【摘要】： 膜聯(lián)蛋白是一類(lèi)鈣依賴性磷脂結(jié)合蛋白,形成進(jìn)化上保守的多基因家族,在動(dòng)、植物中廣泛表達(dá)。在細(xì)胞各種生理功能中起重要作用。膜聯(lián)蛋白A1(Annexin A1,簡(jiǎn)稱(chēng)AnxA1)是哺乳動(dòng)物膜聯(lián)蛋白家族中的一個(gè)重要成員,它與細(xì)胞增殖、分化、信號(hào)傳遞、炎癥等密切相關(guān),前期研究已發(fā)現(xiàn)它在精子中亦有表達(dá),并呈區(qū)域性分布,推測(cè)與精子的一系列鈣依賴事件如運(yùn)動(dòng)、頂體反應(yīng)過(guò)程中的膜融合、受精過(guò)程中的精卵相互作用等有關(guān)。但目前它在生殖系統(tǒng)中的作用尚不明確,在睪丸中的表達(dá)定位尚未見(jiàn)報(bào)道,對(duì)其在精子中的定位研究還相當(dāng)有限,且還未見(jiàn)關(guān)于它在精子中功能的較為直接的研究。本文以小鼠為實(shí)驗(yàn)對(duì)象,研究AnxA1在出生后不同發(fā)育時(shí)期睪丸中的表達(dá)與定位,以期了解它與睪丸出生后發(fā)育及精子發(fā)生的關(guān)系;同時(shí),對(duì)處于不同成熟階段及不同生理功能狀態(tài)的精子進(jìn)行AnxA1定位。此外,制備了AnxA1多克隆抗體并探討AnxA1與體外受精的關(guān)系。本研究旨在為闡明AnxA1在生殖系統(tǒng)中的作用奠定一定的基礎(chǔ),可望為哺乳動(dòng)物及人類(lèi)受精機(jī)制的研究提供一定的理論依據(jù),從而為輔助生殖技術(shù)、不育癥診療與計(jì)劃生育提供一定的理論指導(dǎo)。本實(shí)驗(yàn)分三部分。1膜聯(lián)蛋白A1在小鼠睪丸不同發(fā)育階段的表達(dá)與定位 對(duì)1~8周齡小鼠睪丸進(jìn)行AnxA1間接免疫組化染色及半定量RT-PCR以研究AnxA1的表達(dá)水平及其定位。結(jié)果顯示:AnxA1在各級(jí)生精細(xì)胞中均有表達(dá)。其中精原細(xì)胞染色最強(qiáng),主要分布在細(xì)胞核內(nèi);初級(jí)精母細(xì)胞多為弱陽(yáng)性或陰性;圓形精子細(xì)胞AnxA1分布在細(xì)胞一側(cè)頂體泡、頂體帽中,精子的AnxA1分布在頂體中。精子形成后留下的殘余體亦呈強(qiáng)陽(yáng)性染色。支持細(xì)胞未見(jiàn)明顯陽(yáng)性染色。對(duì)1、2、3、4、5、6、8周齡小鼠睪丸AnxA1免疫組化染色陽(yáng)性強(qiáng)度即平均積分光密度進(jìn)行圖像分析結(jié)果分別是:2.94±0.36, 8.53±0.90, 7.32±1.22, 6.87±0.51, 5.59±0.53, 4.53±0.38, 4.64±0.40,單因素方差分析結(jié)果顯示1、2、5周齡組的AnxA1免疫組化染色強(qiáng)度與其余各組均存在顯著性差異(P0.01),3周齡與4周齡間(P=0.26, 0.05)、6周齡與8周齡間(P =0.78, 0.05)無(wú)顯著性差
[Abstract]:Annexin is a kind of calcium dependent phospholipid binding protein, which forms an evolutionarily conserved polygene family and is widely expressed in animals and plants. It plays an important role in various physiological functions of cells. Annexin A1 (AnxA1) is an important member of mammalian cell binding protein family. It is closely related to cell proliferation, differentiation, signal transduction, inflammation and so on. It is supposed to be related to a series of calcium dependent events, such as movement, membrane fusion during acrosome reaction, sperm-egg interaction during fertilization and so on. However, its role in the reproductive system is still unclear, the expression localization in the testis has not been reported, and the study of its localization in spermatozoa is very limited, and there is no direct study on its function in spermatozoa. In this paper, we studied the expression and localization of AnxA1 in testis of different postnatal development stages in mice, in order to understand its relationship with postnatal development and spermatogenesis. The spermatozoa in different maturation stage and different physiological function were located by AnxA1. In addition, AnxA1 polyclonal antibodies were prepared and the relationship between AnxA1 and in vitro fertilization was investigated. The purpose of this study is to lay a foundation for elucidating the role of AnxA1 in the reproductive system, and to provide a theoretical basis for the study of fertilization mechanism in mammals and human beings. Diagnosis and treatment of infertility and family planning provide certain theoretical guidance. The expression and localization of integrin A1 in mouse testis at different stages of development were divided into three parts. AnxA1 indirect immunohistochemical staining of testis of 1-week-old mice was carried out. And semi-quantitative RT-PCR to study the expression level and localization of AnxA1. The results showed that: AnxA1 was expressed in spermatogenic cells at all levels. The primary spermatocytes were weakly positive or negative. The AnxA1 of round spermatocytes was distributed in one side of the cell acrosome vesicles, acrosome cap and sperm AnxA1 in acrosome. The residual bodies left after spermatogenesis also showed strong positive staining. Sertoli cells showed no positive staining. The results of image analysis on the positive intensity of AnxA1 immunohistochemical staining in testis of mice at 8 weeks of age were: 1: 2.94 鹵0.36, 8.53 鹵0.90, 7.32 鹵1.22,6.87 鹵0.51,5.59 鹵0.53, 4.53 鹵0.38, 4.64 鹵0.40. The results of univariate variance analysis showed that the intensity of AnxA1 immunohistochemical staining was 12 weeks old, 8.53 鹵0.90, 7.32 鹵1.22, 4.53 鹵0.38, 4.64 鹵0.40, respectively. There was no significant difference (P0.01) between 3 and 4 weeks of age (P0. 26, 0. 05) and between 6 and 8 weeks of age (P = 0. 78, 0. 05).
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R329

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