【作者】 崔奎青；

【导师】 石德顺；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2002， 硕士

【摘要】 1．系统探讨了兔卵母细胞孤雌激活的方法。兔卵母细胞经7％的乙醇单独激活处理5min后的分裂率为13.0％，显著低于乙醇激活处理后再在2mmol／LDMAP培养3h的卵母细胞(48.1％)和5μmol／L离子霉素激活处理后在DMAP培养3h的卵母细胞(92.2％)；离子霉素+DMAP处理组的囊胚发育率(27.1％)亦显著高于乙醇+DMAP处理组(15.4％，P＜0.05)和乙醇单独处理组(0％)。当用离子霉素和DMAP激活处理时，注射hCG后18h的卵母细胞的分裂率(92.1％)和囊胚发育率(35.3％)最高，注射hCG后24h的卵母细胞，其分裂率(19.0％)和囊胚发育率(4.2％)均显著下降(P＜0.05)。卵母细胞经离子霉素激活处理后，在DMAP中培养1h，3h和5h的分裂率分别为86.1％，88.6％和72.6％，差异显著(P＜0.05)；囊胚发育率分别为28.3％，34.2％和26.4％，差异不显著(P＞0.05)。离子霉素和DMAP处理后再电激活1次对卵母细胞的分裂率(83.3％vs80.0％)和囊胚率(30.0％vs27.0％)无明显影响(P＞0.05)；卵母细胞激活处理前在细胞松弛素B(5mg／L)中培养20min亦对其分裂率(86.0％vs83.3％)和囊胚发育率(36.7％vs33.3％)无显著影响(P＞0.05)。 2．观察了兔卵母细胞的纺锤体在不同条件下的变化，并对不同去核方法的去核效率进行了比较。结果表明，应用Piezo-driven操作系统去核，能有效减少去核对卵母细胞的机械挤压损伤，使卵母细胞去核后存活率达98％。卵母细胞中极体与核的相对距离随着卵龄的增加而加大，注射hCG后16h的卵母细胞只有70.4％的核位于极体附近，而到19h时，这一比例下降到60.5％。用Spindle View系统能有效提高去核效率，使去核率从盲吸法的83.8％提高到98.4％，且发现在注射hCG后19h左右去核较为合适。纺锤体对环境变化敏感，适宜的温度为36～37℃，温度不适容易使纺锤体解聚。 3．系统研究了兔胚胎细胞核移植的有关影响因素。当电场强度为100v／mm，15μs时，电脉冲3次和4次的融合率(81.7％和85.5％)显著高于电脉冲2次(64.1％，P＜0.05)，虽然融合后的分裂率(77.4％，84.9％和80.0％)差异不显著(P＞0.05)。当脉冲次数为3，脉冲时间为15μs时，电压为150v／mm和200v／mm的重组胚分裂率(56.5％和57.6％)显著低于电压为100v／mm的重组胚(76.9％，P＜0.05)，虽然融合率差异不显著(P＞0.05)。融合前激活受体卵母细胞，虽然其融合率(79.8％)显著低于融合后激活的卵母细胞(95.2％，P＜0.05)，但其分裂率(84.0％)和囊胚发育率 兔细胞核移植及其相关技术的研究 （33．6％）则显著高于融合后激活的卵母细胞（62．0％和 21．0％，P＜0．05）。当用 8—16 细胞胚胎的卵裂球为核供体时，其分裂率门．9％）和囊胚发育率（24．0％）均显I＿． 著高于致密桑枢胚的卵裂球（66．7％和9．8％，P＜几05），虽然融合率无显著差异 （76．3％VS 75刀％，P＞0．05）。当重组胚先在含 3％OCS的 TCM199中培养 48h，然 后在含10％FCS的TCM199中继续培养，其囊胚发育率（33石％）显著高于一直 在3％0＊s中培养（8．0呢）和在10％＊＊s中培养（％．8阮）的重组胚（P＜O．05）。 以上研究结果表明：（1）Spindle iew显微镜结合Piezo－diven操作系统可提高兔 卵母细胞去核率和去核后成活率；（2）兔卵母细胞的最佳激活方法是先用离于霉素激 活smin，然后在DMAP中培养3h，应用此法激活融合前受体卵母细胞可提高其核移 植胚胎的发育率；①融合参数对兔胚胎细胞核移植的效果有显著影响，适宜的融合 参数是100v／mm，15呷，电脉冲3次；O）供体胚胎的发育阶段对核移植效果有显著 影响，应选择致密前的胚胎卵裂球作为核移植供体；（5）核移植胚胎的不同发育阶段 对血清种类和浓度具有不同的要求，早期采用低浓度血清，后期采用高浓度血清可提 高胚胎发育率。更多还原

