本文選題：Skp1　切入點(diǎn)：早期胚胎　出處：《南京醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 為了篩選一些在早期胚胎發(fā)育過(guò)程中起作用的蛋白,本實(shí)驗(yàn)室構(gòu)建了小鼠卵細(xì)胞激活前后的蛋白譜系,通過(guò)質(zhì)譜鑒定、生物信息學(xué)分析及相關(guān)文獻(xiàn)查閱,我們選擇了Skp1這個(gè)蛋白進(jìn)行了深入的功能研究。前人有研究報(bào)道了在酵母中Skp1能參與紡錘體checkpoint的激活及信號(hào)的傳遞,此外,它還在許多關(guān)鍵的細(xì)胞通路中起重要作用,這些結(jié)果提示Skp1可能在胚胎早期發(fā)育過(guò)程中起相當(dāng)重要的作用。 在本研究中,我們首先構(gòu)建了小鼠激活前后卵細(xì)胞的蛋白譜系。從2D膠上我們得到以下信息:Skp1在激活前后卵細(xì)胞中表達(dá)較多,其表達(dá)量在激活前后無(wú)明顯變化;Skp1在2D膠上呈不同分子量的上下兩排分布,推測(cè)其可能是一種糖基化的修飾。隨后我們運(yùn)用westernblot方法進(jìn)一步驗(yàn)證了skp1存在于小鼠卵細(xì)胞及孤雌激活卵細(xì)胞中。其表達(dá)情況與2D膠上的情況類(lèi)似,即表達(dá)量不隨激活而改變,呈不同分子量的上下兩行分布,下面一行蛋白的分子量與理論分子量18700Da較接近。免疫組織化學(xué)的結(jié)果顯示Skp1主要定位于各級(jí)卵母細(xì)胞的胞漿中,另外黃體中也有少量分布。免疫熒光的結(jié)果與免疫組化的結(jié)果類(lèi)似,Skp1主要定位于卵細(xì)胞及各級(jí)胚胎的胞漿中,并且其表達(dá)量及表達(dá)定位隨胚胎發(fā)育不發(fā)生改變。 為研究Skp1蛋白在小鼠早期胚胎發(fā)育過(guò)程中發(fā)揮的作用,我們首先構(gòu)建了pEGFP-Skp1-C2超表達(dá)質(zhì)粒,通過(guò)顯微注射的方法將其注射至受精卵原核中然后觀察受精卵的發(fā)育情況,結(jié)果發(fā)現(xiàn)相對(duì)于對(duì)照組胚胎,超表達(dá)skp1蛋白的胚胎的發(fā)育受到不同程度的阻滯,有較多的胚胎不能正常致密化而發(fā)育至囊胚階段,以上結(jié)果提示Skp1蛋白超表達(dá)能阻滯小鼠早期胚胎的發(fā)育。為進(jìn)一步驗(yàn)證以上實(shí)驗(yàn)結(jié)果,我們將Skp1抗體注射至受精卵中然后觀察胚胎的發(fā)育情況。結(jié)果顯示受精卵進(jìn)行Skp1抗體注射后其發(fā)育得到了促進(jìn),在相同的時(shí)間內(nèi)抗體組胚胎的發(fā)育要快于對(duì)照組胚胎的發(fā)育。這些實(shí)驗(yàn)結(jié)果證實(shí)了Skp1蛋白的阻斷能促進(jìn)小鼠早期胚胎的發(fā)育,這也與上面超表達(dá)實(shí)驗(yàn)的結(jié)果是一致的。 得到以上的實(shí)驗(yàn)表型,我們接下來(lái)研究Skp1影響小鼠早期胚胎發(fā)育的機(jī)制。前人有研究顯示在酵母中,Skp1能與紡錘體checkpoint蛋白相互作用從而參與紡錘體checkpoint激活與信號(hào)的傳遞,那么Skp1蛋白的阻斷就會(huì)導(dǎo)致這一監(jiān)督機(jī)制的廢除從而使本該被阻止發(fā)育的非正常細(xì)胞能夠繼續(xù)發(fā)育下去。鑒于我們得到的實(shí)驗(yàn)表型與此相符,即Skp1抗體阻斷后胚胎的發(fā)育得到促進(jìn),因此我們進(jìn)一步研究這些注射過(guò)抗體后的胚胎。 首先我們使用免疫熒光的方法觀察經(jīng)過(guò)Skp1抗體注射處理后的卵細(xì)胞和胚胎的紡錘體形態(tài),結(jié)果顯示卵細(xì)胞經(jīng)過(guò)Skp1抗體注射處理后其紡錘體形態(tài)出現(xiàn)異常的機(jī)率大大提高,異常率接近40%,明顯高于陰性對(duì)照組和空白對(duì)照組的10.3%和5.4%。