GABA信号在妊娠小鼠子宫的表达及功能初探

发布时间：2018-04-19 01:21

本文选题：GABA信号 + 胚胎植入　；参考：《重庆医科大学》2011年硕士论文

【摘要】：目的胚胎植入和胎盘发生是胎生哺乳动物及人类生殖的重要环节。胚胎植入的成功与否是决定胚胎能否进一步发育的门槛,涉及母体全身的激素调控以及局部的胚胎与子宫间复杂的信息交流;植入后,胚胎滋养层细胞进一步侵入子宫内膜和血管,对胎盘形成及母胎血液循环至关重要,此过程具有严格的时空限制和生理自限性。比较研究发现,胚胎植入过程、滋养层细胞的侵袭特性与肿瘤细胞的转移和侵袭十分相似,其调节机制也近似。近年来发现,γ-氨基丁酸(GABA)及其受体GABAAR、GABABR和合成酶在外周多种内分泌组织和器官有特异性表达,参与调节肿瘤细胞的侵袭和转移。已有研究表明人和大鼠子宫组织表达GABA及其受体,特别是GABAA受体的π亚基在人着床窗口期子宫内膜高表达,提示其可能与子宫内膜容受性改变和滋养层细胞侵袭有关。为探索GABA信号(GABA/GABAAR/GABABBR)在胚胎植入和胎盘发生中的作用,本研究采用免疫组织化学、胚胎体外培养、细胞侵袭实验等方法检测GABA信号在小鼠早期胚胎植入和中期胎盘发生过程中的表达情况,并初步探讨其在胚胎发育和滋养层细胞侵袭过程中的作用,为进一步研究GABA信号与胚胎植入和胎盘发生提供线索。方法1.免疫组织化学方法检测GABA、GABRP、GB1蛋白在妊娠小鼠D1-D5子宫的表达情况;2.将小鼠囊胚培养于含不同浓度GABA抗体的培养液中,观察囊胚脱带、粘附和外延生长的情况;3.免疫组织化学方法检测GABA、GABRP、GB1蛋白在妊娠小鼠D8-D12子宫和胚胎的表达及在绒毛膜滋养细胞癌来源的JAR细胞的表达情况;4.Matrigel细胞侵袭模型检测GABA受体激动剂和拮抗剂对JAR细胞侵袭迁移能力的影响。结果1. GABA、GABRP、GB1蛋白在妊娠小鼠D1-D5子宫组织均有不同程度的表达,且均于D4表达很强,于D5特异性表达在胚胎植入位点的基质细胞。2.一定浓度的GABA抗体能明显抑制囊胚的脱带率,提高已脱带胚胎的外延生长面积,且呈剂量依赖效应,抗体浓度越大,作用越明显;但对已脱带囊胚的黏附率和外延生长率无明显影响。3. GABA、GABRP、GB1蛋白在妊娠小鼠D8-D12的胚胎、胎盘锥、滋养层细胞以及子宫蜕膜组织和血管均有不同程度的表达,并随妊娠的进程而变化。4. JAR细胞表达GABA、GABRP、GB1蛋白。5. GABAAR激动剂muscimol和GABABR拮抗剂2-Hydroxysaclofen均显著促进JAR细胞对matrigel的侵袭;GABAAR拮抗剂SR-95531和GABABR激动剂baclofen均显著抑制JAR细胞对matrigel的侵袭。结论1植入早期小鼠子宫表达GABA信号,GABA抗体能影响囊胚的脱带和外延生长,提示GABA信号可能参与植入过程中子宫与胚胎的“对话”,在小鼠胚胎着床中发挥作用。2妊娠中期小鼠子宫、胚胎以及母胎界面表达GABA信号,激活GABAAR或阻断GABABR能显著提高滋养层细胞的侵袭能力,激活GABABR或阻断GABAAR显著降低滋养层细胞的侵袭能力,提示滋养层细胞可能通过自分泌、旁分泌和血液运输GABA,激活相应的受体,协同调节滋养层细胞的侵袭,影响胎盘的发生。
[Abstract]:The purpose of embryo implantation and placentation is an important link of placental mammals and human reproduction. Embryo implantation success is decided whether to further the development of embryonic threshold, hormone regulation relates to maternal systemic and the embryo and the uterus local complex information exchange; implanted embryonic trophoblast cells, further invasion of the uterus and blood vessels of the formation of placenta and fetal blood circulation is crucial, this process has a strict time limit and physiological selflimited. Comparative study found that the process of embryo implantation, invasion and metastasis of tumor cells and invasion of trophoblast cells is very similar, the adjustment mechanism is similar. In recent years, gamma aminobutyric acid (GABA) and its receptor GABAAR, GABABR and synthetase specific expression in a variety of peripheral endocrine tissues and organs involved in the regulation of tumor cell invasion and metastasis. There have been studies The expression of GABA and its receptor in the rat uterus and the Ming Dynasty, especially the PI subunit of the GABAA receptor expression was high in endometrium, suggesting that it may be related to the invasion of endometrial receptivity and trophoblast cells. To explore the change of GABA signal (GABA/GABAAR/GABABBR) in the event of embryo implantation and placenta in the role of the immunohistochemistry, embryo culture, cell invasion assay method to detect the GABA signal in the early mouse embryo implantation and placental expression of medium in the process, and discuss the support layer of cells in the embryonic development and nourishing the role in the invasion process, to provide clues for further study of GABA signal and embryo implantation and placenta.
