液氦玻璃化冷冻牛未成熟卵母细胞的研究

发布时间：2018-04-26 13:43

本文选题：牛 + 未成熟卵母细胞　；参考：《河南科技大学》2015年硕士论文

【摘要】：哺乳动物卵母细胞的冷冻保存是生命科学领域的重要研究内容之一,近年来,尽管人们对此进行了大量的研究,但卵母细胞冷冻保存的效果仍不理想,尤其是未成熟卵母细胞的冷冻效果更差。影响卵母细胞玻璃化冷冻效果的因素很多,其中降温速率被认为是最重要的影响因素。目前,卵母细胞玻璃化冷冻主要采用液氮(-196℃)为冷源,而液氦的温度可以达到-269℃,本课题拟在液氮OPS玻璃化冷冻的基础上,尝试以温度更低的液氦为冷源,通过加快降温速率来改进牛未成熟卵母细胞的冷冻效果,建立并优化牛未成熟卵母细胞的液氦玻璃化冷冻方法。1.牛未成熟卵母细胞液氮OPS玻璃化冷冻方法的建立试验1:OPS管拉制方法研究。分别以水浴锅、普通酒精灯和自制小酒精灯为热源,探究OPS管的拉制方法。结果表明,水浴锅和普通酒精灯均无法拉制出符合要求的OPS管,只有自制小酒精灯可以拉制出内径和管壁厚度分别为0.8mm和0.07 mm左右的OPS管。试验2:细管法和OPS玻璃化冷冻法的对比。将采集到的COCs随机分为3组,对照组、细管组和OPS组。对照组不经冷冻直接进行IVM、IVF和早期胚胎IVC。两个冷冻组分别用细管法和OPS法进行玻璃化冷冻,解冻后分别进行IVM、IVF和早期胚胎IVC。结果显示:与对照组卵母细胞的形态正常率、成熟率、卵裂率和囊胚率(100%、86.1%、69.7%和38.2%)相比,两冷冻组的各项指标均显著降低(P0.05)。在两个冷冻组中,细管组和OPS组卵母细胞的形态正常率分别为65.2%和77.8%,成熟率分别为31.0%和41.6%,卵裂率分别为19.9%和29.8%,囊胚率分别为0和7.0%。OPS组上述指标均明显高于细管组(P0.05)。结论:用自制小酒精灯更易于拉制出适于玻璃化冷冻的OPS管;液氮OPS法玻璃化冷冻牛未成熟卵母细胞的效果优于细管法。2.牛未成熟卵母细胞液氦OPS玻璃化冷冻方法的探索将采集到的COCs随机分为3组,对照组、液氮组和液氦组。对照组不经冷冻直接进行IVM、IVF和早期胚胎IVC。两个冷冻组分别以液氮和液氦为冷源,以OPS管为载体对牛未成熟卵母细胞进行玻璃化冷冻,解冻后分别进行IVM、IVF和早期胚胎IVC。结果显示:与对照组卵母细胞的形态正常率、成熟率、卵裂率和囊胚率(100%、87.0%、69.5%和42.0%)相比,两冷冻组的各项指标均显著降低(P0.05)。在两个冷冻组中,液氦组解冻后卵母细胞的形态正常率(84.7%)显著高于液氮组(78.5%;P0.05)。经过体外成熟后,液氦组卵母细胞的成熟率(43.9%)显著高于液氮组(39.2%;P0.05)。经过体外受精和早期胚胎体外培养后,液氦组的卵裂率和囊胚率(35.4%和6.4%)显著高于液氮组(30.0%和4.1%;P0.05)。结论:液氦OPS玻璃化冷冻法的效果优于液氮法;液氦作为冷源冷冻保存牛未成熟卵母细胞是可行的。3.液氦OPS玻璃化冷冻方法的优化试验试验1:最佳冷冻保护液的筛选。将采集到的COCs随机分为6组,其中一组不经冷冻,作为对照组,其余五组分别使用五种浓度的冷冻保护液(EDS30、EDS35、EDS40、EDS45和EDS50)进行液氦OPS法玻璃化冷冻,解冻后分别进行IVM、IVF和早期胚胎IVC。结果显示:与对照卵母细胞的形态正常率、成熟率、卵裂率和囊胚率(100%、84.5%、69.9%和41.1%)相比,所有冷冻组的各项指标均显著降低(P0.05)。在五个冷冻组中,EDS35组卵母细胞(90.3%、51.9%、41.8%和10.0%)冷冻-解冻后的冷冻效果均显著高于其他四组(P0.05)。EDS50组(75.1%、36.0%、27.2%和2.6%)的冷冻效果最差,显著低于其他四组(P0.05)。EDS30(83.0%、42.0%、33.8%和6.5%)、EDS40(84.9%、43.9%、34.9%和6.4%)、EDS45(82.6%、42.4%、33.2%和5.1%)组之间的差异均不显著(P㧐0.05)。试验2:液氦玻璃化冷冻中两种操作温度的对比。将采集到的COCs随机分为三组,对照组、39℃组和25℃组。对照组不经冷冻直接进行IVM、IVF和早期胚胎IVC。两个液氦OPS法玻璃化冷冻组除操作温度不同外,其他操作均相同,解冻后分别进行IVM、IVF和早期胚胎IVC。结果表明:与对照组卵母细胞的形态正常率、成熟率、卵裂率和囊胚率(100%、84.6%、71.5%和40.6%)相比,两个冷冻组的各项指标均显著降低(P0.05)。两个冷冻组中,39℃组和25℃组中的形态正常率分别为89.2%和85.8%,差异不显著(P㧐0.05)。39℃组卵母细胞的成熟率、卵裂率、囊胚率分别是51.4%、41.2%和10.0%,均显著高于25℃组(45.7%、34.1%和6.3%;P0.05)。试验3:最佳预处理时间的筛选。将采集到的COCs随机分为四组,对照组、1 min预处理组、3 min预处理组和5 min预处理组。对照组不经冷冻直接进行IVM、IVF和早期胚胎IVC。三个液氦OPS法玻璃化冷冻组除在冷冻平衡液(VS1)中的预处理时间不同外,其他操作均相同。解冻后分别进行IVM、IVF和早期胚胎IVC,结果显示:与对照组卵母细胞的形态正常率、成熟率、卵裂率和囊胚率(100%、84.0%、70.6%和40.1%)相比,三个冷冻组的各项指标均显著降低(P0.05)。三个冷冻组中,1 min组、3 min组和5 min组解冻后卵母细胞的形态正常率分别为87.7%、87.2%和86.4%,彼此之间差异不显著(P㧐0.05)。1min组解冻后卵母细胞的成熟率、卵裂率和囊胚率(49.6%、39.6%和10.4%)与3min组(47.4%、37.0%和8.8%)差异不显著(P㧐0.05),但却显著高于5min组(41.0%、32.0%、6.1%;P0.05)。结论:液氦OPS玻璃化冷冻法的最佳冷冻保护液是EDS35,最佳操作温度为39℃,最佳预处理时间为1min。
[Abstract]:The cryopreservation of mammalian oocytes is one of the important research contents in the field of life science. In recent years, although a lot of research has been done on this, the effect of cryopreservation of oocytes is still not ideal, especially the freezing effect of immature oocytes is worse. There are many factors affecting the vitrification effect of oocyte. The cooling rate is considered to be the most important factor. At present, the vitrification of oocyte mainly uses liquid nitrogen (-196 C) as the cold source, and the temperature of liquid helium can reach -269 C. On the basis of the cryopreservation of liquid nitrogen OPS vitrification, we try to use the liquid helium at a lower temperature as the cold source. The freezing effect of the