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研究生:林俊堯
研究生(外文):Chun-yao Lin
論文名稱:大鼠頸動脈內皮細胞氣球擴張損害模式和caspase13表現的關係
論文名稱(外文):Role of Caspase 13 Activation in Carotid artery Balloon Injury
指導教授:戴明泓
指導教授(外文):Ming-Hong Tai
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
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:2009
畢業學年度:97
語文別:英文
論文頁數:37
中文關鍵詞:血管內皮細胞CASPASE 13細胞凋亡動靜脈瘻管血管內膜增生
外文關鍵詞:caspase 13endothelial cellarterioveinous graftneointima hyperplasiaapoptosis
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血管內皮細胞功能失調/凋亡是血管內膜增生(neointima hyperplasia)形成的重要關鍵。 我們前期實驗發現CASPASE 家族中一個新成員,caspase 13 (CASP 13) ,在血管內皮細胞中被氧化低密度膽固醇誘發出,但血管平滑肌細胞則無此現象。 目前CASP 13是人類或是牛的基因仍有爭議。 本研究收集因動靜脈瘻管人工血管感染而切除的人類臂動脈檢體,經西方墨點法及RT-PCR分析發現CASP13 在血管吻合處的表現上升。 此外,大鼠頸動脈氣球擴張損害模式一直廣泛的應用在血管內膜增生研究上,尤其是血管平滑肌細胞增生的研究。 大部分氣球擴張損害研究都在十四天後中止實驗並犧牲大鼠以進行病理分析。 本研究藉由超音波儀器的協助記錄大鼠頸動脈在氣球擴張損害後血流動力學上的變化。 發現擴張損害前的左側大鼠頸動脈內徑的平均值為0.6 ± 0.07 mm而血流流速為28.75 ± 4.45 cm/sec。 氣球擴張損害後第七天及第十四天時,左側大鼠頸動脈內徑的平均值增加至0.77 ± 0.09 mm 及 0.71 ± 0.08 mm。 此外, 血流流速則增加為47.6 ± 9.2 cm/sec及 33.4 ± 10.8 cm/sec。 但在第二十一天後,大鼠頸動脈血流流速則回復至24 ± 3.5 cm/sec。 病理分析發現CASP 13 蛋白質表現在氣球擴張損害側上升,而免疫染色也可在氣球擴張損害側的血管內皮細胞發現CASPASE 13 的免疫反應。 總結, CASP 13 可以在人類與動物動脈硬化模式中偵測,未來可成為新型血管疾病之分子標記。
ABSTRACT
Endothelial dysfunction/apoptosis is critical to the development of neointima hyperplasia. Our pilot study identified a novel member of the caspase subfamily, caspase 13 (CASP 13), which is induced by oxidized low density lipoprotein (ox-LDL) in endothelial cells, but not in vascular smooth muscle cells. It remains controversial whether CASP 13 is a human or bovine gene. In this study, we collected four human brachial artery segments during arterioveinous fistula graft removal surgeries. By western blot and quantitative RT-PCR analysis, CASP13 expression was elevated at the anastomosis of brachial arteries from patients receiving hemodialysis. Rat carotid artery balloon model is widely used as in vivo atherosclerosis model for studies on neointima formation and especially vascular smooth muscle proliferation. However, most of the balloon injury studies terminated on the 14th day to sacrifice the animals for histological studies. In this study, we employed the ultrasonic device to record the hemodynamic changes in rat carotid artery at different time intervals after balloon injury. The pre-operative mean left carotid artery internal diameter and blood flow of Sprauge Dawley rats was 0.6 ± 0.07 mm and 28.75 ± 4.45 cm/sec, respectively. After balloon dilatation, the mean internal diameter of left carotid artery elevated to 0.77 ± 0.09 mm and 0.71 ± 0.08 mm on day 7 and day 14, respectively. Besides, the mean blood flow velocity also increased to 47.6 ± 9.2 cm/sec and 33.4 ± 10.8 cm/sec on day 7 and day 14, respectively. However, the left carotid artery blood flow velocity returned to 24 ± 3.5 cm/sec on day 21. The CASP 13 protein expression was found elevated in the balloon injury sites and mainly localized in the endothelial cells. In summary, CASP 13 is detected in endothelial cells in both human and rat atherosclerosis models and may constitute a novel molecular target for vascular diseases.
CONTENTS
中文摘要 …………………………………………………… 3
Abstract …………………………………………………… 4
1. Introduction ……………………………………………… 6
2. Specific Aim ……………………………………………10
3. Materials and Methods …………………………………… 11
3.1. Human brachial artery specimen analysis ……………… 11
3.2. Rat carotid artery balloon injury model ………………… 11
3.3. Ultrasonic hemodynamics study ……………………… 12
3.4. Western blot analysis ………………………………… 13
3.5. Quantitative RT-PCR …………………………………… 13
3.6. Immunohistochemistry for CASP 13…………………… 14
3.7. Static analysis ………………………………………… 14
4. Results …………………………………………………… 15
4.1 Ultrasonic hemodynamics study ………………………… 15
4.2 Immunohistochemistry for CASP 13 …………………… 15
4.3 Western Blot Analysis ………………………………… 16
4.4 Human vessel CASP 13 expression at transcriptional level
……………………………………………………………… 17
5. Discussion …………………………………………… 18
6. Conclusion …………………………………………… 21
7. Figure legends ………………………………………… 22
Figure 1: CASP 13 induced by ox-LDL in the human cell line
…………………………………………………………… 22
Figure 2: Ultrasound hemodynamics study of the post balloon dilatated rat carotid arteries ………………………………… 23
Figure 3: hemodynamic comparison of the bilateral carotid arteries ……………………………………………………… 25
Figure 4: Compensatory flow raising phenomenon ……… 27
Figure 5: Immunohistochemistry for CASP 13 in human artery specimen …………………………………………………… 28
Figure 6: Immunohistochemistry for CASP 13 in rat artery specimen …………………………………………………… 29
Figure 7: CASP 13 western blot analysis in human artery specimen …………………………………………………… 30
Figure 8: CASP 13 western blot analysis in rat artery specimen
……………………………………………………………… 31
Figure 9: Human vessel CASP 13 expression at transcriptional level……………………………………………………… 32
8. References ……………………………………………… 33
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