跳到主要內容

臺灣博碩士論文加值系統

(3.236.124.56) 您好!臺灣時間:2021/07/31 03:21
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:鍾杜珍
研究生(外文):Chung,Tu-Chen
論文名稱:Oct-2會使體外培養的主動脈平滑肌細胞之ET-1高度表現
論文名稱(外文):Oct-2 Responsible for High Expression of Endothelin -1 in Cultured Aortic Smooth Muscle Cells
指導教授:趙壯飛呂美華呂美華引用關係陳正繹
指導教授(外文):Chung-Faye ChaoMei-Hua LuJang-Yi Chen
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:48
中文關鍵詞:內皮素-1主動脈平滑肌細胞
外文關鍵詞:ET-1Oct-2VSMC
相關次數:
  • 被引用被引用:0
  • 點閱點閱:138
  • 評分評分:
  • 下載下載:14
  • 收藏至我的研究室書目清單書目收藏:0
內皮素主要藉由血管內皮細胞及平滑肌細胞所合成及分泌,其在高血壓及動脈粥狀硬化等心血管疾病方面扮演了一個重要角色。根據本實驗室先前的研究,在正常血壓大白鼠(Wistar-Kyoto rats, WKY)與自發性高血壓大白鼠(spontaneously hypertensive rats, SHR)血管管壁平滑肌細胞(vascular smooth muscle cells, VSMCs)表現ET-1的量上,SHR較WKY高2.5倍。同時也顯示造成SHR VSMCs中ET-1過量表現的作用區域,具有一個growth hormone factor-1(GHF-1)保留序列。然而,在SHR VSMCs中並無GHF-1的表現;但是,與GHF-1同屬於POU homeodomain family成員的Oct-1/2及C/EBPß,可以與GHF-1保留序列結合。先前的研究也發現Oct-1的表現量在SHR及WKY中並無明顯差異;但是在Oct-2及C/EBPß的表現量上,SHR明顯高於WKY。為了證實Oct-2轉錄因子很可能是造成SHR VSMCs之ET-1有較高表現量的主要原因,本論文先利用西方點墨法分析,確定重組質體Oct-2在A10及WKY VSMCs中會表現,再將Oct-2與不同片段之ET-1啟動子共同轉染至A10及WKY VSMCs中進行分析,實驗結果顯示,Oct-2確實會提高A10及WKY VSMCs中ET-1啟動子的活性。所以Oct-2的確是調控A10及WKY VSMCs中ET-1啟動子的重要因素,而C/EBPß在調控ET-1啟動子上,可能只扮演一個co-facter的角色。因此,我們推論Oct-2可能是SHR VSMCs中ET-1高度表現的重要因子。
Endothelin-1 (ET-1), synthesized and secreted by both vascular endothelial and vascular smooth muscle cells (VSMCs), plays an important role in the development of various circulatory disorders, including hypertension and atherosclerosis. According to our previous studies, ET-1 secreted from aortic smooth muscle cell of spontaneously hypertensive rats (SHR) was about 2.5 times higher than that of age-matched Wistar-Kyoto rats (WKY). We also demonstrated that GHF-1 binding sequence of the ET-1 promoter region was responsible for the enhancement of ET-1 expression. However, there was no GHF-1 expression in VSMCs of SHR. But we found Oct-1/2, which belonging to the same POU homeodomain family as GHF-1, could bind to the GHF-1 binding sequence with the co-factor C/EBPß. We also found that the expression of Oct-1 was similar in SHR and WKY but the expression of Oct-2 and C/EBPs were higher in SHR than in WKY. In order to clarify the transcription factor Oct-2 may responsible for the higher expression of ET-1 in VSMCs from SHR, an Oct-2 expressed construct was transfected to A10 and WKY VSMCs. Western blotting analysis was used to detect the expression of Oct-2 protein. Then, the effect of Oct-2 on the regulation of ET-1 promoter was detected by reporter assay. Our data showed the Oct-2 was expressed in both A10 and WKY VSMCs . We also found that Oct-2 could regulate ET-1 promoter activity in A10 and WKY VSMCs. These data demonstrate that Oct-2 is an important factor for ET-1 expression, and C/EBPβ may only play a co-factor role of the ET-1 promoter regulation in A10 and WKY VSMCs. Therefore, we infer that Oct-2 may be an important factor for ET-1 overexpression in SHR VSMCs.
正文目錄 Ⅰ
圖表目錄 Ⅲ
中文摘要 Ⅳ
英文摘要 Ⅴ
第一章 緒言 1
第二章 實驗材料與方法 8
第一節 實驗動物 8
第二節 細胞株 8
第三節菌株 8
第四節質體 8
第五節實驗藥品與試劑 8
第六節實驗儀器 12
第七節實驗方法 14
1. WKY胸主動脈平滑肌細胞的培養(primary culture) 14
2. 細胞之繼代培養 15
3. WKY胸主動脈平滑肌細胞之純度鑑定 16
4. 質體DNA之抽取 16
5. 洋菜膠電泳法 17
6. DNA片段純化 18
7. 重組質體之構築 18
8. 大腸桿菌之轉形作用 19
9. Rapid Screen 19
10. 細胞之轉染作用(transfection) 20
11. 細胞冷光酵素(Luciferase)活性測定 20
12. 總蛋白之萃取 21
13. 細胞核蛋白之萃取 21
14. 蛋白質濃度分析 22
15. 聚丙醯胺(SDS-polyacrylamide)凝膠電泳法 23
16. 西方墨點法 24

