Mechanisms responsible for leptin-regulated cell survival and tight junction permeability of ischemic endothelial cells

已知腦缺血會導致腦細胞壞死，血腦屏障(BBB)結構之受損，及嗜中性白血球浸潤到腦組織引起發炎造成組織之二次傷害。近年來瘦素被發現具有保護神經細胞及腦組織對抗缺血性傷害之功能。本實驗室因此進一步針對其保護機制做探討，發現將瘦素注射於缺血鼠腦部可降低BBB通透性，減少受損腦組織中嗜中性白血球之數量，及減緩腦組織發炎水腫的程度。然而瘦素對BBB通透性的保護機轉及對嗜中性白血球浸潤的抑制機轉，目前並不清楚，本論文因此先針對瘦素對缺血內皮細胞(BBB的重要成員之一)不同活性之調控做探討，希望能更進一步釐清瘦素對缺血腦組織的保護機轉。實驗中係以出生四天新生鼠之心臟血管內皮細胞做為分析平台，用以評估體外缺血/復氧(GOSD/Reoxygenation, G/R)時，瘦素對內皮細胞之存活率、釋放自由基的能力、細胞通透性及吸引嗜中性白血球貼附能力等的調控，及可能參與調控的分子機轉。研究結果顯示，內皮細胞經GOSD的刺激後，復氧，復糖，復血清均會加劇細胞的傷害，其中以復氧對內皮細胞所造成的傷害最大，而這傷害可被瘦素有效抑制。瘦素對G/R內皮細胞存活的保護主要是經由對p-ERK蛋白表現的提升或caspase-3蛋白表現的抑制所致。瘦素對G/R內皮細胞釋放superoxide的能力亦具抑制性，可以降低superoxide所導致的細胞傷害及發炎反應。實驗中同時發現，G/R亦會加劇內皮細胞和嗜中性白血球間貼附的能力，而瘦素對此貼附性的抑制，在G/R 3h及G/R 6h最明顯，可能和G/R內皮細胞中ICAM-1蛋白表現受到瘦素的抑制有關。同樣的G/R亦會加劇內皮細胞之通透性，而瘦素在G/R 0h，G/R 3h，G/R 6h，及G/R 24h均可有效降低此通透性。實驗結果進一步驗證，瘦素對和G/R內皮細胞通透性的抑制作用，可能是經由提升內皮細胞之occludin，ZO-1及VE-cadherin蛋白表現所致，和β-catenin的表現多寡無關。另外，瘦素所提升的occludin的蛋白表現有可能是經由活化PPARγ/PKCδ的訊號傳遞路徑所致。最後，當進一步在缺血腦組織中評估occludin、ZO-1、VE-cadherin、β-catenin和ICAM-1之蛋白表現變化時，發現五種蛋白之表現在缺血大腦皮質區均明顯提升，但腦部注射瘦素時，五種蛋白之表現明顯被抑制，顯示，除了ICAM-1蛋白表現的變化在體內及體外缺血模式中有其相似性外，其它4種緊密連接蛋白(tight junction protein)的蛋白表現反應在兩個缺血模式中完全相反。綜合以上結果，瘦素可透過不同作用機轉改變內皮細胞之特性，以修復BBB之通透性降低其與嗜中性白血球之貼附力，進而阻止嗜中性白血球浸潤至缺血腦組織，達到抑制發炎及保護缺血腦組織之功效。此研究成果除進一步釐清瘦素對缺血腦組織之保護機轉外，亦揭示日後或可考慮以提升內皮細胞“緊密連接蛋白”(例如利用PPARγ或PKCδ活化劑)或抑制其“貼附性蛋白”表現量的方式來保護BBB的結構完整，進而降低缺血腦組織的發炎性傷害。Cerebral ischemia can lead to death of various brain cells, disruption of blood brain barrier (BBB), brain infiltration of neutrophils and server brain inflammation and tissue injury. In recent years, leptin has been suggested to protect brain from ischemic injury the underlying mechanisms however, remain still unclear and to be determined. Early results from our laboratory have demonstrated that brain injection with leptin can significantly reduce the infarct volume, permeability of BBB and number of infiltrating neutrophils in ischemic brain. How leptin can repair the injured BBB and prevent the brain infiltration of neutrophils into ischemic brain subsequently became the primary concerns of this study. In the study, cardiac vascular endothelial cells (ECs) were first isolated and subjected to an in vitro ischemic environment, where the glucose, oxygen and serum were all deprived from the culture medium and also being called as GOSD condition. The impact of GOSD/re-oxygenation (G/R) upon vascular ECs was evaluated in together for the first time to reflect the impact of ischemia/ reperfusion upon ECs of BBB in brain. Leptin-mediated protection of ECs from G/R-caused injury was then evaluated based on ECs’ survival rate, inflammatory potential, cell-cell adhesion with neutrophils and cell permeability. The results showed that GOSD-induced cell damage was further exacerbated by the followed up treatment including supplementation with oxygen, serum, glucose or any two of them in combine. G/R however, led to the highest cell injury in ECs comparing to other treatments and the injury was inhibited by leptin. Leptin-mediated protection of ECs was likely due to the increased expression of p-ERK and decreased expression of caspase-3. Leptin also inhibited the G/R-induced superoxide release from ECs to avoid the inflammation. Cell-Cell adhesion between ECs and neutrophils was significantly increased under G/R condition, which was also interrupted by leptin probably due to the reduction of ICAM-1 expression in G/R-treated ECs. G/R-increased cell permeability in ECs was again inhibited by leptin and the inhibition was likely due to the increased expression of tight junction proteins including occludin, ZO-1 and VE-cadherin but not β-catenin. Leptin may act through the PPARγ/PKCδ signaling pathway to up-regulate the expression of occluding in both GOSD- and G/R-treated ECs. When the protein expression levels of occludin, ZO-1, VE-cadherin, β-catenin and ICAM-1 were determined by immunohistochemistry at tissue level in ischemic brain the outcome was quite different from that observed in the G/R model. The results showed that the protein expression levels of all 5 proteins were significantly increased at the ischemic core area by 90 min blood vessels occlusion (ischemia) followed by 24 h reperfusion. When leptin was given into the ischemic brain at the end of 90 min ischemia, all the protein expression levels were inhibited. In other words, except the ICAM-1, the responses of occludin, ZO-1, VE-cadherin and β-catenin were opposite to that in vitro. The discrepancy remains still unclear. In overall, leptin can act through multiple ways to alter the properties