臺灣博碩士論文加值系統

巴拉刈(Paraquat, PQ)是種全球廣泛使用的除草劑,然而對人類和動物有很高毒性。當攝入後,巴拉刈很快吸收進入血液中然後主要在肺組織內濃縮,然後進展成不可逆肺纖維化,肺纖維化是巴拉刈中毒的主要死因。肺纖維化的特徵有纖維母細胞增生,肌纖維母細胞分化,和細胞外基質堆積在肺組織。雖然一氧化碳(Carbon monoxide, CO)對人類和動物而言有劇毒性,近年來的研究發現 10-500 ppm 低劑量一氧化碳有些生物效果,例如抗發炎,保護缺氧或缺血肺損傷。研究也顯示一氧化碳釋放分子(Carbon monoxide releasing molecules, CORMs)能改善巴拉刈處理過心肌細胞的存活率,也可以減少博萊黴素(Bleomycin)所致的肺纖維。這些發現推測一氧化碳釋放分子在巴拉刈所導致的肺纖維可能扮演一定程度的治療角色。
本研究首先證實了,一氧化碳釋放分子-3 (CORM-3)和一氧化碳釋放分子-A1(CORM-A1)有劑量相關性的體外抗氧化活性(自由基 DPPH 與 ABTS 清除)。體外細胞實驗指出,20 μM CORM-3 可改善因巴拉刈/過氧化氫處理 24 小時後所造成RAW 264.7/A549/MRC-5 等細胞損傷(提高細胞存活率)。當先期給予 20-100 μM 的 CORMs (CORM-3或CORM-A1),可減少因巴拉刈所誘升的A549細胞/MRC-5細胞所釋放的結締組織生長因子(CTGF)與血管張力素 II (ANGII)。體內動物實驗更進一步證實,腹腔注射 CORMs (CORM-3 或 CORM-A1)14 天後,可以提高 14 天前經巴拉刈處理所造成的小鼠低存活率,也可以減少巴拉刈餵食小鼠肺部組織中膠原纖維(PAS 與 Masson 染色檢查)和轉化生長因子-β(TGF-β) (IHC 染色檢查)的蛋白質過度表現。西方墨點法分析顯示,10 mg/kg CORMs (CORM-3或 CORM-A1)可以降低巴拉刈所誘升的小鼠肺部組織中環氧化酶-2 (COX-2)、α-平滑肌肌動蛋白(α-SMA)和腫瘤壞死因子α(TNF-α)的蛋白質表現量,同時我們發現 5 mg/kg CORMs (CORM-3 或 CORM-A1)的投予劑量下,即可顯著地減少小鼠血清中羥脯氨酸的濃度。整體而言,本研究顯示一氧化碳釋放分子對巴拉刈所致的肺纖維的減緩可能有治療角色。

Paraquat (PQ) is a widely used herbicide in the world but highly toxic to both humans and animals. After ingestion, PQ is rapidly absorbed into the bloodstream and concentrates primarily in pulmonary tissue, and then develops into irreversible pulmonary fibrosis, which is the primary cause of death. Pulmonary fibrosis is characterized by fibroblast proliferation, myofibroblast differentiation, and deposition of extra-cellular matrix in the lung parenchyma. Carbon monoxide (CO) is toxic to humans and animals, recent studies suggest low dose, 10 to 500 ppm, CO has anti-inflammatory effects, and protection against hypoxic as well as ischemic lung injury. Studies revealed Carbon monoxide releasing molecules (CORMs) can improve viability of paraquat-treated cardiomyocytes and decrease pulmonary fibrosis induced by bleomycin. These findings suggest CORMs may have therapeutic effect on paraquat-induced pulmonary fibrosis.
This study showed first in vitro CORMs (CORM-3 or CORM-A1) has dose related anti-oxidant effects (free radicals, ABTS+•, DPPH•, scavengers). In vitro cell study, 20 μM CORM-3 can improve cell injury of RAW264.7/A549/MRC-5 cell after paraquat/H2O2-treatment 24 hours (increase survival rate of cells).Pretreatment with 20-100 μM CORMs (CORM-3 or CORM-A1) can decrease high level of CTGF and ANGII which released by paraquat-treated A549/MCR-5. In vivo animal study showed intraperitoneal injection of CORMs improve the survival for 14 days of mice fed with paraquat, and decrease overexpression of collagen fiber (PAS and Masson stain) and TGF-β (IHC stain). By western blot analysis, 10 mg/kg CORMs (CORM-3 or CORM-A1) decrease protein expression of COX-2, α-SMA and TNF- increased by paraquat. We also found prescription with 5 mg/kg CORMs (CORM-3 or CORM-A1) decrease serum level of Hydroxyproline of mice fed with paraquat. Overall, this study indicates that CORMs would be an attractive therapeutic approach to attenuate the progression of pulmonary fibrosis induced by paraquat.