第三章 結果 25
第四章 討論 30
第五章 結論 35
第六章 實驗結果(圖表) 36
第七章 參考文獻 45

圖表目錄:
圖1 : A10細胞株形態圖
圖2 : WKY大鼠胸主動脈平滑肌細胞之純度鑑定
圖3 : Oct-2 subclone 示意圖
圖4 : 酵素切位分析雙重確認pcDNAOct2-9
圖5 : Western blot分析轉染3.1A與Oct-2表現載體至 A10平滑肌細胞株後,Oct-2之表現情形
圖6 : Western blot分析轉染3.1A與Oct-2表現載體至WKY胸主動脈平滑肌細胞後,Oct-2表現情形
圖7 : 轉染不同量的Oct-2的表現載體量對大白鼠胸主動脈A-10平滑肌細胞株中不同種類ET-1啟動子的試驗結果
圖8 : 轉染不同量的Oct-2的表現載體量對四週大WKY胸主動脈平滑肌細胞中不同種類ET-1啟動子的試驗結果
圖9 : Oct-2轉染至A10平滑肌細胞株,觀察對不同種類ET-1啟動子之實驗結果
圖10 : Oct-2轉染至四週大WKY胸主動脈平滑肌細胞株,觀察對不同種類ET-1啟動子之實驗結果
Alberts GF, Peifly KA, Johns A, Kleha JF and Winkles JA: Constitutive endothelin-1 overexpression promotes smooth muscle cell proliferation via an external autocrine loop. J. Biol. Chem. 269: 10112-10118, 1994.
Andersen B and Rosenfeld MG: POU domain factors in the neuroendocrine system: lessons from developmental biology provide insights into human disease. Endocr. Rev. 22(1):2-35, 2001.
Antonio V, Brouillet A, Janvier B, Monne C, Bereziat G, Andreani M and Raymondjean M: Transcriptional regulation of the rat type IIA phospholipase A2 gene by cAMP and interleukin-1beta in vascular smooth muscle cells: interplay of the CCAAT/enhancer binding protein (C/EBP), nuclear factor-kappaB and Ets transcription factors. Biochem. J. 368(Pt2): 415-424, 2002.

Dawson S. J., Yoon S. O., Chikaraishi D. M.. Lillycrop K. A. and Latchman D. S. :The Ott-2 transcription factor represses tyrosine hydroxylase expression via the heptamer motif in the promoter. Nucl. Acids Res. 22: 1023-1028, 1994
Dasen JS, O’Connell SM, Flynn SE, Treier M, Gleiberman AS, Szeto DP, Hooshmand F, Aggarwal AK and Rosenfeld MG: Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types. Cell 97: 587-598, 1999.
De la HM, Vila V, Jimenez O and Castrillo JL: Anterior pituitary development and Pit-1/GHF-1 transcription factor. Cell Mol Life Sci. 54(10): 1059-66, 1998
Folkow B: Physiological aspects of primary hypertension. Physiol. Rev. 62(2):347-504, 1982.
Fukhgott RF and Zawadski JV: The obligatory role of endothelinal cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373-376, 1980.

