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研究生:陳瑤瑜
研究生(外文):Yao-Yu Chen
論文名稱:HtrA家族蛋白透過功能性拮抗作用調控胎盤細胞入侵
論文名稱(外文):Functional antagonism between high-temperature requirement protein A (HtrA) family members regulates trophoblast cell invasion
指導教授:陳宏文陳宏文引用關係
口試委員:張震東李明亭張功耀黃娟娟
口試日期:2014-06-27
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:44
中文關鍵詞:滋養層細胞胎盤HtrA4細胞入侵
外文關鍵詞:trophoblastplacentaHtrA4cell invasion
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人類滋養層細胞入侵蛻膜化的子宮內膜對於胎盤發育扮演重要的角色,此過程透過來自母親的蛻膜細胞與胚胎滋養層細胞之間的交互作用而被緊密地調控。HtrA4為分泌型絲胺酸蛋白&;#37238;,高度表現在具有入侵能力的絨毛外滋養層細胞,可促進蛻膜化的子宮內膜細胞與滋養層細胞之間的交互作用。相反地,HtrA1與HtrA3則被指出可抑制胎盤細胞入侵的能力。本篇研究發現由蛻膜化的子宮內膜細胞所分泌的HtrA1與HtrA3可以拮抗由HtrA4所調控的胎盤細胞入侵能力,我們證實HtrA1與HtrA3會透過與HtrA4之間的交互作用將其降解,進而抑制胎盤細胞的入侵。內生性的HtrA1與HtrA3分別表現在蛻膜化的子宮內膜基質細胞,T-HESCs,以及子宮內膜上皮細胞,Ishikawa。透過RNA干擾試驗,我們證實蛻膜化的T-HESCs和Ishikawa 細胞培養液中內生性的HtrA1與HtrA3可有效抑制會穩定表現HtrA4的胎盤細胞入侵能力。此外,透過滋養層細胞與蛻膜化子宮內膜單一細胞層的共同培養試驗,也證實可抑制由HtrA4調控的胎盤細胞入侵能力,然而此效應可透過將子宮內膜細胞中內生性的HtrA1與HtrA3剔除掉而回復胎盤細胞原本的入侵能力,說明HtrA1與HtrA3對於滋養層細胞和子宮內膜細胞間的交互作用扮演重要的角色,可調控滋養層細胞的入侵能力。綜合上述實驗結果,本篇研究發現在胎盤發育過程中新的調控機制,透過HtrA家族蛋白之間功能性的交互作用來調控胎盤細胞入侵母體組織的能力。

Human trophoblast invasion of decidualized endometrium is essential for placentation and is tightly regulated and involves decidua-trophoblastic interaction. High-temperature requirement A4 (HtrA4) is a secreted serine protease highly expressed in the invasive extravillous trophoblasts and promotes decidua-trophoblastic interaction. In contrast, both HtrA1 and HtrA3 have been shown to inhibit placental cell invasion. Here we provide evidence that decidua-secreted HtrA1 and HtrA3 antagonize HtrA4-mediated placental cell invasion. We demonstrated that HtrA1 and HtrA3 interact with and degrade HtrA4, thereby inhibit placental cell invasion. HtrA1 and HtrA3 expression is upregulated by decidualization in endometrial stromal and epithelial cells, T-HESCs and Ishikawa cells, respectively. By RNA interference, we demonstrated that HtrA1 and HtrA3 are responsible for the suppression of HtrA4-expressing JAR placental cell invasion by conditioned media of decidualized T-HESCs and Ishikawa cells. Co-culture of the HtrA4-expressing JAR cells with decidualized T-HESC or Ishikawa monolayer also impairs HtrA4-mediated JAR cell invasion, which can be reversed by HtrA1 or HtrA3 knockdown, supporting that HtrA1 and HtrA3 are crucial for trophoblast-decidual cell interaction in the control of trophoblast invasion. Our study reveals a novel regulatory mechanism of placental cell invasion through physical and functional interaction between HtrA family members.

目錄 I
圖表目錄 III
中文摘要 IV
英文摘要 V
第一章 緒論
1.1 胎盤 1
1.2 HtrA家族蛋白 5
1.3 研究動機 8
第二章 材料與方法
2.1 構築重組質體 9
2.2 細胞株培養與轉染 12
2.3 慢病毒感染 13
2.4 SDS聚丙烯醯胺凝膠電泳及西方墨點法 14
2.5 共同免疫沉澱法 15
2.6 活體外蛋白質活性分析 16
2.7 免疫螢光染色 17
2.8 細胞入侵能力檢測實驗 18
第三章 實驗結果
3.1 HtrA家族蛋白在人類胎盤組織中的表現量與分布情形 20
3.2 HtrA家族蛋白之間的交互作用 21
3.3 HtrA1與HtrA3透過蛋白&;#37238;水解作用降解HtrA4 22
3.4 外生性HtrA1與HtrA3抑制由HtrA4調控之胎盤細胞入侵能
力 23
3.5 內生性HtrA1與HtrA3抑制由HtrA4調控之胎盤細胞入侵能
力 23
3.6 HtrA家族蛋白調控滋養層細胞與子宮內膜細胞之間的交互作
用 24
第四章 討論與總結 26
第五章 圖表 29
第六章 參考文獻 42


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