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研究生:彭忠衎
研究生(外文):Chung-Kan Peng
論文名稱:低體溫與麩醯胺酸(Glutamine)對於老鼠急性肺損傷之保護角色
論文名稱(外文):Protective Effects of Hypothermia and Glutamine on Acute Lung Injuries in Rat Models
指導教授:朱士傑朱士傑引用關係黃坤崙黃坤崙引用關係吳清平吳清平引用關係
指導教授(外文):Shi-Jye ChuKun-Lun HuangChin-Pyng Wu
口試委員:張宏林恆毅
口試委員(外文):Hung ChangHen-I Lin
口試日期:2010/08/27
學位類別:博士
校院名稱:國防醫學院
系所名稱:醫學科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:99
語文別:英文
論文頁數:74
中文關鍵詞:急性肺損傷低體溫空氣栓塞缺血再灌流麩醯胺酸
外文關鍵詞:Acute lung injuryHypothermiaAir-embolismIschemia-reperfusionGlutamine
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急性肺損傷已成為重症健康照顧下一個重要的議題。輕度的低體溫在空氣栓塞引發的肺損傷仍未探討。肺部空氣栓塞藉由以每分鐘 25 μL速率靜脈注射空氣四十分鐘,而控制組則不給予空氣注射。老鼠隨機分為兩組控制組-正常體溫 (37°C)與輕度低體溫 (34°C)以及三組空氣栓塞組- 注射空氣前及輕度低體溫,正常體溫並接受空氣注射以及輕度低體溫與注射空氣同時。空氣注射後肺部的乾溼重比例、支氣管肺泡沖洗液中肺部蛋白質、lactate dehydrogenase (LDH), and tumor necrosis factor-α 濃度均明顯上升. 肺組織中Myeloperoxidase 活性,嗜中性球浸潤與間質水腫也顯著增加。 此外,肺組織中的nuclear factor-κB 活性也明顯上升。
在我們的研究發現在空氣注射前給予輕度的低體溫可以減少因空氣栓塞所造成的急性肺損傷。這樣的保護機制似乎來自於發炎反應的抑制。
麩醯胺酸(Glutamine)已藉由其調控免疫反應來治療多種疾病。我們研究麩醯胺酸在缺血再灌流所引發之急性老鼠肺損傷是否有所幫助。以缺血60分鐘後再予以60分鐘的灌流達到急性肺損傷。實驗結束後,支氣管肺泡沖洗液與灌注液。在缺血前或缺血後給予麩醯胺酸 (20 mM)。缺血再灌流造成肺微血管通透係數(capillary filtration coefficient),肺重增率,肺重與體重比,肺部乾濕重比,肺動脈壓與支氣管肺泡沖洗液中肺部蛋白質LDH均增加。在灌流液中Tumor necrosis factor-α、cytokine induced neutrophil chemoattractant-1,肺組織中malondialdehyde濃度、carbonyl content 與 myeloperoxidase 活性亦呈現顯著之升高。此外,肺組織的間隔增厚與嗜中性球增加。肺部NF-κB 活性與IκB-α 的降解亦提高。在缺血前或缺血後給予麩醯胺酸可以顯著地減少上述參數的上升。我們的研究顯示麩醯胺酸的治療可減少由缺血再灌流所引發的肺損傷。其保護的機轉可能來自於抑制 NF-κB 的活化與減少氧化壓力的存在。

Acute lung injury has become a major burden of in critical health issues. The role of mild hypothermia in air embolism–induced lung injury has not been explored. Pulmonary air embolism was induced in rat by venous infusion of air at a rate of 25 μL/min for 40 minutes. Control animals received no air infusion. The rats were randomly assigned to 2 control groups of normothermia (37°C) and mild hypothermia (34°C) and 3 air embolism groups of mild hypothermia induced before air infusion, normothermia with air infusion, and mild hypothermia induced synchronous with air infusion. Air infusion elicited a significant increase in lung wet/dry weight ratio and protein, lactate dehydrogenase, and tumor necrosis factor-α concentration of the bronchoalveolar lavage fluid. Myeloperoxidase activity, neutrophil infiltration, and interstitial edema in lung tissue were also significantly increased. In addition, nuclear factor-κB activity was significantly increased in the lungs. In our study suggests that mild hypothermia before air infusion decreases air embolism–induced acute lung injury. The protective mechanism seems to be the inhibition of inflammation.
Glutamine has been used to treat a number of diseases via modulating the inflammatory response. The purpose of this study is to investigate whether glutamine has a beneficial effect in ischemia-reperfusion (IR) induced acute lung injury in an isolated rat lung model. Typical acute lung injury in rats was successfully induced by 60 min of ischemia and 60 min of reperfusion. Glutamine (20 mM) was administrated before ischemia or after ischemia. IR caused a significant increase in the capillary filtration coefficient; lung weight gain; lung weight to body weight ratio; wet to dry weight ratio; pulmonary arterial pressure; and protein concentration and lactate dehydrogenase level in BALF. Tumor necrosis factor-α and cytokine induced neutrophil chemoattractant-1 in perfusate, and malondialdehyde levels, carbonyl content and myeloperoxidase activities in lung tissue were also significantly increased. In addition, the lung tissues showed increased septal thickness and neutrophil infiltration. Furthermore, NF-κB activity and degradation of IκB-α were significantly increased in the lungs. Treatment with glutamine before ischemia or after ischemia significantly decreased the increase in these parameters. Our study showed that glutamine treatment decreased IR-induced acute lung injury. The protective mechanism may be due to the inhibition of NF-κB activation and the attenuation of oxidative stress.

