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系統識別號 U0026-0812200915152213
論文名稱(中文) 探討肺炎披衣菌感染對血管舒張反應之影響
論文名稱(英文) The effects of Chlamydophila pneumoniae infection on vasorelaxant responses
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 97
學期 2
出版年 98
研究生(中文) 陳家羽
研究生(英文) Chia-Yu Chen
學號 t3696406
學位類別 碩士
語文別 英文
論文頁數 66頁
口試委員 口試委員-楊艾倫
口試委員-蔡佩珍
指導教授-林尊湄
口試委員-黃暉升
中文關鍵字 熱休克蛋白六十  血管舒張  肺炎披衣菌 
英文關鍵字 cHSP60  vasorelaxation  Chlamydophila pneumoniae 
學科別分類
中文摘要 血管舒張功能不全是動脈粥狀硬化早期的重要特徵。目前已有許多證據指出,肺炎披衣菌感染參與在動脈粥狀硬化的過程之中,並且會引起嚴重的併發症。然而目前為止,對於肺炎披衣菌到底是如何引發動脈粥狀硬化的致病機轉,尤其是在疾病發生的早期階段,仍然不清楚。因此,本研究的主要目的是藉由動物以及細胞培養的方式,直接探討肺炎披衣菌感染,對於血管舒張反應的影響及其分子機轉。我們將大白鼠分成兩組,分別經由鼻腔滴入純化的肺炎披衣菌或生理食鹽水,每周一次連續三周,在感染後第四周進行犧牲,並取出大白鼠的胸主動脈,利用乙醯膽鹼和類胰島素因子進行血管舒張功能的分析。結果發現肺炎披衣菌感染的大白鼠,對於乙醯膽鹼或類胰島素因子作為血管舒張劑所引發的內皮層依賴的血管舒張反應,都有明顯變差的情形,而給予一氧化氮合成脢的抑制劑或直接刺激一氧化氮,兩組大白鼠血管舒張反應的差異就消失了。此外,人類臍靜脈內皮細胞被肺炎披衣菌感染後,我們發現細胞中的一氧化氮合成脢以及Akt蛋白磷酸化的情形會明顯減少,進而影響內皮細胞產生一氧化氮的能力。除此之外,我們證實這樣的現象是由於肺炎披衣菌生長複製所產生的一種熱敏感蛋白所引起,最後推論可能是披衣菌之熱休克蛋六十經由細胞膜上的第四型類鐸受體(TLR4),刺激內皮細胞所造成的;而且,我們也在肺炎披衣菌感染的大白鼠中測得高價數之抗披衣菌之熱休克蛋六十抗體,間接證明披衣菌之熱休克蛋六十在肺炎披衣菌感染中所扮演的角色。總而言之,我們的實驗結果發現,肺炎披衣菌感染可能會產生披衣菌之熱休克蛋白六十透過第四型類鐸受體的幫助,抑制內皮細胞中一氧化氮合成脢的活性,進而導致血管舒張反應不良的情形發生。
英文摘要 Impaired vasorelaxant response is an important feature of early atherosclerosis. Increasing evidences suggest that Chlamydophila pneumoniae (C. pneumoniae) infection participates in atherosclerosis and its clinical sequelae. However, it is still unclear how C. pneumoniae infection contributes to the pathogenesis of atherosclerosis especially in the early stage. Specific aim of this study is to explore the molecular mechanisms of C. pneumoniae infection on vasorelaxant responses in vivo and in vitro. Male Wistar rats were inoculated intranasally with C. pneumoniae or saline every week over a 3-week period. The rats were sacrificed at the 4th week after the first inoculation and the thoracic aortas were isolated analyzing vasorelaxant responses to the vasodilators, acetylcholine (ACh) and insulin-like growth factor-1 (IGF-1). Our results showed that both ACh- and IGF-1-evoked vasorelaxations were attenuated in the C. pneumoniae-infected rats with an endothelium-dependent manner. After pre-administration with an inhibitor of nitric oxide synthase (NOS), N-nitro-L-arginine methyl ester, but not with which of prostacyclin, indomethacin, the altered ACh- or IGF-1-induced vasorelaxations were abolished. We also demonstrated C. pneumoniae diminished endothelial NOS (eNOS) phosphorylation with an Akt-dependent pathway in human umbilical vein endothelial cells (HUVECs). The heat-sensitive protein from C. pneumoniae proliferation suppressing the eNOS activation was demonstrated to be chlamydial heat shock protein 60 (cHSP60) through TLR4 on HUVECs. The role of cHSP60 was also verified by higher anti-cHSP60 in the plasma of C. pneumoniae-infected rats. To a greater extent, C. pneumoniae infection suppressed eNOS activity by cHSP60 through TLR4 and resulted in the impaired vasorelaxant responses.