紡錘體的異常包括出現(xiàn)多極紡錘體,紡錘絲散亂不規(guī)則,紡錘體形態(tài)畸形甚至出現(xiàn)斷裂。另外,相對(duì)于正常卵細(xì)胞染色體規(guī)則緊密排列于赤道板,注射Skp1抗體后的卵細(xì)胞其染色體出現(xiàn)排列松散紊亂的機(jī)率也有明顯提高。取受精卵進(jìn)行實(shí)驗(yàn)也得到了類(lèi)似的結(jié)果,以上實(shí)驗(yàn)數(shù)據(jù)提示Skp1抗體注射后能明顯提高卵細(xì)胞和受精卵出現(xiàn)紡錘體形態(tài)及染色體排列異常的機(jī)率。同時(shí)我們還進(jìn)行了染色體核型分析實(shí)驗(yàn),取受精卵進(jìn)行Skp1抗體注射后通過(guò)Giemsa染色的方法觀察染色體的數(shù)目,結(jié)果發(fā)現(xiàn)注射抗體的實(shí)驗(yàn)組胚胎出現(xiàn)非整倍體的機(jī)率相對(duì)于對(duì)照組明顯提高,非整倍體率達(dá)到46.4%,這說(shuō)明Skp1蛋白的阻斷會(huì)誘使胚胎出現(xiàn)非整倍體。在體實(shí)驗(yàn)把處理過(guò)的胚胎移植到假孕母鼠子宮內(nèi),發(fā)現(xiàn)實(shí)驗(yàn)組平均產(chǎn)仔數(shù)為3.6個(gè),明顯低于正常對(duì)照組的7個(gè),仔鼠死亡率也有所上升。通過(guò)以上的實(shí)驗(yàn),我們發(fā)現(xiàn)Skp1蛋白的阻斷雖然能促進(jìn)胚胎的發(fā)育,但這些發(fā)育得到促進(jìn)的胚胎出現(xiàn)紡錘體形態(tài)染色體排列異常及非整倍體的機(jī)率也明顯提高,從而導(dǎo)致產(chǎn)仔數(shù)降低與新生鼠的死亡。這與前人研究的結(jié)果是一致的,因此我們推測(cè)在哺乳動(dòng)物中Skp1蛋白可能與紡錘體checkpoint蛋白相互作用從而參與紡錘體checkpoint激活及信號(hào)傳遞。 綜上,我們研究了Skp1蛋白在小鼠卵細(xì)胞及胚胎中的定位表達(dá),發(fā)現(xiàn)Skp1蛋白的超表達(dá)能阻滯胚胎的發(fā)育,而Skp1蛋白的阻斷則能促進(jìn)胚胎的發(fā)育,進(jìn)一步研究發(fā)現(xiàn)這些發(fā)育促進(jìn)的胚胎出現(xiàn)紡錘體形態(tài)異常及非整倍體的機(jī)率明顯提高,從而導(dǎo)致胚胎移植后產(chǎn)仔數(shù)降低及新生鼠死亡率提高。我們的研究證實(shí)了Skp1蛋白在小鼠早期胚胎發(fā)育過(guò)程中起關(guān)鍵作用,并推測(cè)哺乳動(dòng)物中Skp1在激活和維持紡錘體checkpoint通路過(guò)程中發(fā)揮重要作用。
[Abstract]:In order to select some proteins in early embryonic development, we established a pedigree of mice after oocyte activation protein, identified by mass spectrometry, bioinformatics analysis and related literature review, we selected the Skp1 protein for further investigation. Previous studies have reported activation and signal transmission. In yeast Skp1 can participate in the spindle checkpoint in addition, it plays an important role in many key cellular pathways, these results suggest that Skp1 may be in the process of early embryonic development plays a very important role.
