發(fā)布時(shí)間：2018-04-30 14:00

  本文選題：紫草素 + 卵母細(xì)胞��； 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：研究目的和意義:研究紫草素對體外培養(yǎng)的小鼠卵母細(xì)胞、卵丘卵母復(fù)合體成熟以及早期胚胎發(fā)育的影響,初步探討紫草素對卵母細(xì)胞成熟和早期胚胎發(fā)育的作用機(jī)制。研究方法:1.取6-8周的雌性ICR小鼠的卵母細(xì)胞,用IBMX抑制劑抑制含有不同濃度(0μg/ml、5μg/ml、10μg/ml、20μg/ml、50μg/ml)紫草素的培養(yǎng)基培養(yǎng)卵母細(xì)胞12h后,再洗脫抑制劑釋放卵母細(xì)胞,進(jìn)行培養(yǎng),分別在2h和12h統(tǒng)計(jì)各組卵母細(xì)胞的GVBD率和第一極體排出率。2.分別以各種不同濃度的紫草素溶液干預(yù)小鼠卵母細(xì)胞后直接進(jìn)行體外培養(yǎng),分別在2h和12h統(tǒng)計(jì)各組卵母細(xì)胞的GVBD率和第一極體排出率。3.采用激光共聚焦顯微技術(shù)觀察1Oμg/ml紫草素對卵母細(xì)胞紡錘體組裝和形態(tài)、線粒體含量和分布的影響。4、采用Western Blot實(shí)驗(yàn)觀察在0μg/ml和10μg/ml兩種濃度紫草素作用下小鼠卵母細(xì)胞GV期、GVBD期、MⅠ期、MⅡ期PKM2蛋白的不同變化;并采用免疫熒光技術(shù)檢測小鼠卵母細(xì)胞GV期、GVBD期、MⅠ期、MⅡ期PKM2蛋白的含量和分布。5、統(tǒng)計(jì)0μg/ml和10μg/ml紫草素分別作用于未成熟卵丘卵母細(xì)胞復(fù)合體(GV-COCs)后,2h和12h各組卵母細(xì)胞的GVBD率和第一極體排出率。6、檢測0μg/ml和1Oμg/ml紫草素分別作用于小鼠原核細(xì)胞后發(fā)育為2細(xì)胞(注射hCG 36h)、4細(xì)胞(注射hCG48h)和8細(xì)胞(注射hCG 60h)的細(xì)胞比率。結(jié)果:(1)先用IBMX抑制劑處理,再洗脫IBMX抑制劑,然后放入含有不同濃度紫草素的培養(yǎng)基中進(jìn)行培養(yǎng),結(jié)果為:10μg/ml和5μg/ml紫草素組分別與0μg/ml組比較,GVBD率均沒有明顯變化,差異無統(tǒng)計(jì)學(xué)意義(p0.05)。5μg/ml紫草素組卵母細(xì)胞第一極體排出率與Oμg/ml組相比,差異無統(tǒng)計(jì)學(xué)意義。然而10μg/ml紫草素組卵母細(xì)胞第一極體排出率(13.17± 3.16)%與0μg/ml組(85±5.00)%相比,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(2)直接用不同濃度紫草素干預(yù)的各組實(shí)驗(yàn)結(jié)果為:5μg/ml、10μg/ml紫草素組卵母細(xì)胞的GVBD率與Oμg/ml組相比,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。5μg/ml紫草素組的卵母細(xì)胞第一極體排出率與0μg/ml組相比,差異無統(tǒng)計(jì)學(xué)意義(P0.05),然而10μg/ml紫草素組的卵母細(xì)胞第一極體排出率均值為(59.05±1.65)%與Oμg/ml組的均值(86.67±2.89)%相比,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)采用激光共聚焦顯微技術(shù)觀察免疫熒光結(jié)果顯示,10μg/ml紫草素能明顯抑制小鼠卵母細(xì)胞體外成熟發(fā)育,紡錘體形態(tài)異常率高達(dá)81%(98/121);而0g/ml組紡錘體形態(tài)異常率僅為19%(20/103),兩組比較,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)�？梢�10μg/ml紫草素能明顯抑制小鼠卵母細(xì)胞體外成熟發(fā)育。(4)線粒體染色結(jié)果顯示:在0μg/ml組中,小鼠卵母細(xì)胞中的線粒體呈均勻分布并且線粒體信號簇顏色均一,說明卵母細(xì)胞處于第二次減數(shù)分裂中期(metaphase Ⅱ,MⅡ期),而用10μg/ml紫草素處理過的卵母細(xì)胞所呈現(xiàn)的線粒體分布特點(diǎn)則為周邊分布或半周邊分布,并且出現(xiàn)了線粒體信號簇聚集現(xiàn)象。說明紫草素影響了卵母細(xì)胞的線粒體形態(tài)和分布,進(jìn)而影響了卵母細(xì)胞的成熟。10μg/ml濃度的紫草素能夠使卵母細(xì)胞發(fā)育停滯在MⅠ前期或MⅠ期。(5)免疫熒光結(jié)果顯示:PKM2在0μg/ml組的卵母細(xì)胞減數(shù)分裂成熟過程各個(gè)時(shí)期都基本上是均勻分布在胞漿中。在10μg/ml紫草素組,雖然PKM2在各個(gè)不同時(shí)期的卵母細(xì)胞中也基本均勻分布在胞漿中。但是在同一實(shí)驗(yàn)條件下,PKM2在MⅠ期和MⅡ期卵母細(xì)胞中的含量要低于GV期和GVBD期。(6)GV-COCs體外培養(yǎng)結(jié)果顯示:10μg/ml紫草素組卵丘卵母復(fù)合體的GVBD率與μg/ml組相比,差異無統(tǒng)計(jì)學(xué)意義(p0.05);同樣,10μg/ml紫草素組均第一極體排出率與0μg/ml組比較,差異也無統(tǒng)計(jì)學(xué)意義(p0.05)。因此,10μg/ml濃度的紫草素對COC體外成熟無明顯影響。(7)對早期胚胎發(fā)育的影響結(jié)果顯示:10μg/m紫草素干預(yù)組發(fā)育到2細(xì)胞階段的胚胎比率與0μg/ml組比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。10μg/ml紫草素組發(fā)育到4細(xì)胞階段的胚胎比率均值為(59.26±6.52)%,而0g/ml組的比率均值為(79.23±7.19)%,兩組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05);10g/ml紫草素干預(yù)組發(fā)育至8細(xì)胞階段的胚胎比率均值僅為(18.97±2.57)%,而0μg/ml組則高達(dá)(58.65±2.46)%,兩組比較,差異性具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:紫草素可以影響小鼠卵母細(xì)胞第一極體的排放,導(dǎo)致紡錘體的組裝紊亂、不正常紡錘體比率增加,以及線粒體形態(tài)粗大、呈周邊或半周邊分布,從而抑制了卵母細(xì)胞的體外成熟:紫草素對未成熟COC的體外成熟無影響,對小鼠早期胚胎體外發(fā)育具有一定的影響,使其停滯在8細(xì)胞期,無法繼續(xù)發(fā)育到桑葚胚和囊胚。其確切的作用機(jī)制需要進(jìn)一步進(jìn)行探討。
[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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285

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