膜联蛋白IV蛋白差异表达与人类子宫内膜胚胎接受性关系的初步研究

Study of altered expression of annexin IV and human endometrial receptivity

目的探讨种植窗时期人类子宫内膜膜联蛋白IV(annexinIV)蛋白的差异表达与子宫内膜胚胎接受性的关系。方法采用胶内差异双向电泳(2D-DIGE)和基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)技术,鉴定生育能力正常的妇女子宫内膜种植窗前期[黄体生成素(LH)峰第2天(LH+2d)]和种植窗期(LH+7d)内膜差异表达蛋白质;应用免疫组化链酶菌抗生物素蛋白-过氧化物酶连接(SP)法和免疫印迹(westernblot)技术,检测子宫内膜雌激素受体(ER)、孕激素(PR)水平,并对子宫内膜组织进行膜联蛋白IV的定位和半定量表达分析。结果电泳图谱显示等电点约5·8,相对分子质量为36000的蛋白斑点在LH+7d的表达较LH+2d增加2·12倍;经鉴定,此蛋白斑点肽指纹图谱(PMF)为膜联蛋白IV;LH+2d子宫内膜上皮细胞存在膜联蛋白IV蛋白表达,LH+7d子宫内膜上皮和基质细胞中均有膜联蛋白IV蛋白表达,且LH+7d的表达量显著增加(P<0·05)。结论种植窗前期及种植窗期膜联蛋白IV蛋白的差异表达,提示膜连蛋白IV可能是人类子宫内膜向胚胎接受态转化的重要蛋白。

Objective To explore the association of altered expression of annexin IV in human endometrium during the implantation window and endometrial receptivity. Methods A comparative proteomic strategy, in a combination of two-dimensional fluorescence difference gel electrophoresis （2D- DIGE） and matrix-assisted laser desorption/ionization time of flight mass spectrometry （ MALDI-TOF-MS）, was adopted to search for proteome alternations of pre-receptive （ clay LH ＋ 2 ） versus receptive （ LH ＋ 7 ） endometria. The location and abundance of the identified differentially expressed protein- annexin IV were analyzed by immunostaining and western blot. Results By comparing protein profiles of LH ＋ 2 and LH ＋ 7 samples, we found a protein up-regulated 2. 12 times in LH ＋7 samples, with a relative molecular weight of 36 000 and an isoelectric point near pH 5.8. It was characterized using mass spectrometry and was identified as armexin IV. Immunohistochemical analysis revealed an altered localization of annexin IV - in the epithelia on day LH ＋ 2, and both in the epithelia and stroma cells on day LH ＋ 7. Protein levels of annexin IV were up-regulated on day LH ＋ 7 compared with that on day LH ＋ 2 by Western blot. Integrated optical density of the object （OPTDI） was 46. 249 ±32. 376 and 249. 507 ±31. 959, respectively（P =0. 004）. Conclusions Our study indicates endometrial samples obtained by microbiopsy are available for proteomics studies. It seems possible that the increased expression of annexin IV during the implantation window plays an important role in the morphological differentiation of the uterus to the receptive state.