【作者】 刘斌；

【导师】 薛立群； 刘扬；

【作者基本信息】 湖南农业大学 ， 基础兽医学， 2002， 硕士

【摘要】 动物体内活性自由基参与很多生理和病理过程，正常情况下，体内自由基的生成和清除是处于动态平衡的。本研究利用经超数排卵处理后从母鼠生殖道中获得的未受精卵及早期胚胎，用Griess试剂法检测其产生的NO浓度，用自旋捕捉ESR法检测其活性氧自由基生成的情况，以及用氧电极法检测其消耗氧气的情况，主要结果如下： 1．胚胎发育过程中NO的生成 胚胎在Hanks液中培养4小时后，取培养液加入等体积的Griess试剂，用分光光度法检测培养液中亚硝酸盐的浓度，依此得知NO的生成。发育第1天胚胎与未受精卵的NO生成差异不显著(P＞0.05)，而发育第2、3、4天胚胎的NO生成显著上升(P＜0.01)，第3天胚胎的NO生成最高，并于第4天下降(P＜0.05)。这表明N0参与了小鼠的早期胚胎发育。补充维生素E后，未受精卵及第2天胚胎NO的生成显著下降(P＜0.01)，而第1、3、4天胚胎的NO生成变化不显著(P＞0.05)。 2．胚胎发育过程中羟基自由基的生成 用DEPMPO捕捉活性氧自由基，其加合物的信号强度比用DMPO强，而且加合物寿命更长。因此，本研究选择DEPMPO作为捕捉剂，以捕捉胚胎发育过程中产生的氧自由基。首次用DEPMPO在小鼠早期胚胎牛捕捉到了羟基自由基。未受精卵的羟基自由基的水平很低，而胚胎的羟基自由基水平均高于未受精卵。随着胚胎的发育，其水平开始有上升的趋势，8－细胞期胚胎的羟基自由基的水平最高，其后又有下降的趋势。 3．胚胎发育过程中氧消耗的测定 用氧电极法测定胚胎培养液中氧气的浓度。未受精卵耗氧极低，而胚胎培养液中氧的浓度直线下降(P＜0.0001)，而且随着胚胎的发育耗氧率有上升的趋势。发育过程中羟基自由基生成与耗氧率之间有一定的线性相关性(P＜0.25)。 4．胚胎发育过程中NO与羟基自由基之间的相关性 胚胎发育过程NO与羟基自由基生成具有线性相关性(P＜0.025)。 总之，在本实验条件下，小鼠早期胚胎耗氧增加，胚胎发育过程中有NO和羟基自由基的生成，NO与羟基自由基具有线性相关性，胚胎耗氧率与羟基自由基也有一定的线性相关性。更多还原

【Abstract】 Reactive free radicals are involved in many physiological and pathophysiological processes. Under normal conditions, production and clearance of reactive free radicals are in dynamic equilibrium. In this study, unfertilized oocytes and early embryos were obtained by flushing reproductive tracts of superovulated mice; NO concentrations in the conditioned culture medium from mouse embryos were determined indirectly by measurement of nitrites using the method of Griess reagent; reactive oxygen species were assayed by spin-trapping ESR; oxygen exhausting was determined by detecting oxygen concentration in the culture medium with oxygen meter. The results were as follows:1. NO production by mouse embryos Embryos were cultured in Hanks balanced salt solution for 4 hours. Then the culture medium was collected, and equal amount of Griess reagent was added into it. NO concentrations were determined indirectly by spectrophotometry. NO production was not significantly different between unfertilized oocytes and day 1 embryos (P>0.05). However, NO production by day 2, 3, 4 embryos remarkably increased (P<0. 01) and reached the highest level on day 3. Then, it began decreasing (P<0. 05). It seemed that NO was involved in early mouse embryo development. After Vitamin E treatment, NO production by unfertilized oocytes and day 2 embryos decreased significantly (P<0.01) while there was no significant Variation of NO production by day 1, 3, 4 embryos.2. Hydroxyl radical production by mouse embryos DEPMPO is more suitable for studying oxyradicals than DMPO because of its higher signal intensity and longer life of adducts. Therefore, DEPMPO was adopted to trap oxyradicals in our study. Hydroxyl radical was trapped by DEPMPO in embryos for the first time. Hydroxyl radical concentration of unfertilized oocytes was very low,while those of embryos were much higher. With the development of embryos, hydroxyl radical concentration trended to increase and reached its peak in 8-cell embryos. Then, it trended to decrease.3. Oxygen exhausting of mouse embryos There was very low oxygen exhausting in unfertilized oocytes. Meanwhile, oxygen concentration of culture medium containing embryos decreased with time (P<0. 0001). With the development of embryos, oxygen exhausting rate trended to increase. There was certain linear correlation between hydroxyl production and oxygen exhausting (P<0.25).4. Correlation between NO and hydroxyl radical in mouse embryos There was linear correlation between NO production and hydroxyl radical production(P<0.025).In summary, under the present condition, oxygen exhausting rate of mouse embryos is much higher than that of unfertilized oocytes; NO and hydroxyl radical are produced by early embryos; there is linear correlation between NO and hydroxyl radical production; there is certain linear correlation between oxygen exhausting and hydroxyl radicals.更多还原