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研究生:楊式興
研究生(外文):YANG, SHIH-HSING
論文名稱:白介質-33在呼吸器引發肺損傷中的角色
論文名稱(外文):Role of Interleukin-33 in Ventilator-Induced Lung Injury
指導教授:卓貴美王果行王果行引用關係
指導教授(外文):JOW,GUEY-MEIHSU WANG, GUOO-SHYNG
口試委員:李憶菁黃坤崙林佑穗阮婷王果行卓貴美
口試委員(外文):LEE, YIH-JINGHUANG, KUN-LUNLIN, YOU-SHUEIRUAN, TINGHSU WANG, GUOO-SHYNGJOW,GUEY-MEI
口試日期:2016-07-27
學位類別:博士
校院名稱:輔仁大學
系所名稱:食品營養博士學位學程
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:120
中文關鍵詞:呼吸器引發肺損傷IL-33ST2氫化可體松高碳酸血症
外文關鍵詞:Ventilator-induced lung injuryIL-33ST2hydrocortisonehypercapnia
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正壓型呼吸器能輔助急性呼吸衰竭病人呼吸,但呼吸器機械性的過度充氣及重複的應力會造成肺泡水腫及發炎,這稱為呼吸器引發肺損傷(Ventilator induced-lung injury; VILI)。其臨床表徵是肺臟會出現發炎及充血的現象,而且有相當高的死亡率。儘管臨床使用呼吸器時會同時執行肺保護策略,但是對於死亡率的降低仍有限。因此,了解呼吸器引發肺損傷與發炎機制的關係是非常重要的。已知IL-33 是IL-1 家族的成員,為一新發現的促發炎激素,而IL-33 的受器有二種形式,包括穿膜型的ST2L 與可溶性sST2兩型。IL-33/ST2 路徑是調控免疫作用及臨床上的病生理學特性很重要的機制,與肺損傷及氣喘和大腸炎,還有關節炎與心血管疾病等有著密切的相關性。IL-33的表現在肺組織中有組織型與誘導型的功能機制,表現在平滑肌細胞與支氣管上皮細胞等,當受到如流感A病毒感染時,會誘發釋放出IL-33,然而IL-33在心肌纖維母細胞中也被發現,當受到機械性刺激誘發細胞壞死時,原本存在細胞核中的IL-33會轉移到細胞質中形成液泡,同時增加IL-33的釋放,但是IL-33/ST2 路徑是否參與在VILI過程的作用機制中,仍不清楚。因此,本論文的研究目的是探討 IL-33/ST2 路徑是否參與在大鼠使用呼吸器所引發肺損傷的實驗模式中,並探討IL-33/ST2 路徑在肺保護策略氫化可體松及高碳酸血症處理呼吸器引發肺損傷中的表現與作用。實驗是先利用Wistar雄鼠進行,大鼠氣管插管接上G5 呼吸器四小時,並記錄呼吸參數,同時分別在股動脈及股靜脈插管以利每小時記錄血液動力學變化及收集血清。呼吸參數予以收集與分析,並進行動脈血液氣體分析,動物在實驗後犧牲,收集肺臟組織及支氣管沖洗液(Bronchoalveolar lavage fluid; BALF),利用組織學H&E染色觀察肺損傷形態學的改變,利用ELISA方法檢測血清及支氣管沖洗液,還有肺組織均質化液中之TNF-α及IL-1β,還有IL-33的濃度,利用免疫組織染色與西方點墨法分析IL-33/ST2在肺臟組織中的表現。
首先建立大鼠VILI實驗模式,大鼠接受G5 呼吸器使用壓力控制模式,尖峰吸氣氣道壓力分別維持在10 cmH2O (PC10)及20 cmH2O (PC20),結果發現潮氣容積在通氣一至四小時的變化,在PC20組顯著大於PC10組,但兩組分別在四小時時間點上並無顯著差異,平均動脈血壓PC20組顯著表現出受高壓力而降低,但兩組分別在四小時時間點上並無顯著差異,另外心跳變化都無顯著差異。分析VILI引起肺損傷的嚴重度,結果發現二組均有明顯肺泡空間增大及肺泡壁增厚,並有發炎細胞堆積與透明膜生成,以及出現肺水腫和顯著出血的現象,同時VILI也會增加血清及支氣管沖洗液中TNF-α和IL-1β的濃度。而IL-33在VILI大鼠血清中並無明顯變化,但是在肺組織中卻有明顯增加的現象,但是在BALF中IL-33的分泌會因為使用呼吸器而有明顯減少的趨勢。有趣的是,IL-33的ST2L受器在PC10與PC20 組肺組織的細胞膜層呈現高度的堆積,但在細胞質層的ST2L受器是顯著減少; 而在對照組則無此變化。相對地,sST2 受器在PC10與PC20 組肺組織的細胞膜層和細胞質層均是減少的。這些結果顯示ST2L受器從細胞質轉移到細胞膜。接著探討IL-33 細胞激素在類固醇及高碳酸血症治療VILI大鼠的作用,在大鼠經高壓通氣二小時後,給予氫化可體松發現能減低VILI肺損傷的現象,而IL-33 細胞激素因使用呼吸器而減少分泌到BALF中的現象,會因為投予氫化可體松發現在BALF中IL-33 細胞激素的分泌是回復至較多的現象,此結果可以應用在臨床上,檢測BALF中IL-33 細胞激素的量,當做是否造成呼吸器引起肺損傷的指標,提早提供病人肺保護策略。我們再進一步進行大鼠經高容積通氣中使用高碳酸血症 (Hypercapnia) 治療,即給予 5%CO2吸入四小時,結果發現使用5%CO2吸入治療能改善VILI對血液動力學的影響,改善氣體交換,並明顯減少使用呼吸器所引起的肺發炎症狀,同時因使用呼吸器所引起VILI大鼠之血清及BALF中發炎細胞激素TNF-α的產生,也會因為使用5%CO2吸入治療而減少,此外,使用呼吸器減少BALF中IL-33 細胞激素釋放的現象,也恢復成沒有減少的現象產生。肺組織中IL-33增加及其受器ST2 表現增加,也因為使用5%CO2吸入治療而減少,進而使肺損傷改善。總結上述研究:本論文使用臨床呼吸器成功建立大鼠實驗模式,紀錄與分析血液動力學與呼吸器參數,與利用肺組織學及檢測不同來源的發炎細胞激素來確認VILI實驗模式,發現大鼠經VILI,造成IL-33 細胞激素在肺組織中表現量增加,ST2L受器顯著累積在細胞膜上。經臨床肺保護策略之氫化可體松或高碳酸血症處理,可以減少肺中IL-33 細胞激素的生成及ST2L受器在細胞膜上的堆積,而支氣管沖洗液中即可檢測到IL-33表現量增加的差異。這些發現或許可以作為檢測VILI之新生物標的指標,IL-33/ST2訊息反應在機械性刺激的肺損傷中,可能可以作為新的治療標的。

