紫草素对卵母细胞成熟及早期胚胎发育的影响及机制研究

发布时间：2018-04-30 14:00

本文选题：紫草素 + 卵母细胞　；参考：《北京中医药大学》2017年硕士论文

【摘要】：研究目的和意义:研究紫草素对体外培养的小鼠卵母细胞、卵丘卵母复合体成熟以及早期胚胎发育的影响,初步探讨紫草素对卵母细胞成熟和早期胚胎发育的作用机制。研究方法:1.取6-8周的雌性ICR小鼠的卵母细胞,用IBMX抑制剂抑制含有不同浓度(0μg/ml、5μg/ml、10μg/ml、20μg/ml、50μg/ml)紫草素的培养基培养卵母细胞12h后,再洗脱抑制剂释放卵母细胞,进行培养,分别在2h和12h统计各组卵母细胞的GVBD率和第一极体排出率。2.分别以各种不同浓度的紫草素溶液干预小鼠卵母细胞后直接进行体外培养,分别在2h和12h统计各组卵母细胞的GVBD率和第一极体排出率。3.采用激光共聚焦显微技术观察1Oμg/ml紫草素对卵母细胞纺锤体组装和形态、线粒体含量和分布的影响。4、采用Western Blot实验观察在0μg/ml和10μg/ml两种浓度紫草素作用下小鼠卵母细胞GV期、GVBD期、MⅠ期、MⅡ期PKM2蛋白的不同变化;并采用免疫荧光技术检测小鼠卵母细胞GV期、GVBD期、MⅠ期、MⅡ期PKM2蛋白的含量和分布。5、统计0μg/ml和10μg/ml紫草素分别作用于未成熟卵丘卵母细胞复合体(GV-COCs)后,2h和12h各组卵母细胞的GVBD率和第一极体排出率。6、检测0μg/ml和1Oμg/ml紫草素分别作用于小鼠原核细胞后发育为2细胞(注射hCG 36h)、4细胞(注射hCG48h)和8细胞(注射hCG 60h)的细胞比率。结果:(1)先用IBMX抑制剂处理,再洗脱IBMX抑制剂,然后放入含有不同浓度紫草素的培养基中进行培养,结果为:10μg/ml和5μg/ml紫草素组分别与0μg/ml组比较,GVBD率均没有明显变化,差异无统计学意义(p0.05)。5μg/ml紫草素组卵母细胞第一极体排出率与Oμg/ml组相比,差异无统计学意义。然而10μg/ml紫草素组卵母细胞第一极体排出率(13.17± 3.16)%与0μg/ml组(85±5.00)%相比,差异具有统计学意义(P0.05)。(2)直接用不同浓度紫草素干预的各组实验结果为:5μg/ml、10μg/ml紫草素组卵母细胞的GVBD率与Oμg/ml组相比,差异无统计学意义(P0.05)。5μg/ml紫草素组的卵母细胞第一极体排出率与0μg/ml组相比,差异无统计学意义(P0.05),然而10μg/ml紫草素组的卵母细胞第一极体排出率均值为(59.05±1.65)%与Oμg/ml组的均值(86.67±2.89)%相比,差异具有统计学意义(P0.05)。(3)采用激光共聚焦显微技术观察免疫荧光结果显示,10μg/ml紫草素能明显抑制小鼠卵母细胞体外成熟发育,纺锤体形态异常率高达81%(98/121);而0g/ml组纺锤体形态异常率仅为19%(20/103),两组比较,差异具有统计学意义(p0.05)。可见10μg/ml紫草素能明显抑制小鼠卵母细胞体外成熟发育。(4)线粒体染色结果显示:在0μg/ml组中,小鼠卵母细胞中的线粒体呈均匀分布并且线粒体信号簇颜色均一,说明卵母细胞处于第二次减数分裂中期(metaphase Ⅱ,MⅡ期),而用10μg/ml紫草素处理过的卵母细胞所呈现的线粒体分布特点则为周边分布或半周边分布,并且出现了线粒体信号簇聚集现象。说明紫草素影响了卵母细胞的线粒体形态和分布,进而影响了卵母细胞的成熟。10μg/ml浓度的紫草素能够使卵母细胞发育停滞在MⅠ前期或MⅠ期。(5)免疫荧光结果显示:PKM2在0μg/ml组的卵母细胞减数分裂成熟过程各个时期都基本上是均匀分布在胞浆中。在10μg/ml紫草素组,虽然PKM2在各个不同时期的卵母细胞中也基本均匀分布在胞浆中。但是在同一实验条件下,PKM2在MⅠ期和MⅡ期卵母细胞中的含量要低于GV期和GVBD期。(6)GV-COCs体外培养结果显示:10μg/ml紫草素组卵丘卵母复合体的GVBD率与μg/ml组相比,差异无统计学意义(p0.05);同样,10μg/ml紫草素组均第一极体排出率与0μg/ml组比较,差异也无统计学意义(p0.05)。因此,10μg/ml浓度的紫草素对COC体外成熟无明显影响。(7)对早期胚胎发育的影响结果显示:10μg/m紫草素干预组发育到2细胞阶段的胚胎比率与0μg/ml组比较,差异无统计学意义(P0.05)。10μg/ml紫草素组发育到4细胞阶段的胚胎比率均值为(59.26±6.52)%,而0g/ml组的比率均值为(79.23±7.19)%,两组比较,差异有统计学意义(P0.05);10g/ml紫草素干预组发育至8细胞阶段的胚胎比率均值仅为(18.97±2.57)%,而0μg/ml组则高达(58.65±2.46)%,两组比较,差异性具有统计学意义(P0.05)。结论:紫草素可以影响小鼠卵母细胞第一极体的排放,导致纺锤体的组装紊乱、不正常纺锤体比率增加,以及线粒体形态粗大、呈周边或半周边分布,从而抑制了卵母细胞的体外成熟:紫草素对未成熟COC的体外成熟无影响,对小鼠早期胚胎体外发育具有一定的影响,使其停滞在8细胞期,无法继续发育到桑葚胚和囊胚。其确切的作用机制需要进一步进行探讨。
[Abstract]:The purpose and significance of this study: the effects of Zac on oocyte, oocyte complex maturation and early embryonic development in vitro, and the mechanism of the effect of Zac on oocyte maturation and early embryonic development. Methods: 1. the oocytes of 6-8 weeks of female ICR mice were taken, and IBMX inhibitors were used to inhibit the content of oocytes. The oocyte culture medium with different concentrations (0 g/ml, 5, g/ml, 10 mu g/ml, 20 g/ml, 50 mu g/ml) was cultured on the oocyte 12h, then the elution inhibitor release oocyte and culture, and the GVBD rate and the first polar body discharge.2. of