翻译控制肿瘤蛋白（TCTP）在早孕小鼠子宫内膜围着床期的表达及意义

发布时间：2018-05-15 00:17

本文选题：翻译控制肿瘤蛋白TCTP + 胚胎着床　；参考：《重庆医科大学》2011年硕士论文

【摘要】：背景和目的翻译控制肿瘤蛋白(The translationally controlled tumor protein,TCTP)最初发现于肿瘤组织,后来又从其他组织中分离出来。TCTP广泛表达于动物、植物及酵母中,其基因序列高度保守。最初认为TCTP仅是一类生长相关蛋白,近年来有研究表明,TCTP具有多种生物学功能:可作为一种组织胺释放因子,参与细胞的生长、分裂和分化等;也可作为一种小分子热休克蛋白,以分子伴侣的形式保护细胞蛋白免受热休克损伤;同时还可作为一种钙结合蛋白,在人类滋养层细胞中起钙运输的作用。另外,课题组在小鼠胚胎着床相关基因文库研究中发现TCTP基因在小鼠胚胎着床窗口期子宫内膜组织中呈现高表达。结合文献和前期研究工作,我们推测TCTP可能参与了胚胎着床和早期胚胎发育过程。鉴于TCTP在小鼠胚胎围着床期的研究还未见报道,本实验运用qRT-PCR、Western blot和免疫组化等方法对小鼠胚胎围着床期子宫内膜TCTP基因表达进行了分析,并采用子宫角注射实验初步探讨了TCTP基因的功能。这不仅有助于深入理解TCTP在胚胎着床中的作用,而且为探索胚胎着床的分子机制提供相关实验依据。方法分别采用qRT-PCR法、免疫组化和Western blot法检测分析孕D1, D2, D3, D4, D5, D6和D7小鼠子宫内膜中TCTP mRNA和蛋白的表达情况。然后以假孕D4和D6的小鼠子宫内膜为对照,分别采用qRT-PCR、免疫组化和Western blot法检测TCTP在着床前(孕D4)和着床后(孕D6)子宫内膜中的表达。最后对孕D3.5小鼠实施子宫角注射TCTP反义寡聚脱氧核苷酸,24 h后取出子宫内膜组织,再分别用qRT-PCR、Western blot和免疫组化法检测TCTP mRNA和蛋白表达的差异,同时观察术后孕D8小鼠子宫中胚胎数目和形态的变化以期探讨TCTP基因的功能。结果 qRT-PCR结果显示TCTP mRNA在孕D1~D5天小鼠子宫内膜中随孕天数的递增其表达量逐渐升高,孕D5(着床窗口期)达到顶峰,随后孕D6和D7表达下降。Western blot和免疫组化检测结果与qRT-PCR一致。TCTP主要表达于子宫内膜的腔上皮、腺上皮和基质细胞中。TCTP mRNA和蛋白在正常妊娠小鼠着床前(孕D4)和着床后(孕D6)子宫内膜中的表达均高于假孕D4和D6小鼠(*P0.05)。在小鼠子宫角一侧注射TCTP反义寡聚脱氧核苷酸后,与另一侧注射TCTP正义寡聚脱氧核苷酸和蒸馏水相比,TCTP mRNA和蛋白的表达均受到抑制,并且小鼠胚胎着床数目明显减少(*P0.05)。结论 TCTP mRNA和蛋白在早孕小鼠子宫内膜围着床期的表达规律与胚胎着床时间和部位高度一致,且在着床窗口期(孕D5)呈现出高表达现象;对小鼠实施宫角注射TCTP反义寡聚脱氧核苷酸后,TCTP的表达受到抑制,且小鼠胚胎着床数目明显下降,提示TCTP与胚胎着床密切相关,在小鼠胚胎着床过程中可能发挥着重要的作用。
[Abstract]:Background and purpose The translationally controlled tumor protein (TCTP) was first found in the tumor tissue, then isolated from other tissues. TCTP was widely expressed in animals, plants and yeast, and its gene sequence was highly conserved. It was initially thought that TCTP is only a kind of growth-related protein. In recent years, it has been shown that TCTP has many biological functions: it can be used as a histamine releasing factor to participate in cell growth, division and differentiation. It can also be used as a small molecular heat shock protein to protect cell proteins from heat shock damage in the form of molecular chaperones and to act as a calcium binding protein in the transport of calcium in human trophoblast cells. In addition, the study of mouse embryo implantation related gene library showed that TCTP gene was highly expressed in the endometrium of mouse embryo implantation window. We speculate that TCTP may be involved in embryo implantation and early embryonic development. In view of the fact that the study of TCTP in the period of mouse embryo surrounding bed has not been reported, the expression of TCTP gene in the endometrium of mouse embryo around the bed was analyzed by using qRT-PCRG Western blot and immunohistochemical method. The function of TCTP gene was preliminarily studied by the experiment of uterine horn injection. This will not only help to understand the role of TCTP in embryo implantation, but also provide experimental basis for exploring the molecular mechanism of embryo implantation. Method The expressions of TCTP mRNA and protein in endometrium of pregnant D1, D2, D3, D4, D5, D6 and D7 mice were detected by qRT-PCR, immunohistochemistry and Western blot. Then the expression of TCTP in preimplantation (D4) and postimplantation (D6) endometrium was detected by qRT-PCR, immunohistochemistry and Western blot method. TCTP antisense oligodeoxynucleotides (TCTP antisense oligodeoxynucleotides) were injected into the uterus of pregnant D3.5 mice for 24 hours. The expression of TCTP mRNA and protein was detected by qRT-PCRG Western blot and immunohistochemistry. At the same time, the number and morphology of embryo in the uterus of pregnant D8 mice after operation were observed in order to investigate the function of TCTP gene. Result QRT-PCR results showed that the expression of TCTP mRNA in endometrium of D1~D5 mice increased gradually with the increase of pregnant days, and the peak of D5 (implantation window period) was observed. Subsequently, the expression of D6 and D7 decreased. Western blot and immunohistochemistry showed that D6 and D7 were mainly expressed in the luminal epithelium of endometrium. The expression of. TCTP mRNA and protein in glandular epithelium and stromal cells in normal pregnant mice before and after implantation (D4) and postimplantation (D6) were higher than those in pseudo-pregnant D4 and D6 mice. After injection of TCTP antisense oligodeoxynucleotides into one side of mouse uterus horn, the expression of mRNA and protein of TCTP sense oligodeoxynucleotides and distilled water were inhibited, and the number of embryo implantation in mice was significantly decreased than that of TCTP sense oligodeoxynucleotides and distilled water. Conclusion The expression of TCTP mRNA and protein in endometrium of early pregnant mice was highly consistent with the time and location of embryo implantation, and the expression of TCTP mRNA and protein was high in the implantation window (D5). The expression of TCTP antisense oligodeoxynucleotides was inhibited after injection of antisense oligodeoxynucleotides into mouse uterus horn, and the number of mouse embryo implantation decreased significantly, suggesting that TCTP is closely related to embryo implantation and may play an important role in mouse embryo implantation.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346;R714.1

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