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研究生：	黃次雄 Huang, Tzu-Hsiung
論文名稱：	血管內皮生長因子和發炎性Ly6C high單核球在呼吸器導致肺損傷之角色探討 Investigating the roles of vascular endothelial growth factor and inflammatory Ly6C high monocytes in ventilator-induced lung injury
指導教授：	張志欽 Chang, Chih-Ching
學位類別：	碩士 Master
系所名稱：	醫學院 - 環境醫學研究所 Department of Environmental and Occupational Health
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	58
中文關鍵詞：	呼吸器導致肺損傷、血管內皮生長因子、單核球
外文關鍵詞：	ventilator-induced lung injury, vascular endothelial growth factor, monocyte
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肺泡微血管通透性增加是呼吸器導致肺損傷主要的特徵。血管內皮生長因子是潛在促進血管通透性及細胞生長的因子，也可能是造成呼吸器導致肺損傷的原因。Ly6C high 單核球目前已知在呼吸器導致肺損傷過程中，會大量聚集到肺臟，並造成肺臟順應性的降低，但是否藉由分泌血管內皮生長因子促進肺泡微血管通透性的增加，其中的機轉並不清楚。本研究目的探討Ly6C high單核球與其血管內皮生長因子表現在呼吸器引起之肺損傷所扮演的角色。實驗採用脂多醣體合併機械通氣的雙侵襲性模式。 C57BL/6 公鼠分別暴露於高潮氣容積或低潮氣容積通氣達不同時間點。測量肺泡沖洗液及血液中發炎性細胞激素(如:TNF-α和IL-6),全蛋白質,和血管內皮生長因子以建立肺損傷模式，及利用流式細胞儀分析Ly6C high單核球於肺臟的聚集。在另一部分的實驗小鼠則利用clodronate liposomes去除 Ly6C high單核球以確認這類單核球在呼吸器肺損傷中所扮演的角色。研究結果顯示脂多醣體合併高潮氣容積通氣造成肺泡沖洗液中的全蛋白質含量顯著持續性的增加，並且誘導肺泡沖洗液中血管內皮生長因子含量在通氣6小時之後達到高峰。脂多醣體合併高潮氣容積通氣組別比上未機械通氣及低潮氣容積通氣組別有顯著Ly6C high單核球的聚集，同時也顯示Ly6C high單核球細胞內血管內皮生長因子表現的增加。但經由 clodronate liposomes 特異性去除Ly6C high 單核球之後呼吸器導致肺損傷情形則被弱化。因此，在呼吸器導致肺損傷中，發炎性Ly6C high單核球會聚集到肺臟並藉由其血管內皮生長因子的表現促進肺泡微血管的通透性增加。

The increase of alveolar-capillary permeability is thought to be the prominent features of ventilator-induced lung injury (VILI). Vascular endothelial growth factor (VEGF) is the most potent factor in regulating vascular permeability and mitogenic activity, and it might be responsible for the development of VILI. Furthermore, Ly6C high monocytes are recruited to the lungs and contribute to the decrease of lung compliance in VILI. It is not known whether Ly6C high monocytes are involved in alveolar-capillary permeability through their VEGF production. The aim of this study is to explore the role of Ly6C high monocytes and VEGF in VILI. A “two-hit model” was used, with lipopolysaccharide (LPS) administration combined with mechanical ventilation. Male C57BL/6 mice were exposed to high tidal volume (HTV) or low tidal volume (LTV) mechanical ventilation with room air for different timeline. Inflammatory cytokine (TNF-α and IL-6), total proteins, and VEGF in bronchoalveolar lavage fluid (BALF) and blood were measured. Flow cytometry analysis was used to quantify the recruitment of Ly6C high monocytes to the lungs. The depletion of Ly6C high monocytes with clodronate liposomes was used to establish the role of Ly6C high monocytes in the development of VILI. The results demonstrate that LPS administration combined with HTV caused a significant and sustained increase of total proteins, and induced a significant increases of VEGF protein in BALF with a peak increase at 6 hr. LPS administration combined with HTV resulted in significant pulmonary infiltartion of Ly6C high monocytes with elevated VEGF production (vs. non-ventilated and LTV group). Clodronate liposomes depleted Ly6C high monocytes significantly attenuated VILI. Our results indicate that Ly6C high monocytes infiltrate the lung and contribute to the increase of alveolar-capillary permeability through VEGF production.

I.Abstract---1
II.Chinese Abstract---2
III.Acknowledgments---3
IV.Content Table---4
V.Introduction---8
 A.Acute Respiratory Distress Syndrome (ARDS)---8
 B.Ventilator-Induced Lung Injury (VILI)---10
 C.Vascular Endothelial Growth Factor (VEGF) in ARDS---11
 D.The role of Ly6C high monocytes in VILI--- 13
VI.Specific Aims---17
VII.Materials and Methods---18
 A.Animal model of VILI---18
 B.Quantitation of total protein in BALF---19
 C.Detection of VEGF, IL-6, TNF-α in BALF and blood by ELISA    
   ---20
 D.Histopathological studies of the lung in mice---22
 E.Mouse lung single cell suspension---22
 F.Flow cytometric analysis---23
 G.Depletion of Ly6C high monocytes in vivo---24
 H.Statistical Analysis---25
VIII.Results---26
 1.Physiologic data---26
 2.Time-dependent effect of VILI---26
 3.Time-dependent effect of VEGF during VILI---27
 4.Time-dependent effect of other related cytokines during 
   VILI---27
 5.Ly6C high monocytes recruited to the lung during VILI
   --- 28
 6.Intercellular expression of VEGF in Ly6C high monocytes  
   ---29
 7.Clodronate liposomes depleted Ly6C high monocytes---29
 8.Clodronate liposomes decreased intercellular expression 
   of VEGF in Ly6C high monocytes---30
 9.Depleted of Ly6C high monocytes attenuated alveolar-
   capillary permeability---30
 10.Depleted of Ly6C high monocytes improve oxygen---31
IX.Discussion---32
X.References---38
XI.Figures and Legends---44
 1.Design of experimental groups for study during VILI---44
 2.Physiologic conditions at the beginning and 6 hour of 
   ventilation---45
 3.Expression levels of total protein---45
 4.Expression levels of VEGF protein---46
 5.Expression levels of TNF-α protein---47
 6.Expression levels of IL-6 protein---	48
 7.Flow cytometric analysis of Ly6C high monocyte and 
   Ly6C low monocyte---49
 8.Intercellular expression of VEGF in Ly6C high monocytes-
   50
 9.Clodronate liposomes depleted Ly6C high monocytes---51
 10.Clodronate liposomes treatment had some effect on 
    neutrophil numbers---52
 11.Clodronate liposomes decreased intercellular expression 
    of VEGF in Ly6C high monocytes---53
 12.Depletion of Ly6C high monocytes with clodronate 
    lipsomes attenuate VILI---54
XII.Reagents, Drugs and Chemicals---55
XIII.Instruments---57
XIV.Abbreviations---58

                                    

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校內：2016-08-20公開
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