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研究生:莊美珍
研究生(外文):Mei Jane Chuang
論文名稱:阿茲海默症蛋白中多酸性區域具誘導神經膠細胞自發性死亡之探討
論文名稱(外文):The acidic-rich domain of the - beta amyloid precursor protein induced glial cell apoptosis
指導教授:唐世杰
指導教授(外文):Shye Jye Tang
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:68
中文關鍵詞:阿茲海默症自發性死亡神經膠細胞
外文關鍵詞:Alzheimer's diseaseAmyloid precursor proteinApoptosisMTT assayLPSThioredoxin
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中文摘要
阿茲海默症 (Alzheimer's Disease) 是癡呆症中常見的一種,通常發生在中、老年時期。Amyloid precursor protein (APP)在阿茲海默症形成中所扮演的角色,已經在分子基因層次與細胞生物學上被廣泛的研究。推測疾病發生原因之一的Amyloid-b peptide (Ab),是由APP所衍生而來的,且Ab在細胞外的沉積,會促進神經突向外分支,產生活氧化物質,促成細胞內的有毒氧化環境,進而活化微神經膠細胞。以上所述,被認為是阿茲海默症發生的主要原因。
根據過去的研究已經發現分泌型和膜結合型的APP,在不同的細胞株中可以調控細胞的生長,以及神經突向外分支與細胞基質的黏附。然而,APP的生物功能與對於阿茲海默症的形成機制尚未完全釐清。在本論文中,我們發現可溶性APP(sAPP)的酸性胺基酸區域(acidic-rich domain,amino acid 188-220),對於神經膠細胞的生物活性有很重要的影響。我們將這段區域與硫氧化還原蛋白(Trx)基因接合,構築到表現載體pET-23a,利用IPTG誘導此重組蛋白(rAPP)在BL21(DE3)pLysS中大量表現,並使用離子交換樹脂來進行rAPP的純化。
經由實驗証實,此重組蛋白Trx-rAPP187-288具有誘發神經膠細胞RBA-1,C6神經膠瘤及BV-2微神經膠細胞程式化死亡的生物特性。將APP187-288進行deletion實驗,發現APP187-288的活性區域位於胺基酸序列220-266。再者,我們證實了神經細胞的程式化死亡,並非由
E.coli脂多醣(LPS)引起,也不是APP和Trx融合後所產生新的重組蛋白造成。另外,在實驗中,若以重組蛋白Trx-rAPP220-288預先處理細胞,其細胞的程式化死亡率將會提高。因此,我們推測rAPP會干擾細胞與細胞間的黏附(cell-cell adhesion)。最後,由上述結果與過去文獻比較中發現,APP胺基酸220-288片段導致神經膠細胞的程式化死亡,可能在阿茲海默症中扮演重要的角色。

Abstract
Alzheimer's disease (AD) is the most common case of dementia occurring in mid- to late-life. Amyloid-b peptide (Ab) is the principal component of the extracellular deposits in AD. Ab promotes neurite outgrowth, generates reactive oxygen intermediates, induces cytotoxic cellular oxidant stress, and promotes microglial activation. However, Ab is derived from the transmembrane protein amyloid precursor protein (APP). A role for APP in the development of AD has been extensively researched in the genetic and cellular approach.
Reports obtained from different cell lines have shown that the secreted or membrane-associated forms of APP regulate cell growth, neurite out-growth and cell matrix adhesion. Nevertheless, function of APP and the relationship between APP and AD remain to be uncovered. Here we focus a acidic-rich domain (amino acid 188-220) in sAPP on the impact of biological activities in glial cell. For functional expression in E. coli, the acidic-rich domain was fused with thioredoxin (Trx) and cloned into the expression vector pET-23a. The expression vectors harboring the acidic-rich domain, rAPP; or Trx ; were transformed into BL21(DE3)pLysS. The transformants were induced with IPTG for overexpression, and the recombinant proteins were purified by ion-exchange chromatography.
The recombinant Trx-rAPP187-288 were demonstrated the biological activity that induced apoptosis of glial cells including RBA-1, C6 glioma and BV-2 microglial. The activating domain, between residue 220 to residue 288, was identified by deletion experiment of APP187-288. The recombinant APP220-288 showing EC50 = 1 μM were demonstrated a potent apoptotic activity. In this report we rule out the possibility that the apoptotic activity was resulted from the contaminant of LPS or creating a novel new protein by fusion with Trx. The apoptotic activity was enhanced by the pretreatment of the recombinant Trx-rAPP220-288, suggesting that apoptotic activity of rAPP may result in the interference of cell adhesion. Our results show a new finding for APP220-288 biological activity, inducing glial cell apoptosis, that may play an important role in Alzheimer's Disease.

目錄
英文摘要 ………………………………………………………………1
中文摘要 ………………………………………………………………3
前言 ……………………………………………………………………5
材料 ……………………………………………………………………12
方法 ……………………………………………………………………15
I.基因重組
A. Plasmid construction ………………………………….15
B. DNA ligation …………………………………………..15
C. Competent cell preparation…………………………..15
D. Transformation …………………………………………………….16
E. Minipreparation of the plasmid DNA for fast quick screening-17
F. 目標質體的確認…………………………………………………….18
II.重組蛋白的表現
A.蛋白質的誘導……………………………………………………….18
B. Sonication………………………………………………………….18
C. Bradford microassay……………………………………………. 19
D.SDS-PAGE……………………………………………………….20
E. Protein purification ………………………………………………….21
F. Western Blotting………………………………………………………………22
III. Boiactivity
1. Cell culture……………………………………………………..23
2. MTT assay………………………………………………………….23
3. NO…………………………………………………………………..24
4. Flow cytometry-PI Cell cycle-analysis……………………. 24
結果…………………………………………………………………….25
1. 基因重組蛋白的表現與純化………………………………………...25
2.APP187-288具有促使腦神經細胞死亡的活性………………………...26
3. APP187-288上的活性區域介於Residue 220-266 ……………………26
4.APP220-288對RBA-1細胞形態的改變……………………………….27
5.APP220-288 EC50的測定……………………………………………….27
6.APP220-288抑制 RBA-1 cells的生長並造成死亡……………………28
6.APP220-288抑制 RBA-1 cells的生長並造成死亡 …………………28
7.熱處理使Trx-rAPP220-288喪失致死活性 ………………………….28
8.Trx-rAPP220-288可有效的模擬APP220-288生物活性…………………29
9.APP220-288可抑制其它神經膠細胞生長 …………………………..29
10. 細胞死亡可能與細胞的附著有關 …………………………….30
11.APP220-288不改變RBA-1的細胞週期……………………………..31
討論.……………………………………………………………………32
1. 關於APP上hydrophilic fragment 具有生物活性的可能性…….32
2. Trx-rAPPs活性之關係…………………………………………….32
3. APP220-288 誘導microglial cell產生nitrite..………………….…..33
4. FACS analysis與MTT assay結果不吻合之可能原因……………33
5. RBA-1 apoptosis現象在treatment 72 hr後才顯著……………….34
6. APP220-288誘導細胞死亡的可能mechanism……………………….34
Reference……………………………………………………………..38
圖表…………………………………………………………………….47
附圖……………………………………………………………………62
Appendix

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