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研究生:陳志俊
研究生(外文):Chih-Chun Chen
論文名稱:心血管細胞隙連結的研究:富含膽固醇的飲食與酒精對連接素的影響,以及連接素抗體的製作
論文名稱(外文):Studies on Cardiovascular Cell Gap Junctions: Effects of Cholesterol-Enriched Diet and Alcohol on Connexin Expression, and Production of Antibody Against Connexin
指導教授:王成德葉宏一
指導教授(外文):Cheng-Teh WangHung-I Yeh
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
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:2002
畢業學年度:90
語文別:中文
論文頁數:130
中文關鍵詞:隙連結連接素膽固醇酒精抗體製造免疫組織化學染色共軛焦雷射掃描顯微鏡西馬他丁
外文關鍵詞:gap junctionconnexincholesterolalcoholantibody productionimmunohistochemistryconfocal laser scanning microscopeSimvastatin
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隙連結 (gap junction) 為細胞膜上的管道群,它橫跨相鄰細胞間的間隙,在細胞與細胞之間提供直接溝通的路徑。每個隙連結管道由一對連接半管 (connexon) 所構成,連接半管又由六個連接素 (connexin) 所組成,而連接素則是屬於多基因家族。不同家族成員的連接素所構成的隙連結管道,其性質相異。另外,隙連結管道掌管代謝和電流訊息進出毗鄰細胞,與許多生命現象與隙連結息息相關,例如心臟與血管功能正常運作的維持。
本研究利用抗體結合高解像力的共軛焦雷射掃描顯微鏡術來探討兩種常見的臨床疾病─高血脂症和酗酒對心血管系統隙連結的影響。高膽固醇血症是動脈粥狀硬化的危險因子之一,動脈粥狀硬化已知會影響動脈內皮細胞的隙連結。在大白鼠主動脈內皮細胞的隙連結是由connexin37、connexin40及connexin43所構成。然而膽固醇與內皮細胞各種連接素間的關係卻不清楚,本論文使用餵食高膽固醇飼料在小鼠身上引起高膽固醇血症,再給予短期降膽固醇藥劑 simvastatin,來探究高膽固醇血壓對隙連結的影響。結果顯示:i) 在高膽固醇的環境中,內皮細胞的connexin37及connexin40會降低;ii) 餵食simvastatin七天後,connexin37有顯著的回升,connexin40則否;iii) 小鼠的主動脈並不表現connexin43,即使是餵食膽固醇或加上simvastatin的治療均沒有偵測到connexin43的情況。
另一方面,已知過量酒精會影響心臟的功能,然而,酒精是否會改變心臟的隙連結並不了解,本論文利用餵食動物飲用水中添加不同濃度的酒精及飲用時間的長短,來觀察心臟分布最廣的連接素─connexin43的變化。結果顯示,餵食12%酒精一個月,connexin43沒有明顯的變化,而餵食12%酒精或36%酒精二個月,其心臟connexin43的量有上升的趨勢,此外,餵食36%酒精二個月,還會造成左心室隙連結分布的混亂。
欲探究心血管系統中隙連結扮演的角色,最重要的是必須擁有具專一性的抗連接素抗體。本論文嘗試利用大白鼠來生產多株抗體,並使用親和力膠管柱來純化抗體,最後用西方轉漬法和免疫細胞化學染色法來檢查抗體的專一性。結果顯示,大白鼠在生產抗connexin37、抗connexin40及抗connexin45抗體方面,皆有不錯的成效。這些抗體可以作為往後研究隙連結的利器。
以上結果顯示,心血管疾病會伴隨有隙連結分布與連接素表現的改變,而且每種連接素所受到的影響並不相同,這暗示每種連接素在心血管疾病的致病過程中扮演不同的角色。
Gap junctions are clusters of cell membrane channels that cross the gap between the adjacent cells and provide pathway for direct intercellular communication. Each channel consists of a pair of connexons, each of which is made of six connexin molecules. The connexins belong to a multigene family. Channels composed of different members of the family possess unique properties. Since exchange of metabolic and electrical signals between adjacent cells depend on gap junction, a variety of biological activities are intimately linked to the junctions, such as maintenance of cardiac and vascular function.
