臺灣博碩士論文加值系統

血液透析是台灣以及世界許多國家常使用的腎臟替代療法(renal replacement therapy)。自體動靜脈廔管則是血液透析最常使用、也被列為首要建議考量的透析通道。動脈端血流流速減少以及合併糖尿病則是廔管不成熟以及廔管功能不良的最重要相關因子。在台灣每年新發生的末期腎衰竭案例當中，有約百分之四十合併患有糖尿病。動靜脈廔管出現問題就直接影響到病患及家屬的生活與工作。因此，改善動靜脈廔管的成熟率以及血管功能是很重要的議題。然而，截至目前為止相關的研究仍無法提出有效的藥物來改善廔管的功能或增加血流。尤其針對糖尿病族群，廔管相關的研究更少。本博士論文主旨在探究糖尿病造成廔管功能不良的機轉，並且主張以藥物的多型性效益(pleiotropic effects)來解決這個問題，在文獻搜尋下，選擇使用rosuvastatin降血脂藥物與exendin-4，一種昇糖素類似胜肽(glucagon like peptide) type 1的類似物，治療糖尿病個體的動靜脈廔管印證此假說，首先以動物實驗證實以rosuvastatin能夠改善廔管血管張力反應功能，並增加廔管中血流流速。這個研究使用streptozotocin (60毫克/公斤)在大白鼠體內引發第一型糖尿病，一周後投予安慰劑或rosuvastatin給予糖尿病鼠服用。服用後第三天施以主動脈與下腔靜脈的廔管手術，在手術後持續藥物治療兩週為止， 將動物犧牲並分析血流與血管功能。最終證實rosuvastatin的藥物多型性效益主要包含抗自由基、抗發炎能作用在糖尿病大白鼠動靜脈廔管以改善其血管功能與血流。此外，內皮前驅細胞(endothelial progenitor cells)也受到rosuvastatin的激發而在糖尿病鼠的週邊血液循環中數目上升。由此研究的發現，藥物能同時包含抗自由基、抗發炎以及激發內皮前驅細胞的多型性效益可在糖尿病個體中提供廔管功能性保護作用。而第二個實驗使用exendin-4雖改善廔管動脈端之內皮細胞功能，但並未增進廔管血流，蛋白分析發現exendin-4並未緩和磷酸化p47phox(NADPH oxidase protein的重要次單位蛋白之一)，而且血管平滑肌細胞出現活化(synthetic phenotype)促進血管型變的現象，由此二實驗顯示，多型性效益保護糖尿病動靜脈廔管機制當中，緩解自由基的功能扮演重要的角色。接續此轉譯醫學的重要發現，我首先執行一項探究臨床動靜脈廔管發生未成熟或狹窄機率的觀察性研究以界定出本院廔管失敗的發生率及其變異性，同時研究也探討內皮前驅細胞的數目與動靜脈廔管血流的相關性。透過此前驅研究可設定合理的臨床試驗的病人樣本數，而進一步的執行雙盲隨機臨床試驗，對於糖尿病患者接受手腕動靜脈廔管手術術前給予rosuvastatin或安慰劑，為期一個月的治療，後續定期追蹤廔管血流、血中自由基含量、發炎指數等等。目前總收案患者人數為53人，距設定的總樣本數80人仍約需兩年時間收納新患者，而此試驗的發現可能為末期腎衰竭合併糖尿病患者帶來重要的影響。
總結，我的博士論文研究結果證明，對於糖尿病動靜脈廔管的功能影響除了可提供血管內皮細胞保護、減少發炎因子産生、活化血管生成因子外，最大的應該在於抑制血管內自由基的生成，而藥物多型性效益若能有效抑制糖尿病個體自由基的生成，則有可能改善動靜脈廔管的血管功能並增進血流。這個發現與概念已進入臨床試驗並期待能改善血液透析合併有糖尿病患者的生活品質。