論文目次 Abstract I
中文摘要 II
誌謝 III
Contents IV
Figure List VII
Appendix List VIII
Instruments and Reagents IX
1. Introduction 1
1.1 Background of bacteria chlamydiae 1
1.1.1 The bacteria family Chlamydiaceae 1
1.1.2 Chlamydiae biology 1
1.1.3 Infection and isolation of Chlamydophila pneumoniae 2
1.2 Atherosclerosis as a chronic inflammatory disease 3
1.2.1 Progression of atherosclerosis 3
1.2.2 Association of C. pneumoniae with atherosclerosis 4
1.2.3 Models of C. pneumoniae involved in atherosclerosis 5
2. Materials and Methods 7
2.1 Propagation of C. pneumoniae 7
2.1.1 Inoculation, harvest and purification of C. pneumoniae 7
2.1.2 Determination of C. pneumoniae inclusion forming units 9
I. Chlamydial infection 9
II. Immunofluorescence staining for C. pneumoniae inclusion forming units 10
2.2 C. pneumoniae infection on vasorelaxant responses in vivo 11
2.2.1 Animals and infection protocol 11
2.2.2 Confirmation of C. pneumoniae infection in Wistar rats 12
I. Purification of DNA from rat lung tissues 12
II. Purification of RNA from rat lung tissues 14
III. DNase treatment 15
IV. Reverse transcription (RT) 16
V. Polymerase chain reaction (PCR) 17
VI. Digoxigenin (DIG)-labeling DNA probe 18
VII. Dot blotting, hybridization and detection 19
2.2.3 Evaluation of vasorelaxant responses 21
2.2.4 Examination of the signaling pathway of vasorelaxant responses 22
2.2.5 Determination of plasma cHSP60 antibodies by enzyme-linked immunosorbent assay (ELISA) 23
2.3 Statistical analysis 24
2.4 C. pneumoniae infection on eNOS activation in vitro 25
2.4.1 Cell culture and infection protocol 25
I. Cell subculture 25
II. Preparation of frozen cells 27
III. Culture of frozen-defrozen cells 28
2.4.2 Viability of C. pneumoniae in endothelial cells 29
I. Infectivity assay 29
II. Immunofluorescence staining 30
2.4.3 Western blotting analysis 30
I. Whole cell lysate extraction 30
II. SDS-PAGE 31
III. Western blotting 33
3. Results 35
3.1 Establishment of C. pneumoniae infection in Wistar rats 35
3.2 Effects of C. pneumoniae infection on vasorelaxant responses 35
3.3 Roles of endothelium-derived nitric oxide in the C. pneumoniae- impaired vasorelaxant responses 36
3.4 Establishment of C. pneumoniae infection in HUVECs 37
3.5 Effects of C. pneumoniae infection on eNOS activation 38
3.6 Virulence factors of Chlamydiae for attenuating eNOS activity in HUVECs 38
3.7 Chlamydial heat shock protein 60 (cHSP60) for attenuating eNOS activity in HUVECs 39
3.8 Plasma levels of anti-cHSP60 in the C. pneumoniae-infected Wistar rats 40
4. Discussion 41
5. References 47
6. Figures 53
7. Appendixes 64
8.自述 66
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