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研究生:沈國芳
研究生(外文):Kuo-Fang Shen
論文名稱:人類胎盤融合性蛋白syncytin基因調控之探討
論文名稱(外文):Gene regulation of the human placental fusogenic protein, syncytin
指導教授:陳宏文陳宏文引用關係
指導教授(外文):Hungwen Chen
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:2002
畢業學年度:90
語文別:中文
中文關鍵詞:胎盤人類內生性反轉錄病毒
外文關鍵詞:placentaHERV
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HERV-W是最近被確認的人類內生性反轉錄病毒,其轉錄所產生的mRNAs包含著gag、 pol及env序列。HERV-W的envelope基因轉譯產生功能性蛋白質稱之為syncytin。Syncytin基因主要表現在胎盤的滋養葉融合細胞(syncytiotrophoblast)。其功能與調控胎盤滋養葉胞層細胞(cytotrophoblast)融合生成滋養葉融合層細胞有關。在人類胎盤形態發生過程中,syncytin可能扮演著重要的角色。
GCMa轉錄因子高度的表現在labyrinth層滋養葉細胞,GCMa對於胎盤發育是必需的,在老鼠模式GCMa基因被破壞掉而導致無法生成labyrinth層及滋養葉細胞無法融合生成滋養葉融合層細胞。本篇實驗報告,我們提供證據,指出在轉染實驗中確定了人類融合性蛋白syncytin基因可藉由GCMa轉錄因子加以調控。EMSA實驗中,我們確定了GCM蛋白質是會和syncytin基因形成複合物,而在DNase I footprinting實驗中,我們進一步的確定了GCMa蛋白質的結合位置25538/25547與28026/28033。最後,在共同轉染實驗中我們將這兩個結合位置剔除,GCMa轉錄因子則會失去誘導轉活化syncytin基因啟動子之能力。綜合以上實驗結果,我們成功的證實GCMa轉錄因子可以調控人類融合性蛋白syncytin基因及將GCMa在滋養葉融合層細胞發育過程之生理功能與syncytin所造成之細胞融合關連起來。

HERV-W is one of recently identified human endogenous retrovirus and it was found to transcribe three kinds of mRNAs including gag, pol and env. The envelope gene of HERV-W encodes a functional protein that was named as syncytin. Expression of syncytin gene is restricted to placental syncytiotrophoblast. The ability of human cytotrophoblast cells to fuse into syncytiotrophoblast is mediated by syncytin. Therefore, sync- ytin may play an important role in human placental morphogenesis.
The transcription factor, GCMa, is highly expressed in the lab-yrinthine trophoblast cells. GCMa is required for placenta develop-ment because genetic ablation of mouse GCMa leads to a failure oflabyrinth layer formation and no fusion of trophoblasts to syncytiot-rophoblasts. In this study, we confirmed that GCMa transcription factor can regulate human syncytin gene in the transient expression assay. Our results also indicated that GCMa protein can bind to thepromoter of syncytin gene by electrophoretic mobility shift assay. We further identified two GCMa binding sites in the promoter regi-on, 25538/25547 and 28026/28033, using DNase I footprinting anal-ysis. In transient expression assay, the transactivation of GCMa on syncytin promoter could not be found when these two GCMa bindi-ng sites were deleted. Overall, we successfully demonstrated that GCMa can regulate human syncytin gene expression and linked thephysiological function of GCMa in the development of syncytiotrop-hoblast to syncytin-mediated fusion.

目錄
目錄……………………………………………………………………..Ⅰ
中文摘要………………………………………………………………..Ⅳ
英文摘要………………………………………………………………..Ⅴ
第一章 前言………………………………………………….……… …1
一、胎盤……………………………………………………….………..1
二、融合細胞滋養胚細胞的分化(syncytiotrophoblast
differentiation)……………………………………………………...4
三、轉錄因子GCM (glial cells missing)……………………………5
四、人類融合性蛋白Syncytin基因…………………………………...9
第二章 材料與方法………………………………………………… …11
壹、實驗材料:………………………………………………………..11
貳、實驗方法:………………………………………………………..11
一:聚合酉每連鎖反應 (Polymerase chain reaction)
………………….11
二:勝任細胞製備 ( Competent cells preparation)
…………………..11
三:DNA和質體之接合反應 (Ligation of DNA insert to
expression plasmid) ……………………………………………....12
四:質體之轉形反應 (Transformation of competent cell)
……………12
五:小量DNA之製備純化 (Mini-scale DNA preparation )……… 12
六:中量DNA之製備純化(Midi-scale DNA preparation )…………13
七:洋菜凝膠電泳分析 (Agarose gel electrophoresis)
……………….14
八:洋菜凝膠電泳純析 (Elution of DNA in agarose gel)
…………….14
九:大量DNA之製備純化 (Large-scale DNA
preparation:CsCl banding)…….…………………………………14
十、 SDS聚丙烯醯胺凝膠電泳分析
( SDS-PAGE electrophoresis)……………………………………...15
十一、西方墨點法分析 (Western blotting)……………………… 16
十二、質體構築 (plasmid constructs)…………………………… 16
(1) pCAT3 E1b表現質體: (13243/18725, 18726/23397,
22473/26413, 25468/30953, 25468/28839, 25468/28066,
28067/30953, 28840/30953, 28067/28839, 37553/38281,
38263/43418)…………………………………………………………16
1. (25468/30953 ) pE1bCAT3,( 25468/28839) pE1bCAT3,
(25468/28066) pE1bCAT3, (28067/30953) pE1bCAT3,
(28840/30953) pE1bCAT3, (28067/28839) pE1bCAT3, (37553/38281) pE1bCAT3表現質 體……………………………...…16
2. (22473/26413) pE1bCAT3表現質體…………………………..……17
3.( 13243/18725) pE1bCAT3, (18725/23397) pE1bCAT3,
(38263/43418) pE1bCAT3表現質體……………….………………..18
(2) △d(25468/30953)pE1bCAT3、△p(25468/30953) pE1bCAT3
and △dp(25468/30953) pE1bCAT3表現質體……………….……..18
十三、Cell culture and transfection 細胞培養及轉染作用
……….…..19
十四、CAT (chloramphenicol acetyl transferase) assay分析方法
.……19
十五、Electrophoretic mobility shift assay (EMSA)分析方法…….…..20
十六、DNaseⅠFootprinting………………………………..……….….22
第三章 結果……………………………………………………………25
第一節 GCMa轉錄因子對各片段融合性蛋白syncytin基因
報告構築質體的影響………………………………..…….25
第二節 在體外分析人類融合性蛋白syncytin基因預測的
GCMa結合位置是否會和GCMa蛋白質結合……………26
第三節 在體外分析GCMa和人類融合性蛋白syncytin基因27978/28077與25488/25587 片段的直接結合位置……...27
第四節 在體外分析人工合成pGCMa與dGCMa序列與
是否會和GCMa蛋白質結合……………………………...27
第五節 GCMa轉錄因子對人類融合性蛋白基因syncytin之
GCMa結合位置突變之影響…………………………...….28
結果圖表…………………………………………………………...30
第四章 討論與結語…………………………………………….…..….36
第五章 參考文獻…………………………………………………...….41

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