CONTENTS
Contents ……………………………………………………………... I
List of Figures ………………………………………………………. V
Abstract in Chinese ………………………………………………... VII Abstract in English ……………………………………………….... IX
Introduction
■ Acute lung injury and acute respiratory distress syndrome ….………. 1
■ Management of ALI/ARDS ………………...…………….…….….. 3
■ Ischemia reperfusion lung injury ………………………….………. 4
■ Air embolism ………………………………………..….………… 5
■ Hypothermia ………………………………………….………….. 6
■ Glutamine …………………………………………….………….. 8
■ The objectives of this study ……………………………………….. 11

Materials and Methods
■ Animals ………………………...……………………………….. 12
Study 1. Mild hypothermia in air embolism-induced acute lung injury
■ Pulmonary air embolism- induced acute lung injury ……...……….. 12
Experimental protocol …………………………………… 13
Study 2. Ischemia-reperfusion induced acute lung injury in isolated rat lungs
Preparation of isolated and perfused rat lungs …………………. 15 Induction of lung ischemia and reperfusion ……………………….…. 16
Experimental protocol ……..…………………..……….. 16
■ Capillary filtration coefficient (Kf) ……………………….……….. 17
█ Measurement of lung weight/body weight (LW/BW) and wet/dry (W/D) weight ratio …………………………………………………….. 18
█ Bronchoalveolar Lavage Fluid ……………………………………. 18
█ Protein concentration and lactate dehydrogenase (LDH) activity in lung lavage fluid ……………………………………………………... 18
█ Measurement of TNF-α in BALF and perfusate, and CINC-1 (cytokine induced neutrophil chemoattractant) in perfusate ………………… 19
█ Determination of malondialdehyde (MDA) level and protein carbonyl content in lung tissue ………………………………………..…... 19
█ Determination of myeloperoxidase (MPO) activity ………….……… 20
█ Western blot analysis for IκB-α …………………............................ 21
█ NF-κB p65 activity assay ……………………………...…………... 22
█ Lung Histopathology ……………………………………………... 22
█ Statistical analysis ……………………………………………….. 23
Results
Study 1. The effect of mild hypothermia in air embolism-induced acute lung injury
█ Lung W/D ratio ………………...................................................… 24
█ Protein Concentration and LDH Level in BALF …………………… 24
█ TNF-α in the BALF …………………………………………….… 24
█ Oxygenation …………………………………………………...… 25
█ NF-κB Activation ………………………………………....……… 25
█ Myeloperoxidase Activity ………………………………………… 25
█ Histopathological findings of the lungs ……………….…………… 26

Study 2. The effect of glutamine in ischemia-reperfusion induced acute lung injury in isolated lung model
█ Kf ……………………………………………………………… 27
█ Pulmonary arterial pressure (PAP) …………………………….… 27
█ Lung weight gain …………………………………...…………… 28
█ LW/BW and wet/dry ratios ……………………….……………… 28
█ Lung lavage protein concentration and LDH activity ……....……… 28
█ TNF-α and CINC-1 levels in perfusate ………………………..…… 29
█ MDA level, carbonyl content, and MPO activity …………………… 29
█ NF-κB activation ………………………………………………… 29
█ Histopathological findings of lung ………………………………… 30

Discussion
█ The effect of mild hypothermia in air-embolism induced lung injury ... 31
█ The effect of glutamine in ischemia-reperfusion induced acute lung
injury in isolated lung model ………………………….………… 36
Conclusions ………………………………………...……………… 42
Figures …………………………………………………...………… 43
References ……………………………………………………….… 57

List of Figures
 Figure 1. Alterations in lung wet/dry (W/D) weight ratios.in mild hypothermia in air embolism-induced acute lung injury
 Figure 2. Effect of mild hypothermia on the tumor necrosis factor (TNF)-α and protein concentration, and lactate dehydrogenase (LDH) level in bronchoalveolar lavage fluid (BALF)
 Figure 3. Change in oxygenation during the experiment with treatment with mild hypothermia and normothermia
 Figure 4. Nuclear factor (NF)-_B p65 and myeloperoxidase (MPO) activity assay in lung tissue with treatment with mild hypothermia and normothermia
 Figure 5. Effect of mild hypothermia and normothermia on lung histology.
 Figure 6. The effect of glutamine on the filtration coefficient of the pulmonary vasculature (Kf) during the experiment
 Figure 7. The effect of glutamine on the pulmonary hypertension in IR lung injury
 Figure 8. The changes in lung weight gain, W/D and LW/BW ratios in IR lung injury
 Figure 9. The effect of glutamine on the protein concentration and LDH level of bronchoalveolar lavage fluid (BALF) in IR lung injury.
 Figure 10. The effect of glutamine on the TNF-α and CINC-1 concentration in the perfusate in IR lung injury
 Figure 11. The effect of glutamine on MDA concentration, carbonyl content, and MPO activity in lung tissue n IR lung injury
 Figure 12. The effect of glutamine on the IκB-α level of rat lung (A) as determined by western blot analysis of lung tissue cytoplasmic protein in IR lung injury.
 Figure 13. NF-κB p65 activity assay of the effect of glutamine in IR lung injury.
 Figure 14 Histological appearance of the effect of glutamine lung tissue in IR lung injury

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