Positive-pressure type ventilator could assist artificial breath to the acute respiratory failure patient. It is also well known that ventilator led to lung a repetitive strain and a mechanical overdistension resulted in alveolar edema and inflammation, that is termed ventilator-induced lung injury (VILI), the clinical features of VILI are including lung inflammation and congestion phenomenon, cause to high mortality. Despite the lung protective strategy is implemented to the situation of used ventilator in clinical but reduce the mortality rate is still limited. Therefore, it is very important to understand the VILI-associated with the mechanism of inflammatory mechanism and lung injury. The IL-33 is a newly identified pro-inflammatory cytokine of the IL-1 family. There are two isoforms of the IL-33 receptor including the transmembrane ST2 (ST2L) and soluble ST2 (sST2) isoforms. The IL-33/ST2 pathway is a very important mechanism which regulates immunity effect and involved in pathophysiological mechanism of clinical diseases. It is closely associated with such as acute lung injury, severe asthma, colitis, arthritis, and cardiovascular diseases. The expression of IL-33 in lung tissue appears both constitutive and inducible. The constitutive expression of IL-33 is described in smooth muscle cells and bronchial epithelial cells in lung. The expression of IL-33 is clearly induced if murine was infected by influenza A virus in lungs. Damage to cardio fibroblasts can induce necrosis and release of IL-33 by mechanical strech, IL-33 newly synthesized that is initially existed the nucleus transit into the cytoplasmic space and reside in membrane-bound vesicles. However, it is unclear what does the IL-33/ST2 pathway act to VILI mechanism. The purpose of this study was to study whether the IL-33 and its ST2 receptor to involving in acute lung injury induced by a mechanical ventilator in rats. And, we also to explore IL-33/ST2 pathway participate in the lung protective strategies by hydrocortisone and hypercapnia administration in VILI. Male Wistar rats were intubated after tracheostomy and received ventilation at 10/20 cm H2O of inspiratory pressure PC10/PC20 by a G5 ventilator for 4 hours. And catheters were inserted in femoral artery and vein were collected hemodynamic change and serum sampling. The hemodynamic and respiratory parameters were collected and analyzed. Collecting lung tissue and bronchoalveolar lavage fluid (BALF) when animals were sacrificed after the experiment completed. The morphological changes of lung injury were also assessed by histological H&E stain. The dynamic changes of lung injury markers TNF-α, IL-1β and IL-33 level were measured by ELISA assay in serum, BALF, and lung tissue. Using the immunochemistry and western blotting assay to determine the IL-33 and its receptor ST2 changes in lung tissue.
