臺灣博碩士論文加值系統

C/EBP-β是CCAAT/增強子結合蛋白的一員，為一含basic leucine zipper區域的轉錄因子之成員。與C/EBP-α相似，C/EBP-β為無介入子基因，能產生三種N端不同長度的異構物：分別為38-kDa的全長C/EBP-β (C/EBP-β1)，35-kDa的肝轉錄活化蛋白(LAP;C/EBP-β2)，20-kDa的肝轉錄抑制蛋白(LIP; C/EBP-β3)。根據我們實驗室先前的研究推測，C/EBP-β可能藉由參與結合於內皮素-1(ET-1)啟動子-1294~-1281 bp之間的區域，而促使初代培養的自發性高血壓大白鼠(SHR)血管平滑肌細胞(VSMCs)中的ET-1表現。為釐清C/EBP-β各異構物如何參與調控ET-1啟動子，我們分別建構出全長C/EBP-β，LAP，LIP的表現載體，轉染至VSMC中。實驗結果發現過度表現C/EBP-β2 (LAP)在初代培養的正常血壓大白鼠(WKY)之VSMC，與平滑肌細胞株A10中，均對ET-1啟動子產生抑制效果；C/EBP-β1 (全長C/EBP-β)與C/EBP-β3 (LIP)則在平滑肌細胞WKY與A10對於ET-1啟動子的調控則並不明顯。另外，藉由缺損與點突變之ET-1啟動子，與各C/EBP-β表現載體共同轉染，可以發現轉染表現的C/EBP-β可能並非經由ET-1啟動子-1294~-1281 bp區域，而是透過其他位置來調控ET-1的表現。此顯示C/EBP-β似乎並不參與該片段區域的調控，至於是透過何處，則需做進一步研究。

The CCAAT/enhancer-binding protein-β (C/EBP-β), one of the C/EBP family, is a basic leucine zipper transcription factor. As C/EBP-α, C/EBP-β is an intronless gene which could produce several N-terminally truncated isoforms: the 38-kDa full-length C/EBP-β (C/EBP-β1), the 35-kDa liver transcriptional activating protein (LAP;C/EBP-β2), the 20-kDa liver transcriptional inhibitory protein (LIP;C/EBP-β3). According to our previous studies, C/EBP-β may be involved in binding the region between -1294~-1281 bp of ET-1 promoter which may be responsible for the increase of ET-1 expression in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertension rat (SHR). In order to clarify which C/EBP-β isoform could regulate ET-1 promoter, the expression vectors of full-length C/EBP-β, LAP, and LIP were cloned and then transfected to VSMCs. Our results indicate that C/EBP-β2 (LAP) could repress ET-1 promoter reporter activity both in VSMC cell line A10 and primary cultured VSMC from Wistar-Kyoto rat (WKY). C/EBP-β1 (the full-length C/EBP-β) and C/EBP-β3 (LIP) had no obviously difference in regulation of ET-1 promoter between VSMC from WKY and A10 cell line. Additionally, through co-transfecting the C/EBP-β expression vectors with the truncated and mutated ET-1 promoters, the exogenous C/EBP-β which regulated ET-1 expression wasn’t through the region between -1294~-1281 bp, but through the other site of the promoter. It implicated that C/EBP-β wouldn’t participate in the crucial space. The precise position of C/EBP-β bound element at ET-1 promoter will be clearified in the future studies.

目錄
中文摘要
英文摘要
壹、緒論
貳、材料與方法
一、實驗材料
1. 實驗動物
2. 細胞株
3. 菌株
4. 質體
5. 實驗藥品
6. 操作儀器
二、實驗方法
1.WKY胸主動脈平滑肌細胞的初代培養
2.細胞之繼代培養
3.質體DNA之抽取
4.選殖DNA片段的製作
5.洋菜膠電泳法
6.DNA片段純化
7.重組質體之構築
8.大腸桿菌之轉形作用
9. Rapid Screen
10.細胞之轉染作用
11.細胞冷光酵素活性測定
12.細胞核蛋白之萃取
13.蛋白質濃度分析
14.聚丙醯胺(SDS-polyacrylamide)凝膠電泳法
15.西方墨點法
參、結果
1. 胸主動脈平滑肌細胞培養
2. 全長C/EBP-β cDNA之分析
3. C/EBP-β異構物subclone
4. 將C/EBP-β表現載體轉染至A10 cell，以western blot觀察C/EBP-β異構物之表現情形
5. 將C/EBP-β表現載體轉染至WKY胸主動脈平滑肌細胞，以western blot觀察C/EBP-β異構物之表現情形
6. 藉A10 cell轉染，觀察C/EBP-β異構物調控ET-1啟動子之情形
7. 藉WKY胸主動脈平滑肌細胞轉染，觀察C/EBP-β異構物調控ET-1啟動子之情形
8. 利用外生性C/EBP-β2 tag的抑制效果，預測C/EBP-β在ET-1啟動子之調控位置
9. ET-1啟動子上C/EBP結合序列的可能位置
肆、討論
伍、實驗圖表
陸、參考文獻

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