Gordon DF, Lewis SR, Haugen BR, James RA, McDemott AS, Wood WM and Ridgway EC: Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin -subunit promoter. J. Biol. Chem. 272:24339-24347, 1997.
Guyton AC: Blood pressure control-special role of the kidneys and body fluids. Science 252(5014): 1813-1816, 1991.
Hatada EN, Chen-Kiang S and Scheidereit C: Interaction and functional interference of C/EBP with octamer factors in immunoglobulin gene transcription. Eur. J. Immunol. 30:174-184, 2000.
Hsu W and Chen-Kiang S: Convergent regulation of NF-IL6 and Oct-1 synthesis by interleukin-6 and retinoic acid signaling in embryonal carcinoma cells. Mol. Cell. Biol. 13(4): 2515-2523, 1993.
Ingraham HA, Chen R, Mangalam HJ, Elsholtz HP, Flynn SE, Lin CR, Simmons DM, Swanson L and Rosenfeld MG: A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55(4): 519-529, 1988.
Karin M, Castrillo JL and Theill LE: Growth hormone gene regulation: a paradigm for cell-type-specific gene activation. Trends Genet. 6: 92-96, 1990.
Kawana M, Lee ME, Quertermous EE and Quertermous T: Cooperative interaction of GATA-2 and AP-1 regulates transcription of the endothelin-1 gene. Mol. Cell. Biol. 15(8): 4225-4231, 1995.

Latchman, D. S.: The Oct-2 transcription factor. J. Biochem. CeN Biol. Vol. 28: No. 10: 1081-1083, 1996
Lee ME, Dhadly MS, Temizer DH, Clifford JA, Yoshizumi M and Quertermous T: Regulation of the endothelin-1 gene expression by fos and jun. J. Biol. Chem. 266: 19043-19039, 2001.

Lillycrop K. A., Dent C. L., Wheatley S. C., Beech M. N., Ninkina N. N., Wood J. N. and Latchman D. S. : The octamer binding protein Ott-2 represses HSV immediate early genes in cell lines derived from latently infectable sensory neurons. Neuron 7: 381-390, 1991.
Lillycrop K. A. and Latchman D. S.: Alternative splicing of the Ott-2 transcription factor is differentially regulated in B cells and neuronal cells and results in protein isoforms with opposite effects on the activity of octamer/TAATGARAT-containing promoters. J. Biol.
Chem. 267: 24960-24966, 1992
Lu MH, Chao ZC, Chang LT and Chao CF: Paracrine function of cultured aortic endothelial cells in spontaneously hypertensive rats. Chin. Med. J. 64: 373-381, 2001.
Lu MH, Chao CF, Huang CG and Chang LT: Coculture of vascular endothelial cells and smooth muscle cells from spontaneously hypertensive rats. Clin. Exp. Hypertens. 25:413-425, 2003.
Owens GK and Schartz SM: Alteration in vascular smooth muscle mass on the spontaneously hypertensive rat. Circ. Res. 51: 280-289, 1982.
Paul M, Zintz M, Bocker W, Dyer M: Characterization and functional analysis of the rat endothelin-1 promoter. Hypertension 25: 683-687, 1995.
Ross MH, Kaye GI and Pawlina W: Histology: A Text and Atlas 4th, 2002, pp.326-347.
Schmitz-Spanke S. and Schipke JD: Potential role of endothelin-1 and endothelin antagonists in cardiovascular diseases. Basic. Res. Cardiol. 95: 290-298, 2000.
Sharma RV and Bhalla RC: Calaium and abnormal reativity of vascular smooth muscle in hypertension. Cell Calcium. 9:267-274, 1988.
Yanagisawa MA, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K and Masaki T : A novel potent constrictor peptide produced by vascular endothelial cell. Nature 332: 411-415, 1988.

蘇偉碩,自發性高血壓大白鼠主動脈平滑肌細胞內皮素基因表現升高其順式調節元件之探討,國防醫學院生物及解剖學研究所碩士論文,1999。

郭佳惠,自發性高血壓大白鼠在腦下垂體前葉中腦下垂體專一轉錄因子之研究,國防醫學院生物及解剖學研究所碩士論文,2001。

楊添鈞,自發性高血壓大白鼠主動脈平滑肌細胞內皮素基因表現升高的相關轉錄因子之確認,國防醫學院生物及解剖學研究所碩士論文,2004。

陳信良,探討C/EBP-β異構物對ET-1啟動子調節之影響,國防醫學院生物及解剖學研究所碩士論文,2005。
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