A VILI study model of rat were established, rats received a G5 ventilator and used a pressure control mode, the peak inspiration gas channel pressure maintains separately at 10 cmH2O (PC10) and 20 cmH2O (PC20). The results found that tidal volume was significantly higher in PC20 than PC10 group, but was no difference within group during VILI for 4 hours in PC10 or PC20 groups. The mean arterial blood pressure was decreased by higher pressure in PC20 group, but was no difference within group during VILI for 4 hours in PC10 or PC20 groups. The heart rate was not significant difference. Analyzed the severity of lung injury in VILI, the obvious alveolar space increase, alveolar wall thicker, inflammatory cell accumulation, hyaline membrane generation, pulmonary edema, and alveolar hemorrhage were found in the PC10 and PC20 groups. Moreover, the concentration of TNF-α and IL-1β was increased in BALF and serum in the VILI groups. There is no significant of serum IL-33 in both groups. But, the concentration of IL-33 in BALF were obvious reduced in both group. In addition, the IL-33 expression was increased in lung tissue with VILI. Interestingly, the data showed that ST2L membrane form was highly accumulated in the membrane fraction of lung tissue in the PC10 and the PC20 group compared to control group, but the ST2L was dramatically decreased in cytosol fraction. Conversely, the sST2 soluble form was slightly decreased in the membrane and cytosol fractions in the VILI group compared to the control group. These results indicated ST2L receptor translocated to membrane from cytosol in VILI rats. Then, whether the IL-33 cytokine and its receptor ST2 involved in the hydrocortisone and hypercapnia treated VILI were explored. Rats were administered by hydrocortisone after high-pressure ventilation 2 hours could reduce lung injury. Moreover, the VILI-induced the reduction of IL-33 in BALF were restored to more by hydrocortisone. Take together, examination of the IL-33 in BALF as a biomarker of VILI may provide a new concept of lung protection strategy. Furthermore, the VILI rats were received high volume ventilation and were giving hypercapnia, it is 5% CO2 inhalation for four hours. Those consequences of 5% CO2 inhalation could improve hemodynamic changes and gas exchange. And, the VILI-induced pulmonary inflammation symptom was significantly reduced. In addition, the VILI-induced the increase of TNF-α in BALF was reduced by using the 5% CO2 inhalation. In addition, the reduction of IL-33 cytokine release to BALF also was restored to rich. The VILI-induced the increase of IL-33 and its receptor ST2 were reduced by 5%CO2 inhalation, then ventilator-associated lung injury was improved.