In this study, we first constructed the protein spectrum before and after the activation of mouse oocytes. We get the following information from 2D gel: Skp1 expressed in activated oocytes before and after more, the expression did not change significantly before and after activation in Skp1; showed different molecular weight in 2D gel on the two rows that distribution. It may be a glycosylation modification. Then we use Westernblot method to further verify the existence of Skp1 in mouse oocytes and parthenogenetic egg cells. The expression of a similar situation with 2D glue on the situation, which is expressed with activation and change, is not the same molecular weight on the two line distribution molecular weight and molecular weight, the following line of 18700Da protein is closer to the cytoplasm. Immunohistochemistry results showed that Skp1 was mainly localized in all oocytes, another corpus luteum in a small amount of the distribution. The results of immunofluorescence and immunohistochemistry The results are similar to that Skp1 is mainly located in the cytoplasm of egg cells and at all levels of embryo, and the expression and expression orientation of the embryo do not change with the development of the embryo.
For the play of Skp1 protein in the process of early embryonic development in mice, we constructed a pEGFP-Skp1-C2 expression plasmid by microinjection method will be developed and then observe the pronuclei of fertilized oocytes injected into the fertilized egg, embryo results compared with the control group, over expression of Skp1 protein in embryo development by different block the degree of more densification and not normal embryo development to the blastocyst stage, these results suggest that Skp1 protein overexpression can block the development of early mouse embryos. In order to further verify the above experimental results, then we will observe the embryonic development of Skp1 antibody injected into fertilized eggs. The results showed that the fertilized eggs were Skp1 antibody after injection its development was to promote, at the same time the antibody group embryo development faster than in the control group embryos. These results confirmed that Skp The blocking of 1 protein can promote the development of early mouse embryos, which is also in accordance with the results of the above overexpression.
Above phaenotypes, we next studied the mechanism of early embryonic development of Skp1 mice. Previous studies have shown that in yeast, Skp1 and checkpoint proteins interact and participate in the spindle spindle checkpoint activation and signal transmission, then Skp1 will block protein lead to the abolition of the supervision mechanism so that the development is blocked the non normal cells can continue to develop it. In view of the phenotype we get in line with this, Skp1 antibody after blocking the development of embryos is promoted, so we further study the antibody injection after the embryo.
First, we use immunofluorescence observation after oocyte and embryo Skp1 antibody injection after the treatment of spindle morphology, results showed that oocytes after Skp1 antibody injection after treatment the spindle abnormal rate is greatly improved, the abnormal rate of close to 40%, significantly higher than the negative control group and blank control group and 10.3% 5.4%. spindle including the emergence of abnormal multipolar spindle spindle, scattered irregular, abnormal spindle morphology or even fracture. In addition, compared with the normal oocyte chromosome rules closely at the equatorial plate, injection of Skp1 antibody after oocyte chromosome was arranged in disorder probability can be improved significantly. The fertilized eggs were also obtained similar experiments the above data suggest that Skp1 antibody injection can significantly improve the egg cells and zygotes appeared spindle morphology and chromosome alignment Abnormal probability. At the same time, we also conducted a number of experiments of karyotype analysis, observation of chromosome methods of zygote Skp1 antibody after injection by Giemsa staining, the experiment group showed embryo injection antibodies appear non probability of aneuploidy compared with the control group improved significantly, the aneuploidy rate reached 46.4%, indicating that Skp1 protein blocking will induce embryonic aneuploidy in vivo. The treated embryos were transferred to pseudopregnant females in the uterus, found that the experimental group average litter size was 3.6, significantly lower than 7 in normal control group, rats mortality rate also increased. According to the above results, we found that blocking Skp1 although the protein can promote the development of the embryo, but the growth promoting embryonic arrangement spindle morphology of chromosome abnormality and the probability of aneuploidy were significantly increased, resulting in reduced litter size and The death of neonatal rats is consistent with previous studies. Therefore, we speculate that in mammals, Skp1 protein may interact with spindle checkpoint protein and participate in spindle checkpoint activation and signal transduction.
In summary, we studied the localization of Skp1 protein in oocytes and embryos in expression, showed that overexpression of Skp1 proteins can block the development of embryos, while blocking Skp1 protein can promote embryo development, further promoting the development of the study found that these embryos appeared spindle shaped and abnormal state of aneuploidy rate increased significantly, which leads to embryo transfer and reduced litter size in newborn rats increased mortality. Our study confirmed that Skp1 proteins play a key role in the process of early embryonic development in mammals, and speculated that Skp1 play an important role in the activation and maintenance of spindle checkpoint pathway in the process.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R321

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本文編號(hào)：1708110