γ-氨基丁酸通过其B受体影响小鼠植入前胚胎的发育  

γ-aminobutyric acid could affect the development of preimplantation embryos through its B-type receptor in mice

作　　者：田娜[1] 曹科[1] 杨根岭[1] 陈文豪[1] 王晓杰[1] 张倩[1] 谭毅[1] 谭冬梅[1] 

机构地区：[1]重庆医科大学实验动物中心,重庆400010

出　　处：《重庆医科大学学报》2017年第8期970-975,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金面上资助项目(编号:31601206;31171436)

摘　　要：目的:探讨γ-氨基丁酸(γ-aminobutyric acid,GABA)对小鼠植入前胚胎发育的影响及其机制。方法:(1)收集不同发育阶段的小鼠植入前胚胎,通过激光共聚焦检测GABA及其B受体B1亚基(B receptor B1 subunit,GB1)、A受体pi亚基(A receptor pi subunit,GABRP)以及GABA合成酶(L-glutamate decarboxylase,GAD67)的表达。(2)不同发育阶段的胚胎分别培养在含10,20,30,40,50μg/μL GABA和40μmol/L B型受体抑制剂(3-Amino-2-(4-chlorophenyl)-2-hydroxypropanesulfonic acid,2-hydroxysaclofen)的培养液中,48~72 h时观察囊胚的形成并计算囊胚形成率。结果:(1)GABA与其受体GB1蛋白在小鼠胚胎中的表达模式一致,在2-细胞和4-细胞阶段,主要表达在细胞膜上,而在8-细胞至囊胚阶段,则转移到细胞核中表达;而另一受体GABRP在植入前胚胎的各个阶段均不表达。GAD67在胚胎发育的各个阶段均有表达。(2)各发育阶段胚胎的囊胚形成率均随着GABA浓度的增加而下降,且呈剂量依赖性;10μg/μL GABA可显著降低2-细胞胚胎的囊胚形成率(92%vs.99%,χ2=5.70,P=0.035)。(3)40μmol/L 2-hydroxysaclofen能够有效逆转30μg/μL和40μg/μL GABA对囊胚形成的抑制作用(99%vs.45%,χ2=72.32,P=0.000;99%vs.4%,χ2=180.66,P=0.000)。结论:GABA通过其B受体抑制小鼠植入前胚胎的发育。Objective：To investigate the effects and possible mechanisms of γ-aminobutyric acid（GABA）on preimplantation embryo development in mice. Methods：（1）The embryos at different development stages were collected and the localization of GABA,B-type receptor protein（GB1）,the π subunit of A-type receptor（GABRP）,and L-glutamic acid decarboxylase 67（GAD67）was detected by immunofluorescence staining.（2）The embryos at different development stages were cultured in vitro in KSOM medium containing 10,20,30,40,or 50 μg/μL GABA and 40 μmol/L 2-hydroxysaclofen（GABA B-type receptor antagonist）for 48-72 h,respectively,and blastocyst formation rate was counted. Results：（1）GABA and GB1 are mainly expressed in the membranes of 2-cell and 4-cell embryos,while in the 8-cell to blastocyst stages,they are localized in the nucleus. However,GABRP was not detected in preimplantation embryos at any stage. In addition,GAD67,a key GABA synthetase,was mainly localized in the cytoplasm in preimplantation embryos.（2）The blastocyst formation rate of each development stage was significantly declined in a dose-dependent manner. In addition,the blastocyst formation rate of 2-cell embryos stage was remarkably inhibited by 10 μg/μL GABA（92% vs. 99%,χ^25.70,P=0.035）.（3）40 μmol/L 2-hydroxysaclofen could effectively reverse the inhibitory effect of 30 μg/μL and 40 μg/μL GABA on blastocyst formation（99% vs. 45%,χ^272.32,P=0.000;99% vs. 4%,χ^2180.66,P=0.000）. Conclusion：GABA could affect the development of preimplantation embryos through its B-type receptor in mice.

关 键 词：Γ-氨基丁酸 植入前胚胎 2-hydroxysaclofen 囊胚形成 

分 类 号：Q954.4[生物学—动物学]