Skp1蛋白在小鼠早期胚胎发育过程中的功能研究

发布时间：2018-04-04 03:11

本文选题：Skp1　切入点：早期胚胎　出处：《南京医科大学》2008年硕士论文

【摘要】： 为了筛选一些在早期胚胎发育过程中起作用的蛋白,本实验室构建了小鼠卵细胞激活前后的蛋白谱系,通过质谱鉴定、生物信息学分析及相关文献查阅,我们选择了Skp1这个蛋白进行了深入的功能研究。前人有研究报道了在酵母中Skp1能参与纺锤体checkpoint的激活及信号的传递,此外,它还在许多关键的细胞通路中起重要作用,这些结果提示Skp1可能在胚胎早期发育过程中起相当重要的作用。在本研究中,我们首先构建了小鼠激活前后卵细胞的蛋白谱系。从2D胶上我们得到以下信息:Skp1在激活前后卵细胞中表达较多,其表达量在激活前后无明显变化;Skp1在2D胶上呈不同分子量的上下两排分布,推测其可能是一种糖基化的修饰。随后我们运用westernblot方法进一步验证了skp1存在于小鼠卵细胞及孤雌激活卵细胞中。其表达情况与2D胶上的情况类似,即表达量不随激活而改变,呈不同分子量的上下两行分布,下面一行蛋白的分子量与理论分子量18700Da较接近。免疫组织化学的结果显示Skp1主要定位于各级卵母细胞的胞浆中,另外黄体中也有少量分布。免疫荧光的结果与免疫组化的结果类似,Skp1主要定位于卵细胞及各级胚胎的胞浆中,并且其表达量及表达定位随胚胎发育不发生改变。为研究Skp1蛋白在小鼠早期胚胎发育过程中发挥的作用,我们首先构建了pEGFP-Skp1-C2超表达质粒,通过显微注射的方法将其注射至受精卵原核中然后观察受精卵的发育情况,结果发现相对于对照组胚胎,超表达skp1蛋白的胚胎的发育受到不同程度的阻滞,有较多的胚胎不能正常致密化而发育至囊胚阶段,以上结果提示Skp1蛋白超表达能阻滞小鼠早期胚胎的发育。为进一步验证以上实验结果,我们将Skp1抗体注射至受精卵中然后观察胚胎的发育情况。结果显示受精卵进行Skp1抗体注射后其发育得到了促进,在相同的时间内抗体组胚胎的发育要快于对照组胚胎的发育。这些实验结果证实了Skp1蛋白的阻断能促进小鼠早期胚胎的发育,这也与上面超表达实验的结果是一致的。得到以上的实验表型,我们接下来研究Skp1影响小鼠早期胚胎发育的机制。前人有研究显示在酵母中,Skp1能与纺锤体checkpoint蛋白相互作用从而参与纺锤体checkpoint激活与信号的传递,那么Skp1蛋白的阻断就会导致这一监督机制的废除从而使本该被阻止发育的非正常细胞能够继续发育下去。鉴于我们得到的实验表型与此相符,即Skp1抗体阻断后胚胎的发育得到促进,因此我们进一步研究这些注射过抗体后的胚胎。首先我们使用免疫荧光的方法观察经过Skp1抗体注射处理后的卵细胞和胚胎的纺锤体形态,结果显示卵细胞经过Skp1抗体注射处理后其纺锤体形态出现异常的机率大大提高,异常率接近40%,明显高于阴性对照组和空白对照组的10.3%和5.4%。纺锤体的异常包括出现多极纺锤体,纺锤丝散乱不规则,纺锤体形态畸形甚至出现断裂。另外,相对于正常卵细胞染色体规则紧密排列于赤道板,注射Skp1抗体后的卵细胞其染色体出现排列松散紊乱的机率也有明显提高。取受精卵进行实验也得到了类似的结果,以上实验数据提示Skp1抗体注射后能明显提高卵细胞和受精卵出现纺锤体形态及染色体排列异常的机率。同时我们还进行了染色体核型分析实验,取受精卵进行Skp1抗体注射后通过Giemsa染色的方法观察染色体的数目,结果发现注射抗体的实验组胚胎出现非整倍体的机率相对于对照组明显提高,非整倍体率达到46.4%,这说明Skp1蛋白的阻断会诱使胚胎出现非整倍体。在体实验把处理过的胚胎移植到假孕母鼠子宫内,发现实验组平均产仔数为3.6个,明显低于正常对照组的7个,仔鼠死亡率也有所上升。通过以上的实验,我们发现Skp1蛋白的阻断虽然能促进胚胎的发育,但这些发育得到促进的胚胎出现纺锤体形态染色体排列异常及非整倍体的机率也明显提高,从而导致产仔数降低与新生鼠的死亡。这与前人研究的结果是一致的,因此我们推测在哺乳动物中Skp1蛋白可能与纺锤体checkpoint蛋白相互作用从而参与纺锤体checkpoint激活及信号传递。综上,我们研究了Skp1蛋白在小鼠卵细胞及胚胎中的定位表达,发现Skp1蛋白的超表达能阻滞胚胎的发育,而Skp1蛋白的阻断则能促进胚胎的发育,进一步研究发现这些发育促进的胚胎出现纺锤体形态异常及非整倍体的机率明显提高,从而导致胚胎移植后产仔数降低及新生鼠死亡率提高。我们的研究证实了Skp1蛋白在小鼠早期胚胎发育过程中起关键作用,并推测哺乳动物中Skp1在激活和维持纺锤体checkpoint通路过程中发挥重要作用。
[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.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R321

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