The work of this thesis is to investigate the influence of hyperlipidemia and alcoholism on cardiovascular gap junction by using anti-connexin antisera in conjunction with high-resolution confocal laser scanning microscopy. Hypercholesterolemia is well-known to be one key atherogenic factor. Previous reports had shown that hypercholesterolemia affect the gap junctions in the arterial endothelial cells, which contain connexin37, connexin40 and connexin43. However, the effect of hypercholesterolemia on endothelial connexins is not clear. To answer this question, hypercholesterolmia was induced in mice by long-term feeding of cholesterol-enriched diet. In addition, a cholesterol-lowering drug, simvastatin, was given in a short time. The results showed that: i) in hyperlipidemic mice, endothelial connexin37 and connexin40 are downregulated; ii) 7-days use of simvastatin, restores connexin37, not connexin40, nearly to the normal range; iii) connexin43 was not expressed in the aortic endothelia in all animals of different feeding and treatment.
On the other hand, excessive intake of alcohol is known harmful to the heart function. However, whether alcohol change the myocardial gap junctions was not clear. I therefore conduct an experiments investigating the change of connexin43, the major connexin expressed in heart, in rats administrated with different concentrations of alcohol in the drinking water for one or two months. The results showed that while the myocardial connexin43 remained stationary in rats drinking 12% alcohol for one month, by those drinking 12% or 36% alcohol for two months had an increase. In addition, the distribution of the connexin43 gap junctions is disorganized in rats drinking 36% alcohol for two months.
To investigate the role of gap junction in the cardiovascular system, the first thing is to have antiserum specific to individual connexins. In the thesis, I therefore try to generate anti-connexin polyclonal antibodies in rats followed by affinity purification and characterization by Western blotting plus immunocytochemistry. The results showed the antiserum specific to connexin37, connexin40 and connexin45 are successfully produced. These antisera are useful tools for future gap junction study.
These findings demonstrate that cardiovascular diseases are associated with alterations of gap junction distribution and connexin expression. In addition, during the disease process, individual connexins are differentially regulated, suggesting that each member of the connexin family in the cardiovascular system plays an unique role in the pathogenesis of diseases.
中文摘要.................................................Ⅰ
英文摘要.................................................Ⅲ
致謝辭...................................................Ⅵ
目錄.....................................................Ⅶ
第一章 緒論...............................................1
1.1前言...................................................2
1.2隙連結管道的分子組成...................................3
1.3隙連結主要的功能是作為細胞之間溝通的管道...............10
1.4隙連結溝通的調節.......................................12
1.5細胞、組織和器官的隙連結...............................17
1.6隙連結與疾病發生的關聯性...............................28
1.7關於本論文.............................................34
圖1.1....................................................5
第二章 膽固醇對主動脈內皮細胞隙連結的影響.................35
2.1前言...................................................36
2.2材料與方法.............................................36
2.3結果...................................................44
2.4討論...................................................49
表2.1....................................................41
圖2.1....................................................40
圖2.2....................................................42
圖2.3....................................................45
圖2.4....................................................47
圖2.5....................................................48
圖2.6....................................................50
圖2.7....................................................51
第三章 酒精對心臟隙連結的影響.............................55
3.1前言...................................................56
3.2材料與方法.............................................57
3.3結果...................................................65
3.4討論...................................................71
表3.1....................................................66
表3.2....................................................71
圖3.1....................................................67
圖3.2....................................................69
圖3.3....................................................70
圖3.4....................................................72
第四章 抗連接素抗體的製造、純化和鑑定.....................75
4.1前言...................................................76
4.2材料與方法.............................................77
4.3結果...................................................90
4.4討論.............................................. ....103
表4.1....................................................78
表4.2....................................................91
表4.3....................................................103
表4.4....................................................104
表4.5....................................................105
圖4.1....................................................84
圖4.2....................................................92
圖4.3....................................................93
圖4.4....................................................95
圖4.5....................................................96
圖4.6....................................................97
圖4.7....................................................98
圖4.7 (續) ..............................................99
圖4.8....................................................100
圖4.8 (續) ..............................................101
圖4.9....................................................102
圖4.10...................................................107
第五章 結論...............................................108
參考文獻..................................................111
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