【Abstract】 1. Parthenogenetic activation of rabbit oocytes was investigated in this study. The cleavage rate of oocytes activated in 7% ethanol for 5 min (13.0%) were significantly lower than that of oocytes activated in 7% ethanol (48.1%) or 5 \unao\JL ionomycin (92.2%) for 5 min following in 5mmol/L DMAP for 3 h. The percentage of cleaved oocytes developing to blastocysts (27.1%)was also significantly higher (P<0.05) when oocytes actvated in ionomycin and DMAP, in comparison with oocytes activated in ethanol alone (0%) or ethanol plus DMAP (15.4%). When oocytes were activated using ionomycin and DMAP at 18 h after hCG injection, 92.1% of oocytes cleaved and 35.3% of cleaved oocytes developed to blastocysts, which were significantly higher than that of oocytes activated beyond 24 h post-hCG injection (19.0% and 4.2% resp, p<0.05). Three hours was proved to be appropriate for oocytes culturing in DMAP after ionomycin treatment, in which 88.6% of oocytes cleaved and 34.2% of cleaved oocytes developed to blastocysts, which was higher than that of oocytes culturing in DMAP for 1 h (86.1% and 28.3% resp) or 5 h (72.6% and 26.4% resp). Additional electronic pulse to oocytes activated using ionomycin and DMAP did not affect their cleavage rate (83.3% vs 80.0%) and blastocyst development (30.0% vs 27.0%). Exposure of oocytes to cytochalasin B (5mg/L) for 20 min prior to ionomycin and DMAP treatment also did not influence their subsequent cleavage rate (86.0% vs 83.3%) and blastocysts developed (36.7% vs 33.3%).2. The change of rabbit oocyte spindles in different condition was measured, and different methods for enucleation of oocytes were also compared in this study. Results indicate that the Piezo-driven system can reduce the mechanical damage to oocytes, and then improve the survival rate of oocytes after enucleation up to 98.0%. The space between nuclear and the first polar body increase as the oocyte aged, the proportion of oocytes with the first polar body near to spindle deceased from 70.4% to 60.5% when the age of oocytes increased from 16h to 19h post-hCG injection; The spindle view system can significantly increase the enucleation efficiency (98.4%), compared with the blind enucleation method (83.8%, P<0.05). The optimum age for enucleation was proved to be 19h post-hCG injection, and the optimum temperature of manipulation was proved to be36~37癈.3. Factors affecting the efficiency of nuclear transfer in rabbits were examined in the present study. When 100 v/mm of pulse intensity and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells (81.7% and 85.5% resp) compared with 2 electronic pulses (64.1%, P<0.05),42but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups (77.4%, 84.9% and 80.0% resp, P>0.05). When the duration and number of electronic pulse were fixed at ISjis and 3, increase of pulse intensity from 100 v/mm to 150 v/mm and 200 v/mm resulted in a significantly decrease in the cleavage rate of reconstructed embryos (76.9% vs 56.5% and 57.6% resp, P<0.05), although the fusion rate did not significantly differ among the three groups (79.4%, 82.1% and 86.8% resp, P>0.05). Significantly more reconstructed embryos cleaved (79.8%) and developed to blastocysts (24.0%) when they were derived from donor embryos at the 8-16 cell stage, in comparison with the reconstructed embryos derive from donor embryos at the compact morula stage (66.7% and 9.8% resp, P<0.05), although the fusion rate was similar (76.3% vs 75.0, P>0.05). Activation of cytoplasts prior to fusion increased the cleavage rate (84.0% vs 62.0%, p<0.05) and blastocyst development (33.6% vs 21.0%, P<0.05) of reconstructed embryos, but decreased the fusion rate (9.8% vs 95.2%, P<0.05), compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts (33.6%) when they were cultured in TCM-|-3%OCS at the first 48h and then cultured in TCM199 + 10%FCS, in comparison with the recon更多还原