Establishment of transgenic mice which reported the NF-κB activation to study the hyperoxia-induced lung injury

罹患ARDS（急性呼吸窘迫症）的病人多以機械性通氣與高濃度氧（氧氣濃度大於95％）的支持療法來延續生命。然而，機械性通氣與高氧治療法會導致急性肺臟損傷與肺臟發炎反應。目前已知NF-κB 為一群參與調控細胞存亡與分化及發炎反應的轉錄因子。雖然體外實驗早已證實NF-κB 的轉錄活化能力，但體內的NF-κB 轉錄活化模式卻鮮為人知。為了探討體內NF-κB 的活化作用，於高濃度氧誘發之肺臟損傷中所扮演的角色，因此我們將產製具FVB/NJNarl 之背景，而冷光的表現受NF-κB 所調控NF-κB/luciferase 的基因轉殖小鼠，並且利用即時活體影像系統觀察動物體內NF-κB 活化狀態。我們將wild type FVB/NJNarl 品系小鼠暴露於高濃度氧氣大於95％的環境下，血液中的嗜中性球與淋巴球的數目，於暴露在高氧環境24 小時後明顯地開始增加。此外顯微鏡觀察到肺臟中之發炎細胞數目於暴露於高氧環境後72 小時有增加之趨勢。與正常小鼠相較之下，暴露於高氧環境48 至72 小時的小鼠，肺臟細胞之核內NF-κB 蛋白質含量有增加的現象。同時，將NF-κB/luciferase 暴露於高氧環境中，肺臟細胞之核內NF-κB 蛋白有核易位的現象，且帶動其調控的luciferase 之表現。以非侵入性的活體影像系統偵測，活體動物之生物體暴露於高氧24 小時後開始有發光訊號。但為何種器官所散發的生物體發光訊號仍需要進一步調查。綜合上述，我們成功建立利用基因轉殖小鼠報導NF-κB 的活化作用，並且利用高濃度氧誘發之肺臟損傷的模式，可發現NF-κB 的活化具帶動冷光的表現，因此，此小鼠品系為提供一個可貴NF-κB 活化作用的分析工具，且未來可應用在許多與NF-κB 相關的疾病模式上。In patients with acute respiratory distress syndrome (ARDS), supportive therapy with mechanical ventilation and hyperoxia (O2> 95%) is often life saving. However,further acute lung injury is an unfortunate consequence of oxygen therapy as well as mechanical injury secondary to ventilator induced/associated lung inflammation.Nuclear factor kappa B (NF-κB) is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-κB has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the NF-κB activation on the hyperoxia-induced lung injury in vivo, the NF-κB/luciferase transgenic mice, FVB/NJNarl background, that express luciferase under the control of NF-κB was generated, which enabling real-time in vivo imaging of NF-κB activation in intact animals. The wild type FVB/NJNarl mice exposed to >95% oxygen in a hyperoxic chamber were first under investigated. Numbers of lymphocytes and neutrophils in blood were significantly increased started 24 hours hyperoxia exposure. Microscopic analysis revealed that hyperoxic exposure for 72 hours was associated with increased inflammatory cells in the lungs. NF-κB translocation in lung was increased and reached a maximum between 48 and 72 hours compared to the control mice. Additionally, treating NF−κB/luciferase transgenic mice with hyperoxia,the correlation of luciferase expression and NF-κB nuclear translocation was confirmed in lung homogenate. Using noninvasive optical image system, the bioluminescence signals were observed in the dorsal of live animal started from 24 hours exposure. The specific organ emitted bioluminescence signals need to be further investigation. Taken together, using hyperoxia induced lung injury model, we successfully established transgenic mice which reported the NF-κB activation via luciferase expression and this mouse should provide a valuable tool to analyze NF-κB activation in many disease models.目錄 致謝............................................................... i 中文摘要........................................................... ii 英文摘要........................................................... iii 目錄............................................................... v 圖目錄............................................................. viii 表目錄............................................................. ix 緒論............................................................... 1 第一章、文獻探討................................................... 2 第一節： 活性氧分子的產生......................................... 2 第二節： 活性氧分子過量對核酸、蛋白質及細胞的影響................. 2 第三節： 活性氧產生的疾病......................................... 2 第四節： 高濃度氧對肺臟造成的影響................................. 2 第五節： 發炎性轉錄因子Nuclear factor-κB (NF-κB) ....................3 第六節： 高濃度氧與發炎性反應..................................... 3 第七節： 醫學影像-生物活體影像系統(In vivo bioluminescence)的應用..... 3 第八節： 研究目的................................................. 4 第二章、材料與方法................................................. 5 第一節： 供胚用小鼠之飼養......................................... 5 第二節： 注射用基因之純化......................................... 5 第三節： 供胚用小鼠超級排卵處理................................... 