Methods: 1. immunohistochemical detection of GABA, GABRP, GB1 protein expression in D1-D5 mice uterine pregnancy; 2. cultured mouse blastocysts cultured in different concentration of GABA antibody, observe the blastocyst adhesion and removal, epitaxial growth of 3.; immunohistochemical detection of GABA, GABRP, GB1 protein expression in the expression of D8-D12 in mice uterus and embryo pregnancy and JAR cells in chorionic trophoblast cell carcinoma derived 4.Matrigel cells invasion model; effect of detection of GABA receptor agonists and antagonists on the invasion and migration of JAR cells.
Results 1. GABA, GABRP, GB1 protein expression in uterine tissues of pregnant D1-D5 mice at different levels, and on the expression of D4 is very strong, in the D5 specific expression of GABA antibody in the stromal cells of.2. certain concentration of embryo implantation sites can significantly inhibit the removal rate of blastocysts with, improve embryo growth area with the extension off and, in a dose-dependent manner, antibody concentration increases, the effect is more obvious; but to have off with the blastocyst adhesion rate and epitaxial growth rate has no obvious effect on.3. GABA, GABRP, GB1 protein in placenta cone of pregnant mouse D8-D12 embryos, trophoblast cells and decidual tissue and blood vessels have different levels of expression, and as the pregnancy process and the changes of.4. JAR cells expression of GABA, GABRP, GB1 protein.5. GABAAR agonist muscimol and GABABR antagonist 2-Hydroxysaclofen significantly promotes the invasion of JAR cells to Matrigel; GABAAR antagonists SR-95531 and GABABR The agonist baclofen significantly inhibited the invasion of JAR cells to Matrigel.
Conclusion implantation of mouse uterus 1 early expression of GABA signal, GABA antibody can affect the removal and epitaxial growth of blastocysts, suggesting that GABA signaling may be involved in the uterus and embryo implantation in the process of "dialogue", play the role of.2 in mouse uterine pregnancy mouse embryo implantation, embryo and maternal fetal interface expression of GABA signal, GABAAR activation or blockade of GABABR can significantly improve the trophoblast cell invasion ability, activation of GABABR or blocking GABAAR significantly reduced the invasive ability of trophoblast cells, suggesting that trophoblast cells via autocrine, paracrine and blood transport GABA, activate the corresponding receptor, CO regulation of trophoblast invasion, the effect of placenta.

【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】：R321

【参考文献】