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系統識別號 U0026-0812200911430448
論文名稱(中文) 大白兔動脈硬化發生初期之內皮細胞功能失調
論文名稱(英文) Endothelial Dysfunction in Early-Stage Atherosclerotic Rabbit Arteries
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 93
學期 2
出版年 94
研究生(中文) 黃怡嘉
研究生(英文) Yi-Chia Huang
學號 s3692406
學位類別 碩士
語文別 英文
論文頁數 78頁
口試委員 指導教授-任卓穎
召集委員-陳麗玉
口試委員-陳洵瑛
中文關鍵字 內皮細胞功能失調  動脈硬化  鈣離子訊號 
英文關鍵字 endothelial dysfunction  calcium signaling  atherosclerosis 
學科別分類
中文摘要   初期動脈硬化形成過程中,內皮細胞的功能失調伴隨著脂肪紋的形成被視為是最顯著的特徵。到目前為止,早期動脈硬化發生時初期脂肪紋形成的進程仍不清楚。因此本研究將利用in situ方法偵測,在初期脂肪紋形成過程中,內皮細胞的功能狀態和不同動脈硬化相關指標性分子的表現,它們在空間上與時間上的相關性。利用紐西蘭大白兔經由短時期高膽固醇餵食引發其胸主動脈 (TA) 與頸動脈 (CA) 生成初期脂肪紋,我們透過分析乙醯膽鹼所誘發內皮細胞內的鈣離子濃度變化 ( EC [Ca2+]i signaling, 視為內皮細胞功能性指標) 來描述初期脂肪紋在這兩種動脈中的發展模式。另外,我們也觀察不同促進發炎的分子其 en face 分佈情形 (P-selectin and NF-kB)。結果顯示,在正常餵食的兔子中,其胸主動脈內皮細胞內鈣離子濃度的變化顯著低於頸動脈內皮細胞內鈣離子濃度的變化。在高膽固醇餵食的兔子中,其胸主動脈脂肪紋在膽固醇飼料介入後兩週即形成;且脂肪紋廣泛分佈在四週的胸主動脈上,此時在頸動脈上僅可測得少量的脂肪紋。有趣地,鈣離子訊號上升的現象,不僅發生於覆蓋在胸主動脈脂肪紋上的內皮細胞,也發生於覆蓋在脂肪紋鄰近平滑區域的內皮細胞。這種內皮細胞功能改變的現象,發生在所有長度小於500 μm 的脂肪紋中。在遠離脂肪紋的區域,內皮細胞鈣離子訊號顯示出異質性,特別在局部小範圍內顯示出未來可能進一步形成脂肪紋的位置。當脂肪紋更進一步廣泛分佈於胸主動脈時,覆蓋在團簇脂肪紋上的內皮細胞其鈣離子訊號不再有上升的現象。相反的,當脂肪紋在頸動脈上形成時,內皮細胞內鈣離子的變化呈現一個完全不同的型態。無論是覆蓋在微脂肪紋上 (50~150 μm, 5~15 cells) 或是覆蓋在微脂肪紋鄰近平滑區域的內皮細胞,其細胞內鈣離子訊號未有改變;覆蓋在微小脂肪紋上 (150~350 μm, 15~30 cells) 或覆蓋在小脂肪紋上 (350~500 μm, 30~90 cells) 的內皮細胞,其細胞內鈣離子訊號顯示下降狀態。此外,這種頸動脈內皮細胞功能失調的狀態,似乎由覆蓋在小脂肪紋上的內皮細胞蔓延到小脂肪紋鄰近的平滑區域。初代培養的內皮細胞,經由四週膽固醇餵食之兔子胸主動脈取得。此時的NF-kB部份的分佈在細胞核內,這與NF-kB在正常內皮細胞中,主要呈現細胞質的分佈截然不同。頸動脈en face免疫染色圖片顯示,P-selectin的表現早於脂肪紋的形成。NF-kB和P-selectin的表現量伴隨著脂肪紋的增長而增加。覆蓋在頸動脈鄰近脂肪紋平滑區域的內皮細胞,其P-selectin呈現較微量且無定型的表現。總體而言,高膽固醇飼料所誘發的內皮細胞功能失調,不僅可以透過血管結構的改變來描述,像是脂肪紋的出現和不同防止-發炎的分子所表現的模式,同時也可透過血管局部內皮細胞內鈣離子訊號的改變提供更多初期內皮細胞功能失調的資訊。
英文摘要  Endothelial dysfunction associated with fatty streak formation is considered as the hallmark during early-stage atherosclerosis. As the fatty streak-forming process remains unclear at present, it is desirable to monitor the functional behavior of endothelial cells in situ as well as the spatial/temporal relationship among various atherosclerotic markers. Using New Zealand White rabbits fed with high-cholesterol diet for short periods of time, we characterized early-stage fatty streaks formed in their thoracic aortas (TA) and carotid arteries (CA) by examining the acetylcholine-evoked endothelial intracellular calcium ion changes ( EC [Ca2+]i signaling, an indicator for endothelial functional performance). In addition, we also characterized the en face distribution of pro-inflammatory molecules (P-selectin and NF-kB). In rabbits under normal diet, the EC [Ca2+]i signaling in TA was lower than that in CA. In hypercholesterolemic rabbits, TA fatty streaks appeared as early as 2 weeks after diet intervention; and they spread widely across the whole TA in 4 wks when only a few CA fatty streaks became detectable. TA endothelial calcium signaling remained unchanged in 1-wk hypercholesteremic rabbits. Interestingly in 2-wk atherosclerotic TA, [Ca2+]i signaling was elevated not only in ECs covering fatty streaks but also in adjacent ECs covering the smooth regions. This kind of endothelial functional alteration happened around any fatty streak less than 500 μm in length. The ECs far away from any detectable fatty streak showed heterogeneous calcium signaling, especially in small localized area that presumably represent future fatty streak sites. When TA fatty streaks became widely spread at later stages, ECs covering