小鼠孤雌激活胚胎早期发育过程中胞吞作用相关蛋白的动态表达分析

Dynamic Expression Analysis of Endocytosis Related Proteins during Early Development of Parthenogenetic Activated Embryos in Mice

为探索哺乳动物孤雌激活胚胎早期发育过程中胞吞作用相关蛋白的动态表达,通过体外生产小鼠孤雌激活胚胎,采用实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹(Western blot)技术检测不同发育时期小鼠孤雌激活胚胎胞吞作用相关蛋白Cav1、Cav2基因和蛋白相对表达水平,并采用免疫荧光技术进行不同发育时期小鼠孤雌激活胚胎中Cav1、Cav2表达定位分析。结果表明,在小鼠孤雌激活胚胎不同时期均可检测到Cav1、Cav2基因及其蛋白的表达,其中囊胚和桑椹胚中该2个基因的表达水平最高,4-8细胞时期次之,2细胞最低;各发育时期孤雌激活胚胎中Cav1、Cav2蛋白主要定位在胚胎细胞胞质内;囊胚中内细胞团(ICM)细胞中Cav1的荧光强度高于滋养层细胞。说明胞吞作用相关蛋白Cav1、Cav2可能参与小鼠早期胚胎发育的生理调控,桑椹胚和囊胚时期可能是其发挥生理功能的重要时期,并且在囊胚时期对内细胞团发育的调控作用更为显著。研究结果可为探索胞吞作用参与哺乳动物胚胎早期发育的调控机制提供重要参考。

In order to explore the dynamic expression of endocytosis related proteins during the early embryonic development of mammalian parthenogenesis, mouse parthenogenetic activated embryos were cultured in vitro,real-time fluorescence quantitative PCR(QRT-PCR) and Western blot(WB) were used to detect the relative expression levels of Caveolin-1(Cav1) and Caveolin-2(Cav2) genes and proteins related to parthenogenetic activation of embryo enucleation in mice at different development stages, and to analyze the expression localization of Cav1 and Cav2 in parthenogenetic activation embryos of mice at different development stages by immunofluorescence technology.The results showed that the expressions of Cav1 and Cav2 genes and their associated proteins could be detected at different stages of parthenogenetic activation in mouse embryos, and the expression levels of Cav1 and Cav2 genes were the highest in blastocyst and morula, followed by 4-8 cell stage and 2 cell stage.The expression of Cav1 and Cav2 proteins in parthenogenetic activated embryos at various developmental stages is mainly located in the cytoplasm of embryonic cells.Cav1 in the inner cell mass(ICM) exhibits a higher fluorescence intensity than that in tropheetoderm(TE).These results indicated that Cav1 and Cav2,which are related to endocytosis, may participate in the physiological regulation of early embryonic development in mice, and their important functions may play an important role in the stage of morula and blastocyst, and the regulation effect of endogenous cells developing in the blastocyst stage is more significant.The results provided an important reference for exploring the regulatory mechanism of endocytosis involved in early embryonic development in mammals.