5 第四節： 胚之基因注射與胚移殖..................................... 6 第五節： 轉基因動物之分析......................................... 6 第六節： 高濃度氧損傷動物模式..................................... 6 第七節： 肺部組織外觀觀察......................................... 7 第八節： 石蠟包埋組織............................................. 7 第九節： 組織免疫化學染色......................................... 7 第十節： 組織免疫螢光化學染色..................................... 7 第十一節： 血液中血球計數......................................... 8 第十二節： 組織核內蛋白萃取....................................... 8 第十三節： 蛋白質定量............................................. 8 第十四節： 蛋白質電泳（SDS-PAGE）................................9 第十五節： 西方墨點法(Western blot) .................................9 第十六節： ECL 呈色...............................................9 第十七節： 肺沖洗液中細胞激素測定................................. 9 第十八節： 放射性條帶雜合分析(Slot-blot hybridization) .................10 第十九節： 及時影像分析IVIS (In-vivo Imaging System) .................10 第二十節： 統計方法及繪圖軟體..................................... 10 第三章、結果....................................................... 11 第一節： 觀察高濃度氧引發動物行為表現與肺部損傷情形............... 11 第二節： 組織病理變化觀察......................................... 11 第三節： 組織免疫化學染色偵測NF-κB 的表現.........................11 第四節： 組織免疫螢光化學染色偵測NF-κB 是否有進入細胞核內.........12 第五節： 小鼠經高濃度氧處理後血液學上的分析比較................... 12 第六節： 高濃度氧誘發之小鼠肺臟組織中NF-κB 核易位現象.............12 第七節： 肺沖洗液中細胞激素測定................................... 13 第八節： 產製NF-κB-luc 基因轉殖鼠與表現載體........................13 第九節： 利用高濃度氧處理之NF-κB 基因轉殖鼠的即時影像............ 13 第十節： 比較純合子與雜合子的NF-κB 基因轉殖鼠之冷光反應...........13 第四章、討論....................................................... 14 第五章、結論....................................................... 17 參考文獻........................................................... 18 圖................................................................. 23 表................................................................. 37 附錄............................................................... 40 圖目錄 Figure 1 The hyperoxia animal model................................... 23 Figure 2. Hyperoxia induced lung injury animal........................... 24 Figure 3. Histological evaluation of hyperoxia-induced lung injury............ 25 Figure 4. Histological evaluation of hyperoxia-induced liver damage........... 26 Figure 5. Nuclear translocation of the NF-κB in lung tissue lung sections were examined by immunohistochemical staining....................... 27 Figure 6. Nuclear translocation of the NF-κB in liver tissue liver sections were examined by immunohistochemical staining....................... 28 Figure 7. Differential count of white blood cell in mice exposed to hyperoxia for 0-72hrs.................................................... 29 Figure 8. Physilogical results of red blood cell and platelets in mice after exposure to hyperoxia for 0-72hrs...................................... 30 Figure 9. Nuclear translocation of NF-κB in lung tissues in hyperoxia-induces mice...................................................... 31 Figure 10. The levels of NF-κB and luciferase expression in lung tissue were evaluated by Western blot analysis .............................. 32 Figure 11. The levels of KC cytokines in BAL fluid from animals after exposure to hyperoxia for 0, 24, 48 and 72h, respectively ..................... 33 Figure 12. The preparation of NF-κB-luc transgene.......................... 34 Figure 13. In vivo imaging of NF-κB-luc transgenic mice response to hyperoxia... 35 Figure 14. In vivo imaging of hyperoxia - treated NF-κB-luc+/- and NF-κB-luc+/+ transgenic mice............................................. 36 表目錄 Table 1. Sequence of the primers used in this study........................ 37 Table 2. Differential count of white blood cell in mice exposed to hyperoxia for 0-72hrs................................................... 38 Table 3. Physilogical results of red blood cell and platelets in mice after exposure to hyperoxia for 0-72hrs...................................... 3