mature oocyte, the establishment and optimization of the liquid helium vitrification of the immature oocytes of cattle.1. the establishment of the liquid nitrogen OPS vitrification method for the immature oocytes of cattle. A study on the method of drawing the 1:OPS tube. The method of drawing the OPS tube was investigated with water bath, ordinary alcohol lamp and self-made small alcohol lamp as the heat source. It shows that the water bath pot and the ordinary alcohol lamp can not make the OPS tubes that meet the requirements. Only the self-made small alcohol lamp can pull out the OPS tubes with the inner diameter and the thickness of the tube wall about 0.8mm and 0.07 mm respectively. The comparison of the 2: tube method and the OPS vitrification method. The collected COCs is divided into 3 groups, the control group, the tubule group and the OPS group. Two cryosurgery groups of IVM, IVF and early embryo IVC. were vitrified without freezing. The results of IVM, IVF and early embryo IVC. after thawing respectively showed that compared with the normal rate, maturation rate, cleavage rate and embryo rate (100%, 86.1%, 69.7% and 38.2%) of the control group, two freezing groups were compared. The morphological normal rates of the oocytes in the two frozen groups were 65.2% and 77.8%, the maturation rates were 31% and 41.6%, the cleavage rates were 19.9% and 29.8%, respectively, and the blastocyst rates were 0 and 7.0%.OPS respectively higher than those in the fine tube group (P0.05). Conclusion: the homemade small alcohol lamp was more than that of the small tube group (P0.05). It is easy to draw the OPS tube suitable for vitrification, and the effect of liquid nitrogen OPS method for vitrification of immature oocytes is better than that of liquid helium OPS vitrification of immature oocytes of.2. cattle. The collected COCs is randomly divided into 3 groups, the control group, the liquid nitrogen group and the liquid helium group. The control group is directly IVM, IVF and the control group without freezing. The early embryo IVC. two cryosurgery groups used liquid nitrogen and liquid helium as cold sources respectively, and OPS tube was used as the carrier to freeze the immature oocytes of cattle. After thawing, IVM, IVF and early embryo IVC. showed that compared with the control group, the normal rate, maturation rate, cleavage rate and blastocyst rate (100%, 87%, 69.5% and 42%) were compared with that of the control group. The indexes of the two freezing group were significantly decreased (P0.05). In the two frozen groups, the normal rate of oocyte in the liquid helium group (84.7%) was significantly higher than that in the liquid nitrogen group (78.5%; P0.05). The maturation rate of the oocyte in the liquid helium group (43.9%) was significantly higher than that in the liquid nitrogen group (39.2%; P0.05) after in vitro maturation. The cleavage rate and blastocyst rate (35.4% and 6.4%) in the liquid helium group (35.4% and 6.4%) were significantly higher than that in the liquid nitrogen group (30% and 4.1%; 4.1%; P0.05). Conclusion: the effect of liquid helium OPS vitrification is superior to the liquid nitrogen method; the cryopreservation of the immature oocyte by liquid helium as a cold source is a feasible optimization test of the.3. liquid helium cryopreservation method of liquid helium, the optimum