results, the optimal induction enucleation conditionwas treated with 0.4ug/mL Deme for 60min; The rate of enucleation methods-M₁ and M₂was 84.97% and 78.93%, respectively, Although no obvious difference（P＞0.05） wasobserved between M₁ and M₂, But M₂ was better than M₁ in concerns with the damageand utilization rate of oocytes; When 0.4ug/mL Deme worked together with 0.05MSucrose or 5ug/mL CB（Cytochalasin）, the IE rates were no significant different betweenCB Group and Sucrous Group（P＞0.05）, but CB Group and control group（0.4ug/mLDeme） were significant different（P＜0.05）; 78 oocytes with cortical protrusions connected to the plasma membran were cultured in Deme-free medium for 8h, Therecovery of cortical protrusions was determined by every 2h from the start point. Theratios of returned oocytes observed at each time point were 0%（0/78）, 19.23%（15/78）,42.30%（33/78）, 66.67%（52/78） and79.49%（62/78）, correspondingly. The results indicatedthat the enucleation should be done within 2h in case retroposition happened.2. Isolation and purification of bovine oviduct epithelial cells and study of its correlatedbiological characteristicsAccording to the designed five-step purification procedure, the epithelial cells werepurified, The purity of epithelial cells identified by PCK immunological histochemistryreached up to 95%. With the help of observation of growth characteristics andmeasurement of growth curve, it indicates cells growth condition are suitable; The fourthpassage bovine oviductal epithelial cells were cultured with different concentrations ofEGF （0ng/mL, 25ng/mL, 50ng/mL and 75ng/mL, respectively） were added into theculture medium. The percentage of cell at S phage at day 5 after addition of EGF was36.70%, and G₂-M phage cell was 11.50% in control group. The percentage of cells at Sphase of groups supplemented with 25ng/mL, 50ng/mL and 75ng/mL were 30.30%,26.90% and 29.40%, and G₂-M phase were 10.80%, 11.90% and 10.90%, respectively.Compared with the control group, the cell cycle distribution changed significantly,especially the percentage of cell at S phage decreased seriously. The rate of apoptosis in50ng/mL EGF group was 0.86%, which was much lower than that of controlledgroup（3.40%）, indicating that 50ng/mL EGF was in flavor of growth of bovine oviductepithelial cells in vitro.3. Reconstruction of cloned embryos using chemically induced enucleation combinedwith whole-cell intracytoplasmic microinjection and study of following development ofreconstructed embryosA total of 40 reconstucted embryos were cloned by whole-cell intracytoplasmicinjection using oocytes which were enucleated by mechanical assisted enucleation afterinduced by Deme. The cleavage rate of cloned embryos was 62.50%（25/40）, and the rateof blastocyst was 10.00%（4/40）; A total of 71 cloned bovine embryos were reconstructedusing oocytes which were induced and formed protrusion, and the rate of the clonedembryos was 52.11%（37/71）, The rate of blastocyst was 8.45%（6/71）. There was nosignificant difference between the rate of cleavage and blastocyst of the twogroups（P＞0.05）. There were some abnormal development phenomena shared by the twogroups: fragment of embryo, asymmetry of cleavage and blastomere globuloid structure e.g. The results indicated that production of bovine reconstructed embryos usingchemically induced enucleation combined with whole-cell intracytoplasmic injection wasfeasible, and the related mechanism needed further research.更多还原