the oocyte in 2H and 12h were statistically analyzed to interfere with the mouse oocytes with various concentrations of purple grass solution. In vitro culture, the GVBD rate and the first polar body discharge rate of the oocytes in each group were measured in 2H and 12h respectively. The laser confocal microscopy was used to observe the influence of 1O uh g/ml on the spindle assembly and morphology, the mitochondrial content and distribution of the oocyte.4. The Western Blot test was used to observe the two species of the two species, namely, 0 mu g/ml and 10 mu g/ml. GV phase, GVBD stage, M I stage and M II PKM2 protein of mouse oocytes were changed under the action of concentration of purple grass, and the GV phase of mouse oocyte was detected by immunofluorescence technique, GVBD phase, M I stage, M II PKM2 protein content and.5 distribution, and 0 micron g/ml and 10 mu of purple herb were used in the immature oocyte complex. After s), the GVBD rate and the first polar body discharge rate of 2H and 12h were.6, and 0 micron g/ml and 1O mu g/ml were detected in mice prokaryotic cells to develop into 2 cells (injecting hCG 36h), 4 cells (hCG48h) and 8 cells (injecting hCG). The results were as follows: 10 u g/ml and 5 mu g/ml group were compared with 0 mu g/ml group, and the GVBD rate was not significantly changed, the difference was not statistically significant (P0.05).5 mu g/ml herb group oocyte first polar body discharge rate compared with O u g/ml group, the difference was not statistically significant. However, 10 mu g/ml was no significant difference. The difference of the first polar body discharge rate (13.17 + 3.16)% of the oocyte (13.17 + 3.16)% and 0 mu (85 + 5)% was statistically significant (P0.05). (2) the experimental results were 5 micron g/ml, and the GVBD rate of the oocyte in the 10 mu g/ml group was not statistically significant (P0.05).5 micron g/ml. There was no significant difference between the first polar body discharge rate of the oocyte and the 0 g/ml group (P0.05). However, the average discharge rate of the first polar body of the oocyte in the 10 u g/ml group was (59.05 + 1.65)% compared with the mean of the O g/ml group (86.67 + 2.89)%, the difference was statistically significant (P0.05). (3) laser confocal microscopy was used. The results of immunofluorescence showed that 10 mu g/ml could obviously inhibit the mature development of mouse oocyte in vitro, and the abnormal spindle shape rate was up to 81% (98/121), while the spindle shape abnormal rate in 0g/ml group was only 19% (20/103), and the difference between the two groups was statistically significant (P0.05). It was found that 10 uh g/ml could obviously inhibit the oocytes of mice. In vitro maturation. (4) the mitochondrial staining results showed that in the 0 g/ml group, the mitochondria in the oocytes of the mice were evenly distributed