freezing protection of 1: COCs randomly divided into 6 groups, one group without freezing, as the control group, the other five groups using five concentrations of cryopreservation solution (EDS30, EDS35, EDS40, EDS45 and EDS50) for liquid helium OPS method of vitrification, after thawing IVM, IVF and early embryo IVC. results showed: and the shape of the control oocyte Compared with the normal rate of state, maturity, cleavage rate and blastocyst rate (100%, 84.5%, 69.9% and 41.1%), all the indexes of all cryopreservation groups were significantly decreased (P0.05). In the five freezing groups, the freezing and thawing of group EDS35 oocytes (90.3%, 51.9%, 41.8% and 10%) were significantly higher than those of the other four groups (P0.05).EDS50 group (75.1%, 36%, 27.2% and 2.6%). The freezing effect is the worst, significantly lower than the other four groups (P0.05).EDS30 (83%, 42%, 33.8% and 6.5%), EDS40 (84.9%, 43.9%, 34.9% and 6.4%), EDS45 (82.6%, 42.4%, 33.2% and 5.1%) groups are not significant differences (P?). Test 2: liquid helium vitrification cold freezing operation temperature comparison. The collected COCs randomly divided into the group, the control group, the control group, The control group was not frozen directly for IVM, IVF and early embryo IVC. two liquid helium OPS vitrification group, except the operating temperature, the other operations were the same. After thawing IVM, IVF and early embryo IVC., respectively, showed that the normal rate, maturation rate, cleavage rate and blastocyst rate of the control group were 100%. (100% Compared with 84.6%, 71.5% and 40.6%, the indexes of two cryopreservation groups were significantly decreased (P0.05). In the two freezing groups, the normal rate in the 39 and 25 C groups was 89.2% and 85.8%, and the difference was not significant (P? 0.05), the maturation rate, cleavage rate and blastocyst rate were 51.4%, 41.2% and 10%, respectively. 4.1% and 6.3%; P0.05) screening the best pretreatment time for 3:. The collected COCs was randomly divided into four groups, the control group, the 1 min pretreatment group, the 3 min preconditioning group and the 5 min preconditioning group. The control group did not freeze the IVM, IVF and early embryos IVC. three liquid helium OPS method in the cryopreservation group (VS1). The other operations were the same. After thawing, IVM, IVF and early embryo IVC were carried out respectively. The results showed that compared with the normal rate, maturity, cleavage rate and blastocyst rate (100%, 84%, 70.6% and 40.1%) of the control group, the indexes of the three freezing groups were significantly reduced (P0.05). Three freezing groups, 1 min, 3 min and 5. The normal morphologic rates of oocyte in group min were 87.7%, 87.2% and 86.4%, respectively, and there was no significant difference between each other (P? 0.05).1min group after thawing, the maturation rate of oocytes after thawing, cleavage rate and blastocyst rate (49.6%, 39.6% and 10.4%) and 3min group (47.4%, 37% and 8.8%) were not significant (P? 0.05), but significantly higher than those of group 5min (41%, 32%, 6.1%; P0.05). Conclusion: the best cryopreservation solution for liquid helium OPS vitrification is EDS35, the best operating temperature is 39 C, and the best pretreatment time is 1min..

【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

【共引文献】