目錄

目錄............................................................................................................... 5

一、 研究背景與動機........................................................................... 12

1. 巴拉刈.................................................................................................................... 12

1.1 巴拉刈的中毒機制 ........................................................................................... 12

1.2 巴拉刈中毒的臨床症狀 ................................................................................... 16

1.3 巴拉刈中毒的治療 ........................................................................................... 17

2. 肺纖維.................................................................................................................... 18

2.1 纖維化的機轉 ................................................................................................... 18

2.2 肺纖維化發病機轉 ........................................................................................... 19

3. 血管張力素II(angiotensin II, ANGII)簡介.......................................................... 19

4. 結締組織生長因子(connective tissue growth factor, CTGF).......................... 21

5. α-平滑肌肌動蛋白(α-Smooth Muscle Actin,α-SMA).................................. 21

6. 環氧化酶(Cyclooxygenase-2, COX-2)............................................................... 21

7. 羥脯氨酸(Hydroxyproline, Hyp)........................................................................ 22

8 一氧化碳................................................................................................................ 22

8.1 一氧化碳的生理作用 ....................................................................................... 22

8.2 一氧化碳治療的方法 ....................................................................................... 23

二、 研究動機和目的........................................................................... 26

三、 實驗材料與方法........................................................................... 27

1. 實驗材料................................................................................................................ 27

2. 儀器設備................................................................................................................ 28

3. 實驗方法................................................................................................................ 29

3.1 抗氧化能力實驗:(清除 DPPH 自由基能力測定)......................................... 29

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3.2 總抗氧化能力實驗:(清除 ABTS 自由基能力測定)..................................... 29

3.3 RAW 264.7 細胞毒性實驗 .............................................................................. 30

3.4 RAW 264.7 細胞存活實驗 ............................................................................... 30

3.5 MRC-5 細胞毒性實驗 ...................................................................................... 30

3.6 MRC-5 細胞存活實驗 ...................................................................................... 31

3.7 CTGF/ANGII 實驗........................................................................................... 31

3.8 ELISA ................................................................................................................ 31

3.9 動物實驗-肺纖小鼠誘導模式設計.................................................................. 32

3.10 蘇木精(hematoxylin)和伊紅(eosin)染色法 ( H&E Stain )................... 33

3.11 PAS 組織化學染色法....................................................................................... 34

3.12 Masson trichrome stain 組織化學染色法......................................................... 34

3.13 免疫組織化學染色法 (Immunohistochemistry, IHC Stain )........................... 34

3.14 西方墨點法(Western blot)................................................................................ 35

3.15 肺部纖維化相關生化值測試- Hydroxyproline 含量....................................... 36

4. 統計分析................................................................................................................ 37

四、 結果............................................................................................... 38

1. 抗氧化實驗............................................................................................................ 38

2. 細胞實驗................................................................................................................ 38

2.1 RAW 264.7 細胞毒性實驗................................................................................. 38

2.2 RAW 264.7 細胞存活實驗................................................................................. 39

2.3 MRC-5 細胞毒性實驗 ........................................................................................ 39

2.4 MRC-5 細胞存活實驗 ........................................................................................ 40

3. CTGF/ANGII 實驗............................................................................................... 41

3.1 A549 細胞給予 CORM-3/CORM-A1 後 CTGF 的表現 ................................... 41

3.2 MRC-5 細胞給予 CORM-3/CORM-A1 後 CTGF 的表現................................ 41

3.3 A549 細胞給予 CORM-3/CORM-A1 後 ANGII 的表現.................................. 41

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3.4 MRC-5 細胞給予 CORM-3/CORM-A1 後 ANGII 的表現............................... 42

4. 動物實驗:............................................................................................................ 43

4.1 實驗動物存活率 ................................................................................................. 43

4.2 病理 ..................................................................................................................... 43

4.3 H&E Stain............................................................................................................ 43

4.4 PAS Stain............................................................................................................. 44

4.5 Masson’s trichrome Stain .................................................................................... 44

4.6 IHC Stain(TGF-β)................................................................................................ 44

4.7 西方墨點法 ......................................................................................................... 44

五、 討論............................................................................................... 46

六、 結論與展望................................................................................... 48

七、 參考文獻....................................................................................... 92

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