In conclusion, this present study used the clinical ventilators success established the experimental VILI animal model. The hemodynamic parameters and respiratory parameters from the ventilator were collected and analyzed. Using the lung tissue histology and detection of inflammatory cytokines from different sources to confirm VILI experimental model. Rats were through VILI, resulting in IL -33 cytokine expression increases in the amount of lung tissue, ST2L receptors significantly accumulated in the cell membrane. However, the clinical lung protective strategies of hydrocortisone or hypercapnia treatment could reduce IL-33 cytokine generation in lung and reduced the accumulation of ST2L receptors on the cell membrane, and BALF could be detected changes in IL-33 expression increased. Therefore, the IL-33/ST2 pathway can be an examined function as new biomarkers of VILI and probably a biological target in therapy. Moreover, IL-33/ST2 signaling activated by mechanically responsive lung injury may potentially serve as a new therapy target.

目錄 II
圖表目錄 III
中文摘要 IV
Abstract VI
致謝 X
第一章 :前言 1
第一節:呼吸器引發肺損傷之定義 1
第二節:呼吸器引發肺損傷之可能機轉 3
第三節:避免呼吸器引發肺損傷之保護策略 6
第四節:白介質-33 /ST2受器與肺損傷的影響 7
第五節:白介質-33 /ST2受器與機械性刺激的相關機制 11
第六節 : 研究目的 13
第二章 :白介質-33 在呼吸器引發肺損傷的機制表現 14
第一節 :序論 14
第二節 :實驗材料與方法 17
第三節 :實驗結果 22
第四節 :討論 27
第三章 :白介質-33 在氫化可體松、高碳酸血症處理大鼠呼吸器引發肺損傷中的作用 55
第一節 : 序論 55
第二節 : 實驗材料與方法 60
第三節 : 實驗結果 68
第四節 :討論 74
第四章 :結論與未來發展 107
第五章 :參考文獻 111

圖表目錄
圖 一:大鼠高壓通氣之呼吸波形與潮氣容積分析 33
圖 二: VILI對血液動力學之趨勢變化 35
圖 三: VILI大鼠在氣體交換的改變 37
圖 四:大鼠經高壓通氣四小時後之肺損傷嚴重度 39
圖 五: TNF-α細胞激素在VILI大鼠的表現 41
圖 六: IL-1β 細胞激素在VILI大鼠的表現 43
圖 七: IL-33 細胞激素在VILI大鼠的表現 45
圖 八:大鼠暴露高氣道壓力四小時,肺中IL-33 的表現分析 47
圖 九:肺組織的ST2 受器在大鼠暴露於VILI後的分析 49
圖 十: NF-кB 路徑在肺組織中的表現 51
圖 十一:VILI大鼠與經氫化可體松處理之氣道壓力與潮氣容積之監測分析 81
圖 十二:大鼠暴露於VILI在血液動力學之變化 83
圖 十三:VILI大鼠經HC處理肺組織病理變化 85
圖 十四: IL-33 細胞激素在血清及支氣管沖洗液中的表現量分析 87
圖 十五: VILI大鼠經高碳酸血症之PaCO2、PaO2之趨勢分析 89
圖 十六: VILI大鼠經高碳酸血症處理之血液動力學分析 91
圖 十七:大鼠經高壓通氣四小時後之肺組織病理學變化 93
圖 十八: VILI大鼠之發炎細胞激素分析 95
圖 十九: IL-33 濃度在VILL大鼠經高碳酸血症處理的表現 97
圖 二十: IL-33在肺組織中的表現 99
圖 二十一: HCA處理減緩肺組織ST2 受器在VILI大鼠的表現 101
圖 二十二:模式圖IL-33/ST2在VILI的路徑 110

表 一:大鼠在氣道壓力10 cmH2O及20 cmH2O與正常鼠之血液動力學與氣體交換之特性 53
表 二: VILI大鼠經高碳酸血症處理之血液動力學之特性 103
表 三: VILI大鼠經高碳酸血症處理之血液動脈氣體分析 105


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