| dc.contributor.advisor | 趙知章 | zh_TW |
| dc.contributor.advisor | Chao, Chih Chang | en_US |
| dc.contributor.author (Authors) | 曾惠敏 | zh_TW |
| dc.contributor.author (Authors) | Tseng, Hui Min | en_US |
| dc.creator (作者) | 曾惠敏 | zh_TW |
| dc.creator (作者) | Tseng, Hui Min | en_US |
| dc.date (日期) | 2009 | en_US |
| dc.date.accessioned | 8-Dec-2010 02:00:37 (UTC+8) | - |
| dc.date.available | 8-Dec-2010 02:00:37 (UTC+8) | - |
| dc.date.issued (上傳時間) | 8-Dec-2010 02:00:37 (UTC+8) | - |
| dc.identifier (Other Identifiers) | G0096754008 | en_US |
| dc.identifier.uri (URI) | http://nccur.lib.nccu.edu.tw/handle/140.119/49169 | - |
| dc.description (描述) | 碩士 | zh_TW |
| dc.description (描述) | 國立政治大學 | zh_TW |
| dc.description (描述) | 生命科學研究所 | zh_TW |
| dc.description (描述) | 96754008 | zh_TW |
| dc.description (描述) | 98 | zh_TW |
| dc.description.abstract (摘要) | 蛋白質激酶 CK2 是一種多功能的絲胺酸／蘇胺酸蛋白激酶，且普遍存在於哺乳類動物細胞中，CK2 受質眾多，對於細胞週期的發展、轉錄作用以及抗細胞凋亡等過程中扮演很重要的角色。SRF 是一種哺乳類動物的轉錄因子，它會結合到血清反應元素 SRE 上進而調控一些促進細胞存活的基因轉錄作用。Mcl-1歸類於抗細胞凋亡 Bcl-2 家族，具有促進細胞存活的能力。過去研究顯示 SRF 的 DNA 結合活性會受到蛋白激酶 CK2 的磷酸化而增加，且 SRF 對 Mcl-1 的活性調控作用也被描述在其他的研究中，然而，對於細胞的訊息目前還沒有更詳細的研究。在本實驗中，我們探討是否可以藉由 CK2 調控 SRF 的路徑來影響 Mcl-1 的表現以作為抗細胞凋亡的機制。利用 CK2 抑制劑 TBB 處理的結果顯示，在 4 hr 後，phospho-SRF 蛋白質表現的降低具有劑量相關性。而相似的降低也可以從 Mcl-1 的 mRNA 和蛋白質表現量觀察到。處理 24 hr 後，phospho-SRF 的蛋白質表現量有顯著降低，而 Mcl-1 的 mRNA 表現量相較 Mcl-1 的蛋白質影響層面微弱。另一方面，轉染野生型 CK2α 會增加 phospho-SRF，相反的，轉染抑制催化活性的突變型 CK2αA156 則會顯著降低 phospho-SRF 的表現。更進一步，野生型 CK2α 同時增加 Mcl-1 的 mRNA 及蛋白質層級，而 CK2αA156 則會降低 Mcl-1 的表現。突變型的 SRF99A 轉染作用降低 Mcl-1 的 mRNA 及蛋白質，並經由共同轉染的實驗顯示具有抵抗上游野生型CK2α 對 Mcl-1 蛋白質的影響。綜合這些結果我們認為 CK2α對 SRF 的訊息調控影響包括對 Mcl-1 的表現。且這條訊息路徑所促進的 Mcl-1 蛋白質表現可能對魚藤酮處理所引發的細胞凋亡作用具有保護的效果。 | zh_TW |
| dc.description.abstract (摘要) | Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrates and is ubiquitously expressed in mammalian cells to play an important role in cell cycle progression, transcription, and anti-apoptosis. The serum response factor (SRF) is a mammalian transcription factor which binds to serum response element (SRE) and mediates some gene transcriptions relevent to promote the cell survival. The Myeloid cell leukemia 1 (Mcl-1) belongs to the anti-apoptotic Bcl-2 family and its effect are involved in promoting cell viability. Previous studies have revealed that the DNA-binding activity of SRF is enhanced when it is phosphorylated by protein kinase CK2. The activation regulation of Mcl-1 by SRF has also been reported in other studies. However, the detailed cellular signaling has not been studied well. In the present study, we investigate whether the regulation of Mcl-1 expression through CK2-mediated SRF pathway is involved in its anti-apoptotic effects. The results from CK2 inhibitor TBB revealed that the phosphorylated SRF were reduced in a dose-dependent manner after 4 hr of TBB treatments in PC12 cells. The similar decreases were also observed in the mRNA and protein levels of Mcl-1. After a 24 hr exposure of PC12 