and the mitochondrial signal clusters were uniform, indicating that oocytes were at the middle of the second meiotic division (metaphase II, M II), and the oocytes treated with 10 G /ml purple herb were divided into mitochondria. The Burt point is distributed around or around the periphery, and there is a cluster of mitochondrial signal clusters. It is indicated that the morphology and distribution of the mitochondria in the oocyte are affected by the purple grass element, and the mature.10 mu g/ml concentration of the oocyte can stop the development of oocyte in the prophase of M I or M I. (5) the results of immunofluorescence It is shown that the meiotic maturation process of the oocyte in the 0 g/ml group is basically evenly distributed in the cytoplasm at all times. In the 10 mu g/ml herb group, although PKM2 is basically distributed in the cytoplasm of the oocytes at various stages, but under the same experimental conditions, PKM2 is contained in the M I and M II oocytes. The volume was lower than that of GV and GVBD. (6) in vitro culture of GV-COCs showed that the rate of GVBD in the oval oval complex of 10 mu g/ml group was not statistically significant (P0.05), and the difference was not statistically significant (P0.05). The difference between the first polar body discharge rate and the 0 Mu group was not statistically significant (P0.05). Therefore, the concentration of 10 mu g/ml was purple. (7) the effect of COC on the development of early embryo showed that the ratio of embryos developed to the 2 cell stage in the 10 mu g/m group was compared with that of the 0 mu g/ml group, and the difference was not statistically significant (P0.05), the ratio of the embryo ratio of the.10 mu g/ml group to the 4 cell stage was (59.26 + 6.52)%, while the ratio of the 0g/ml group was more than that of the 0g/ml group. The average rate was (79.23 + 7.19)%, and the difference was statistically significant (P0.05). The average rate of embryo in the 10g/ml purple herb intervention group was only (18.97 + 2.57)% (18.97 + 2.57)%, while the 0 g/ml group was (58.65 + 2.46)%. The difference was statistically significant (P0.05). Conclusion: Zac can affect the oocyte of mice. The emission of one polar body leads to the disturbance of the spindle assembly, the increase of the abnormal spindle ratio and the large mitochondria, and the peripheral or semi surrounding distribution, which inhibits the maturation of the oocyte in vitro: the ripening of the immature COC has no effect on the maturation of the immature embryos in vitro, and has a certain effect on the development of the early embryos in the mice and makes it stagnate. 8 cell stage can not continue to develop into morula and blastocyst. Its exact mechanism needs further study.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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