【摘要】：Zhangfei屬于具有堿性亮氨酸拉鏈bZIP結(jié)構(gòu)的轉(zhuǎn)錄因子,它參與調(diào)節(jié)內(nèi)質(zhì)網(wǎng)應(yīng)激和未折疊蛋白反應(yīng),并在胚胎發(fā)育、人類疾病過(guò)程等方面也發(fā)揮著重要作用。至今,國(guó)內(nèi)外對(duì)Zhangfei蛋白的研究甚少,在生殖內(nèi)分泌領(lǐng)域的作用未見報(bào)道。本實(shí)驗(yàn)在抗Zhangfei單克隆抗體大量制備的基礎(chǔ)上,采用超敏感的免疫組織化學(xué)方法和蛋白免疫印跡技術(shù)對(duì)小鼠在發(fā)情周期中卵巢和子宮中的Zhangfei蛋白表達(dá)進(jìn)行了研究。主要結(jié)果如下: 1采用切膠電洗脫的方法對(duì)Zhangfei融合蛋白進(jìn)行純化,可以獲得純度較高的Zhangfei融合蛋白,濃度達(dá)2.002 mg/mL;通過(guò)培養(yǎng)雜交瘤細(xì)胞和小鼠腹水誘生法對(duì)抗Zhangfei單抗進(jìn)行制備,ELISA方法檢測(cè)雜交瘤細(xì)胞培養(yǎng)上清中單抗的效價(jià)為1∶1000,腹水單抗的效價(jià)為1∶2×10~5。 2 Zhangfei在發(fā)情周期各階段小鼠卵巢的卵母細(xì)胞胞漿中均有表達(dá)。原始卵泡和初級(jí)卵泡的顆粒細(xì)胞中未見Zhangfei蛋白陽(yáng)性染色,次級(jí)卵泡階段的顆粒細(xì)胞出現(xiàn)Zhangfei蛋白的陽(yáng)性染色;成熟卵泡的顆粒細(xì)胞中Zhangfei蛋白的陽(yáng)性染色更加明顯。此外,在整個(gè)發(fā)情周期的黃體中均有Zhangfei蛋白陽(yáng)性分布,而在凋亡的顆粒細(xì)胞和各級(jí)卵泡的卵泡膜細(xì)胞中無(wú)表達(dá)。從發(fā)情前期,發(fā)情期,發(fā)情后期到間情期,Zhangfei蛋白的表達(dá)逐漸增強(qiáng),推論其可能與卵巢中的類固醇激素有關(guān),對(duì)卵巢的組織學(xué)變化和協(xié)調(diào)卵泡成熟過(guò)程有重要的生殖生理調(diào)控作用。 3發(fā)情各階段小鼠子宮均見Zhangfei蛋白的表達(dá),主要分布于內(nèi)膜腔上皮和腺上皮細(xì)胞中,其他部位無(wú)表達(dá)。其中,Zhangfei蛋白在間情期表達(dá)較強(qiáng),發(fā)情期表達(dá)較弱,暗示它的表達(dá)可能受到卵巢類固醇激素的調(diào)控,參與子宮周期性變化的調(diào)節(jié)。 綜上所述,本試驗(yàn)在制備抗Zhangfei單克隆抗體的基礎(chǔ)上,利用免疫組化技術(shù)和蛋白免疫印跡技術(shù)對(duì)在發(fā)情周期不同階段小鼠卵巢和子宮中的Zhangfei蛋白表達(dá)分布進(jìn)行研究,為進(jìn)一步研究Zhangfei蛋白在生殖內(nèi)分泌領(lǐng)域的作用奠定了基礎(chǔ)。
[Abstract]:Zhangfei is a transcription factor with basic leucine zipper bZIP structure. It plays an important role in regulating endoplasmic reticulum stress and unfolded protein reaction, and also plays an important role in embryonic development and human disease process. Up to now, there are few studies on Zhangfei protein at home and abroad, and the role of Zhangfei protein in the field of reproductive endocrine has not been reported. Based on the preparation of monoclonal antibodies against Zhangfei, the expression of Zhangfei protein in ovary and uterus of mice during estrous cycle was studied by ultrasensitive immunohistochemical method and Western blotting technique. The main results are as follows: 1 Zhangfei fusion protein with high purity can be obtained by electroelution of Zhangfei with a concentration of 2.002 mg/mL;. Zhangfei monoclonal antibody was prepared by culture of hybridoma cells and mice ascites. The titer of monoclonal antibody in the supernatant of hybridoma cells detected by ELISA method was 1: 1000, and the titer of monoclonal antibody in ascites was 1:2 脳 10 ~ (5). 2 Zhangfei was expressed in oocyte cytoplasm of mouse ovary at all stages of estrous cycle. No positive staining of Zhangfei protein was found in granulosa cells of primordial follicles and primary follicles, but the positive staining of Zhangfei protein was found in granulosa cells of secondary follicles, especially in granulosa cells of mature follicles. In addition, there was a positive distribution of Zhangfei protein in the corpus luteum of the whole estrous cycle, but not in the apoptotic granulosa cells and follicular membrane cells. From early estrus, late estrus to interoestrus, the expression of Zhangfei protein increased gradually, inferred that the expression of Zhangfei protein might be related to steroid hormones in ovary. It plays an important role in regulating ovarian histology and coordinating follicular maturation. (3) the expression of Zhangfei protein was found in the uterus of all stages of estrus, mainly in the epithelial cells of endometrium and glandular epithelium, but not in other parts. Among them, the expression of Zhangfei protein was strong in the interoestrous stage and weak in the estrus stage, suggesting that the expression of Zhangfei protein might be regulated by ovarian steroid hormone and participate in the regulation of the periodic changes of uterus. In conclusion, on the basis of the preparation of monoclonal antibodies against Zhangfei, the expression and distribution of Zhangfei protein in ovary and uterus of mice at different stages of estrous cycle were studied by immunohistochemistry and Western blotting. It lays a foundation for further study on the role of Zhangfei protein in reproductive endocrine field.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R339.2

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 陳風(fēng)雷;小鼠Zhangfei基因過(guò)表達(dá)和shRNA干擾慢病毒載體的構(gòu)建和鑒定[D];西北農(nóng)林科技大學(xué);2013年

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本文編號(hào)：2367722