陳義雄Chen, Yee-Hsiung臺灣大學:生化科學研究所王希超Wang, Hsi-ChaoHsi-ChaoWang2010-05-042018-07-062010-05-042018-07-062008U0001-2407200815431700http://ntur.lib.ntu.edu.tw//handle/246246/178846雌性生殖道內具免疫系統來抵禦病菌等外來物的侵入。而精子需具備抵抗雌性生殖道免疫系統攻擊的能力，才能到達位於輸卵管的壺腹進行受精動作。Carcinoembryonic antigen-related cell adhesion molecule 10 （CEACAM10）係成熟公鼠副屬性腺貯精囊液中的蛋白質，在雄性與雌性小鼠的性腺體中，其特別表現於貯精囊中。CEACAM10為分子量36-kDa的分泌性醣蛋白，雖然在體外實驗（in vitro）已證實CEACAM10可結合於精子的表面並促進其活動力，但於雌性生殖系統中所扮演的角色尚待釐清。由公鼠貯精囊液所純化的CEACAM10 與動情期子宮液反應，發現子宮液內特定蛋白質有減少的趨勢。經過LC/MS/MS鑑定，證實該減少的蛋白質為補體complement 3 component（C3）。進一步的實驗發現CEACAM10能造成子宮液內補體C3 β chain的降解，使得補體C3b、iC3b、C3c、C3dg失去完整的結構。在體內實驗（in vivo）著床前期第1天的小鼠子宮液內有CEACAM10的存在，而其子宮液內補體C3 β chain也有減少情形，提示CEACAM10可能由引起C3 β chain的降解而破壞補體C3的結構，使其喪失補體生理活性，藉此抑制補體活化以致提高精子的存活率。人類補體C3與小鼠補體C3在氨基酸序列以及蛋白質序列上有高度的相似性，所以先將人類補體C3與小鼠貯精囊液CEACAM10反應，檢測補體活性的變化。結果顯示：（1）CEACAM10與人類補體C3單獨反應無法造成補體C3降解；（2）加入缺乏人類補體C3血清於CEACAM10與人類補體C3的反應中，可以引起補體C3 β chain降解；（3）在人類補體C3與缺乏人類補體C3血清中，CEACAM10可以抑制羊紅血球溶血。Innate immunity plays an essential role in the defense of the invaders in the female genital tract. Spermatozoa must therefore evade immunity attack in order to reach the ampulla of oviduct for fertilization. A 36-kDa has been demonstrated carcinoembryonic antigen-related cell adhesion molecule 10（CEACAM10）from adult mouse seminal vesicle secretions. Among the sexual glands of male and female mice, it is exclusively expressed in seminal vesicle. Although, it is able to bind on the entire surface of mouse spermatozoa and enhance sperm motility in vitro, its role in murine female reproductive system has no yet understood. This work aimed to that direction, I incubated purified CEACAM10 with uterine luminal fluid collected from adult female mice during estrus and resolved the protein components by nonreduced SDS-PAGE. A 180-kDa protein was markedly decrease by CEACAM10. This protein was demonstrated to be complement 3 component（C3）based on the peptide sequences determined by LC/MS/MS analysis. Further, I illustrated the degradation of β chain of C3 in the uterine fluid by CEACAM10. As a result, C3b、iC3b、C3c、C3dg became incomplete. In the preimplantation period, CEACAM10 was determined in the uterine fluid and only on day 1. It was accomplished with the degradation of C3 β chain. Taken together, the presence of CEACAM10 in the uterine fluid caused the loss of C3 activity via the degradation of its β chain to prevent the ejaculated spermatozoa in the uterine lumen from the immunity, attacked by the complement system. C3 was highly conserved and I studied the effect of CEACAM10 in the human C3 activity. These results were summarized：（1）Incubation of human C3 and mouse CEACAM10 didn’t cause the C3 degradation；（2）Addition of the human C3 deficient serum to the incubation of human C3 and CEACAM10 caused the human C3 β chain degradation;（3）CEACAM10 was able to inhibit the lysis of sheep blood by human C3 and the C3 deficient serum together.目錄文摘要 I文摘要 II寫表 3一章 概論 4鼠雄性生殖系統 4鼠貯精囊的研究 4鼠雌性生殖系統 5鼠子宮的生理及子宮液的生成 5體系統 7體系統的活化機制 7體的生物活性 9鼠C3的構造 10鼠子宮內C3的表現情形 11要研究方向與實驗背景 12二章 材料與方法 13驗材料 13驗方法 13、小鼠子宮液、輸卵管沖洗液與貯精囊液樣品製備 13.1 自然週期子宮液之取得 13.2著床前期子宮沖洗液之取得 13.3著床前期輸卵管沖洗液之取得 14.4 貯精囊液的收集 14、由貯精囊液純化CEACAM10 的方法 14.1陰離子交換樹脂層析法 14.2 高效液相層析法 15、蛋白質的分析方法 17.1 蛋白質定量 17.2 SDS-聚丙烯醯胺膠體電泳 17.3 Coomassie Blue G-250染色 19.4西方墨點法 19、補體C3溶血活性分析 21三章 實驗結果 25、小鼠貯精囊液CEACAM10蛋白質的純化 25、小鼠CEACAM10對母鼠子宮液內蛋白質的影響 25、小鼠CEACAM10對母鼠子宮液內補體C3的影響 25、著床前期母鼠子宮液與輸卵管液內補體C3的變化 26、小鼠CEACAM10蛋白質對人類補體C3的影響 27、小鼠CEACAM10蛋白質對人類補體C3活化補體傳統路徑的影響 28四章 實驗討論 29、小鼠CEACAM10對小鼠補體C3與人類補體C3的影響 29、小鼠CEACAM10在母鼠生殖系統的生理功能 30次 32一、純化小鼠貯精囊液CEACAM10蛋白的離子交換樹脂層析圖 33二、小鼠子宮液與CEACAM10反應後蛋白質的變化 35三、以質譜分析小鼠子宮液分子量180-kDa位置的蛋白質 37四、免疫偵測小鼠子宮液與CEACAM10反應後補體C3的變化 39五、以質譜分析小鼠子宮液分子量120-kDa位置的蛋白 41六、CEACAM10具有時間遞增性的減少小鼠子宮液內補體C3 43七、CEACAM10具有濃度遞增性的減少小鼠子宮液內補體C3 45八、小鼠著床前期子宮液中補體C3與CEACAM10變化於還原性與非還原性SDS-PAGE的圖形 47九、小鼠著床前期輸卵管液中補體C3與CEACAM10變化於還原性與非還原性SDS-PAGE的圖形 49十、人類補體C3與CEACAM10反應後的變化 51十一、在有缺乏人類補體C3血清的情況下，小鼠CEACAM10具有時間遞增性的減少補體C3 53十二、在有缺乏人類補體C3血清的情況下，小鼠CEACAM10具有濃度遞增性的減少人類補體C3 55十三、人類補體C3活性溶血活性分析法的建立 57十四、小鼠CEACAM10具有時間遞增性的降低人類補體C3溶血反應 59十五、小鼠CEACAM10具有濃度遞增性的降低人類補體C3溶血反應 61考文獻 63application/pdf1214826 bytesapplication/pdfen-US小鼠貯精囊液CEACAM10補體C3補體活性mouseseminal vesicle secretionC3complement activityhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178846/1/ntu-97-R95b46003-1.pdf