一个新的子宫雌激素反应蛋白的分子克隆及功能研究

To further explore the biocharacters and functions of the 250 kDa estrogen-responsive protein. Methods The 250 kDa estrogen-responsive protein were isolated from the uterine luminal fluid (ULF) obtained from estradiol-treated ovariectomized rats and purified by SDS-PAGE. Isoelectric focusing (IEF) analysis, matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and Western blot were used to study the characters and functions of this protein. Polyclonal antibodies were raised against this protein. The immunohistochemical analysis was used to determine the expressin of the 250 kDa protein in different tissues of rats. The cross reactions of the rabbit anti-250 kDa protein antibodies with human myocardium、Hepar、spleen、pancreas、encephalon and et al. were also examined by immunohistochemical assay. The function of the 250 kDa protein in the embryos implantation was evaluated by method of uterine horn single injection. Results The estimated isoelectric point (pI) of this protein is pH 7.1 by using isoelectric focusing (IEF) analysis. The purified 250 kDa protein was digested in-gel by trypsin and the masses of generated peptides were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from PMF were used in protein database search and the protein identification. We did not find match peptides in SWISS-PROT. The results of the embryos implantation experience indicate that the number of embryoes implanted in the horns after the antibody treatment were not significantly less than (p>0.05) those of controls. The 250 kDa protein is only expressed in the glandular and luminal epithelial cells of the rat uterine endometrium. The antibodies also reacted with some unknown protein in human hyperplasia of mammary glands, fibroadenoma, duct carcinoma and lobular carcinoma of mammary glands, but did not react with the normal tissues around the diseased tissues. The further identification of Western blot indicated that this unknown protein with estimated molecular weight of 100 kDa was recognized from the tissues extract of mammary cancer but without from the blood of the patients. Conclusion We presume that the 250 kDa protein is an unknown new protein. It is an uterine special protein. This 250 kDa protein perhaps do not play a directly important role in embryo implantation, but maybe it plays another important roles in uterine. The 100 kDa protein expressed in human mammary glands would be used as a specific marker for the diagnosis and therapy in this kind of diseases in future.

雌激素相关因子直接或间接参与胚胎植入及发育等生殖过程。我们从大鼠子宫腔液中纯化出一250KD雌激素反应蛋白。该蛋白由子宫内膜腺上皮和腔上皮合成，与生殖周期性相关，抗雌激素制剂可抑制其合成。本课题拟研究编码该蛋白的基因和氨基酸序列及其生殖过程的影响，从分子水平阐明雌激素类药物的作用机制，并为开发新型抗着床药物奠定理论基础。.