cells to TBB, a decreased in phosphorylated SRF and Mcl-1 mRNA were observed; a decreased in Mcl-1 protein level was also detected, albeit to a lesser extent. On the other hand, transfection of the wildtype CK2α increased, whereas transfection of the catalytically inactive CK2αA156 mutant decreased phosphorylated SRF. Further, wildtype CK2α increased, whereas CK2αA156 mutant decreased the mRNA and protein levels of Mcl-1. Furthermore, the mutant SRF99A transfection decreased, the mRNA and protein levels of Mcl-1 and antagonized the up-regulatory effects of wildtype CK2α on Mcl-1 protein level in the co-transfection experiments. These results together suggest that CK2α-mediated SRF signaling is involved in the regulation of Mcl-1 expression, and this signaling pathway may involves the anti-apoptotic effects of Mcl-1 against rotenone treatment. | en_US |
| dc.description.tableofcontents | 目 錄 V 圖 次 VIII 縮寫表 IX 第一章 緒論 1 第一節、蛋白激酶 CK2 2 一、蛋白激酶 CK2 對細胞週期的影響 3 二、蛋白激酶 CK2 對轉錄因子的影響 4 三、蛋白激酶 CK2 對細胞凋亡的調控 5 第二節、血清反應因子 (serum response factor) 6 一、血清反應因子對細胞的影響 7 二、血清反應因子參與細胞保護機制 7 第三節、抗細胞凋亡蛋白 Bcl-2 家族 7 第四節、抗細胞凋亡蛋白 Mcl-1 9 第五節、計劃性細胞凋亡 (Apoptosis) 10 一、細胞死亡的形式 10 二、細胞凋亡機制 11 第六節、PC12 細胞株 12 第七節、本論文之研究目的 13 第二章 實驗材料與研究方法 14 第一節、細胞培養 15 一、細胞株種類及來源 15 二、細胞培養 15 三、藥物處理 16 四、細胞轉染 16 第二節、質體製備 17 一、抗生素篩選及養菌 17 二、抽質體 DNA 17 三、菌種保存 18 第三節、西方點墨法 18 一、蛋白質測定 19 二、蛋白質萃取 19 三、配製樣本 19 四、鑄膠 20 五、蛋白質電泳 (SDS-PAGE) 20 六、轉漬 (transfer) 20 七、免疫轉印 (Immunoblotting) 21 八、免疫沉澱法 (Immunoprecipitation, IP) 22 第四節、即時定量聚合酶連鎖反應 23 一、萃取 DNA 23 二、互補鏈 DNA 反轉錄反應 24 三、即時定量聚合酶連鎖反應 (q-PCR) 24 第五節、細胞存活率分析 25 第六節、統計分析 25 第三章 實驗結果 26 第一節、抑制 CK2 表現量會降低細胞的存活率 27 第二節、CK2 抑制劑 TBB 處理 4 hr 對細胞中 phospho-SRF 或 Mcl-1 的mRNA 及蛋白質表現 29 第三節、CK2 抑制劑 TBB處理 24 hr 對細胞中 phospho-SRF 或 Mcl-1 的mRNA 及蛋白質表現 31 第四節、轉染 CK2α 的質體 DNA 影響細胞中 phospho-SRF 或 Mcl-1 的 mRNA 及蛋白質表現 33 第五節、轉染突變型 SRF99A 的質體 DNA 影響 Mcl-1 的 mRNA及蛋白質 表現量 35 第六節、轉染野生型 CK2αWT 與突變型 SRF99A 的質體 DNA 對 Mcl-1 蛋白質的影響 37 第七節、轉染野生型 CK2αWT 的質體 DNA 對魚藤酮 (rotenone) 處理 24hr的細胞存活率影響 39 第八節、轉染野生型 CK2αWT 與突變型 SRF99A 的質體 DNA 對魚藤酮處 理24 hr 的細胞存活率及 Mcl-1蛋白質的影響 41 第四章 討論 44 第五章 結論 50 參考文獻 51 附錄一 XI 附錄二 XII 附錄三 XIII 附錄四 XIV 附錄五 XV | zh_TW |
| dc.language.iso | en_US | - |
| dc.source.uri (資料來源) | http://thesis.lib.nccu.edu.tw/record/#G0096754008 | en_US |
| dc.subject (關鍵詞) | 腎上腺髓質嗜鉻細胞瘤細胞株 | zh_TW |
| dc.subject (關鍵詞) | 蛋白激酶 CK2 | zh_TW |
| dc.subject (關鍵詞) | 血清反應因子 | zh_TW |
| dc.subject (關鍵詞) | 骨髓細胞白血病-1 | zh_TW |
| dc.subject (關鍵詞) | 抗細胞凋亡 | zh_TW |
| dc.subject (關鍵詞) | PC12 cells | en_US |
| dc.subject (關鍵詞) | protein kinase CK2 | en_US |
| dc.subject (關鍵詞) | SRF | en_US |
| dc.subject (關鍵詞) | Mcl-1 | en_US |
| dc.subject (關鍵詞) | anti-apoptosis | en_US |
| dc.title (題名) | 蛋白激酶 CK2 與轉錄因子 SRF 所調控之抗細胞凋亡蛋白 Mcl-1 對 PC12 神經細胞之保護機制的探討 | zh_TW |
| dc.title (題名) | Anti-apoptotic effects of Mcl-1 through CK2-mediated SRF pathway in PC12 cells | en_US |
| dc.type (資料類型) | thesis | en |
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