clusters of fatty streaks no longer showed enhanced EC [Ca2+]i signaling. In contrast, fatty streak-associated EC [Ca2+]i alterations in CA followed a different pattern from those in TA. [Ca2+]i signaling was unaltered in ECs either covering or adjacent to a micro fatty streak (50~150 μm, 5~15 cells); it became retarded in ECs covering a mini (150~350 μm, 15~30 cells) or a small (350~500 μm, 30~90 cells) fatty streak. Moreover, this CA fatty streak-associated endothelial dysfunction extended to areas adjacent to a small fatty streak. In primary cultured endothelial cells obtained from 4-wk atherosclerotic TA, NF-kB was partially localized in EC nucleus, contrasting to mainly cytosolic localization in the normal endothelial cells. Immunostained pictures from en face CA preparations showed that the P-selectin expression was earlier than the fatty streak formation. Immunostaining intensities of NF-kB and P-selectin increased with the growing fatty streaks. Endothelial P-selectin showed mild amorphous immunostaining in regions adjacent to fatty streaks. Taken together, the high-cholesterol diet-induced endothelial dysfunction could be characterized not only by vascular structural changes, such as the appearance of fatty streaks and expression pattern of various pro-inflammatory molecules, but also by localized alterations in EC [Ca2+]i signaling.
論文目次 Chinese abstract ---------------------------- I
English abstract ---------------------------- III
Acknowledgement ----------------------------- V
Contents ------------------------------------ VII
List of table and figures ------------------- IX
List of appendix ---------------------------- XI
Symbols ------------------------------------- XII
I. Introduction ----------------------------- 1
II. Materials ------------------------------- 8
III. Methods -------------------------------- 13
Animals and diet feeding ------------------ 13
Animal anesthetization -------------------- 13
Examination of lipid deposition in blood
vessels ----------------------------------- 14
Vessel preparation and fura-2-AM loading -- 15
Tissue flow chamber assembling ------------ 17
Instrument setup -------------------------- 18
Measurement and calibration of EC [Ca2+]i
signaling --------------------------------- 19
Immunofluorescence staining of primary
cultured aortic endothelial cells --------- 21
En face Immunofluorescence staining of rabbit
arteries ---------------------------------- 22
Statistical analysis ---------------------- 23
IV. Results --------------------------------- 24
Oil red O staining ------------------------ 24
Acetylcholine-induced endothelial calcium
signaling in normal TA or CA -------------- 24
Acetylcholine-induced endothelial calcium
signaling in1-wk hypercholesterolemic TA--- 29
Acetylcholine-induced endothelial calcium
signaling in early-stage atherosclerotic TA- 25
Acetylcholine-induced endothelial calcium
signaling in early-stage atherosclerotic CA- 27
Immunofluorescence staining of primary
cultured endothelial cells ----------------- 28
En face immunofluorescence staining of rabbit
arteries ----------------------------------- 28
V. Discussion ---------------------------------30
VI. References ------------------------------- 40
Tables --------------------------------------- 47
Figures -------------------------------------- 48
Appendix ------------------------------------- 64
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