of vascular ECs under G/R stress, in order to lower down the permeability of BBB, neutrophil adherence to ECs and eventually the number of brain infiltrating neutrophils and brain inflammation in ischemic brain. Results from the study further clarified the underlying mechanisms responsible for leptin-mediated brain protection against ischemia/reperfusion injury; also provided valuable information for future screening or developing of new anti-stroke drugs. Reagents or strategies which can elevate the tight junction proteins (such as PPARγ agonist or PKCδ activator) down regulate the cell adhesion molecules may have their therapeutic values in the treatment of stroke and are worthy of study.致謝.....................................................i 中文摘要..................................................ii 英文摘要..................................................iii 目錄.....................................................v 圖目錄...................................................viii 表目錄...................................................ix 前言.....................................................1 一、中風................................................1 二、腦缺血與發炎..........................................1 三、血腦屏障.............................................2 四、血管內皮緊密連接(tight junction, TJ )之組成及對血腦屏障 之重要性.............................................3 五、缺血性中風與血腦屏障(BBB)..............................5 六、瘦素................................................5 七、瘦素與腦中風..........................................6 八、研究目標.............................................7 實驗材料與方法.............................................9 一、試劑................................................9 二、細胞培養.............................................11 (一) 心臟內皮細胞培養...................................11 (二)嗜中性白血球分離....................................11 三、體外缺血/復氧處理(GOSD/Reoxygenaton)..................12 四、Leptin處理..........................................12 五、細胞存活之分析........................................12 六、細胞免疫染色法(immunocytochemistry)...................12 (一)細胞接種.........................................12 (二)細胞固定.........................................12 (三)細胞免疫染色......................................12 七、蛋白質萃取............................................13 八、蛋白濃度測定(bradford assay)..........................13 九、西方墨點法(Western blot)..............................13 (一) 配製SDS-PAGE 膠體...............................13 (二) SDS 膠體電泳分析...............................14 (三) 蛋白質轉漬....................................14 (四) 免疫雜交......................................14 十、內皮細胞通透性分析(endothelial permeability assay)...14 十一、嗜中性白血球/內皮細胞貼附分析(neutrophil-endothelial adhesion assay)..................................15 十二、Nitrite釋放量之分析...............................15 十三、Superoxide釋放量之分析............................15 十四、缺血性腦中風動物模式之建立...........................16 (一) 實驗動物......................................16 (二) 腦缺血再灌流(ischemia/reperfusion)動物模式之建 立...........................................16 十五、梗塞區域評估.......................................16 十六、免疫組織染色法((immunohistochemistry, IHC).........17 (一)組織固定.......................................17 (二)組織切片.......................................17 (三)組織免疫染色法..................................17 十七、統計分析.........................................17 結果....................................................18 一、心臟血管內皮細胞的生長曲線.............................18 二、體外缺血/再復氧(GOSD/Reoxygenatio)對內皮細胞造成嚴重之傷害， 傷害程度隨復氧時間的增長而加據..........................18 三、在體外缺血/再復氧(G/R)環境下，瘦素對內皮細胞的保護性具有劑量 及時間之依存性.......................................19 四、在體外缺血/再復氧(G/R)環境下，瘦素對內皮細胞之保護有賴p-ERK 的存在.............................................19 五、在體外缺血/再復氧(G/R)環境下，瘦素可抑制內皮細胞中caspase-3之 蛋白表現............................................20 六、在體外缺血/再復氧(G/R)環境下，瘦素對內皮細胞釋放NO之能力不 具影響力............................................20 七、在體外缺血/再復氧(G/R)環境下，瘦素在G/R6h可顯著抑制內皮細胞釋 放超氧陰離子(superoxide)的能力........................21 八、嗜中性白血球對經體外缺血/再復氧(G/R)刺激後之內皮細胞的貼附程 度及內皮細胞中ICAM-1蛋白的表現量均明顯受到瘦素之抑制.......21 九、在體外缺血/再復氧(G/R)環境下，瘦素可顯著降低內皮細胞之通透性.21 十、在體外缺血/再復氧(G/R)環境下，瘦素可提升內皮細胞中緊密連接相 關蛋白(occludin, ZO-1, VE-cadherin)之表現............22 十一、在體外缺血/再復氧(G/R)環境下，瘦素可透過活化PPARγ/PKCδ的訊 號傳遞路徑來提升內皮細胞中occludin蛋白的表現量............23 十二、缺血腦組織中顯著被提升的緊密連接相關蛋白(occludin, ZO-1, VE-cadherin, β-catenin)及貼附蛋白(ICAM-1)之表現量明顯被 瘦素抑制............................................24 討論.....................................................25 參考文獻.......................................................53 一、中文文獻.............................................53 二、英文文獻.............................................53 附錄一....................................................6