Diabetes mellitus, the most common cause of end-stage renal disease (ESRD) in Taiwan, is an independent risk factor of reduced blood flow in arteriovenous (AV) fistula. However, there is currently limited therapeutic strategy to prevent the loss of primary patency and to improve durability of AV access among diabetes. This PhD thesis aimed to identify the mechanism of AV fistula dysfunction induced by hyperglycemia. Insulin-dependent diabetes was induced in Sprague-Dawley rats by a single injection of streptozotocin (60 mg-1·kg-1). One week after induction of diabetes, an AV fistula was created in abdominal aorta and the adjacent inferior vena cava of anesthetized rats. Rats were allocated to control or rosuvastatin-treated groups which receive placebo or rosuvastatin (15 mg·kg-1·day-1) in chow for 2 weeks. It was then acknowledged that the generation of pleiotropic effects including anti-inflammation, anti-oxidant and EPC mobilization could be essential pleiotropic effects to improve AV fistula function. Exendin-4 (Ex4), a glucagon like peptide-1 analogue, presented similar pleiotropic effects as statins. To testify the hypothesis, those diabetic rats received intraperitoneal vehicle or Ex4 (0.5 μg·kg-1·day-1) before creation of AV fistula. The aortic limb of AV fistula was analyzed on day 14 postoperatively. Ex4 was found to present anti-inflammatory effects in aorta tissue and mobilized circulating EPCs diabetic rats. However, Ex4 failed to attenuate abundances of phosphorylated p47phox, a major subunit of NADPH oxidase, and did not improve AV fistula blood flow. These important findings indicated the importance of anti-oxidative effects of rosuvastatin in the AV fistula of hyperglycemic rats. The cyclic guanosine monophosphate (cGMP) was then restored in rosuvastatin treated rats which improve vascular relaxation and fistula blood flow. These findings were pivotal to the following clinical studies in my thesis. Before initiating the clinical trial, a pilot study is conducted to survey the incidence of AV fistula failure among ESRD subjects with diabetes. This prospective cohort study revealed a primary failure rate of 24.1% among those who received AV fistula surgery. In addition, there was a positive correlation between fistula wall stress and circulating EPC numbers. The mobilization of circulating EPCs was statistically significant among patients who developed a compromised radiocephalic fistula within 90 days postoperatively. A double blind, randomized control trial was then conducted in ESRD subjects with diabetes. On preparing this PhD thesis manuscript, a total of 53 cases were included in this trial and we expected promising findings to improve the life quality of this patient group.
In summary, this PhD project identified that rosuvastatin presented non-lipid lowering pleiotropic effects in AV fistula of diabetes. Rosuvastatin restored AV fistula blood flow and vascular function mainly through anti-oxidative mechanism which attenuated the uncoupling of eNOS and promoted the propagation of NO-cGMP pathway. The beneficial effect of rosuvastatin over AV fistula is clinically applicable and is supposed to be evident from the ongoing clinical trial.

Chapter 1. Introduction……………………………….….……..…..1
1.1 Research niche in arteriovenous fistula in end-stage renal disease with diabetes…………………….………...….…3
1.1.1 Arteriovenous fistula in end-stage renal disease……………….………...3
1.1.2 Diabetes is a risk factor predicting failure of arteriovenous fistula in end-stage renal disease……………….……..4
1.1.3 Oxidative stress in diabetes……………………...5
1.2 Vascular protection mechanism of statins …………….7
1.2.1 Hydroxymethylglutaryl-coenzyme A inhibitors………………………….7
1.2.2 Mevalonate pathway and endothelial function………………………….11
1.2.3 Oxidized LDL-c and endothelial function………………………………11
1.2.4 Pleiotropic effects of statins on vascular function………………………14
1.3 Vascular protection in exendin-4, a glucagon like peptide-1 analogue………………………………………………………16
1.3.1 Pleiotropic effects of Glp-1 agonists in clinical studies….….………….17
1.4 Pleiotropic effects in translational research………….20
1.4.1 Research niche of pleiotropic effects in marketed medications………...20
1.4.2 Statins and exendin-4 share common pleiotropic effects in vascular protection…………………………………….20
1.5 Thesis aims……………………………………………………….………21
Chapter 2. Pleiotropic effects of rosuvastatin restore vascular function in a diabetes rat model ………………………23
2.1 Backgrounds and Aims………………………………….…….23
2.2 Materials and Methods………………………….…….….25
2.3 Results……………………………………………………………… .32
2.4 Discussion………………………………………………………….35
2.5 Tables and Figures………………………………………….….39
Chapter 3. Identification of anti-oxidative effects as the main vascular protective mechanism in diabetic rats…………………49
3.1 Backgrounds and Aims………………………………………..…….49
3.2 Materials and Methods………………………………………….….51
3.3 Results………………………………………………………….……..57
3.4 Discussion…………………………………………………..……60
3.5 Tables and Figures…………………………………………….….…66
Chapter 4. Pilot study of arteriovenous fistula among end-stage renal disease patients…………………………………….………..74
4.1 Backgrounds and Aims………………………………….….……74
4.2 Materials and Methods…………………………………….………78
4.3 Results……………………………………………………….……..82
4.4 Discussion…………………………………………………….…85
4.5 Tables and Figures……………………………………….………89
Chapter 5. Conclusion, General discussion and Perspectives……….96
5.1 Experimental findings from bench to bedside…….96
5.1.1 Importance of anti-oxidative effects in the pleiotropism of rosuvastatin……96
5.1.2 Role of endothelial progenitor cells in arteriovenous fistula…....103
5.2 Limitations of animal and clinical pilot studies…….…103
5.3 Perspectives…………………………………105
Bibliography………………………………………………107
Curriculum Vitae………………………………………………………..130
Publication lists…………………………………………………...131

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