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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林淑華(Shu-Wha Lin) | |
| dc.contributor.author | Chia-Lu Hung | en |
| dc.contributor.author | 洪嘉璐 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:30:17Z | - |
| dc.date.available | 2022-09-08 | |
| dc.date.copyright | 2017-09-08 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-18 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68679 | - |
| dc.description.abstract | APAP (acetaminophen) 濫用為已開發國家急性肝衰竭的常見原因，在高劑量下 APAP 會經由細胞色素 (cytochrome) P450 CYP2E1 及 CYP1A2 氧化為親電性高的活性物質 NAPQI (N-acetyl-p-benzoquinone imine)。NAPQI會與細胞內的許多蛋白質形成非特異性的共價鍵結而破壞其功能造成細胞損傷，肝臟藉由glutathione (GSH) 與 NAPQI 共價結合以降低其肝毒性，當 GSH被大量消耗之後，ROS (reactive oxygen species) 及 RNS (reactive nitrogen species) 含量上升。最終使細胞氧化壓力上升、粒線體失去功能和DNA損壞。
Hepsin 為第二型穿膜絲胺酸蛋白酶，主要表現於肝臟。本實驗室先前發現 Hepsin 基因剔除小鼠在注射 400 mg/kg APAP 後，會因嚴重的肝損傷而在8~10小時內死亡。本篇論文首先確認 Hepsin 基因剔除小鼠在投予400 mg/kg APAP 4~6小時後，血清AST、ALT 及 HMGB1 相較野生型小鼠皆大量上升，表示 Hepsin 基因剔除小鼠肝損傷較嚴重。由肝組織 H&E 染色可見野生型及基因剔除小鼠於投予 APAP 2小時後皆發現肝細胞空泡化，而 Hepsin 基因剔除小鼠在投予 APAP 4小時後較野生型小鼠有較高的壞死比例。實驗進一步證明野生型小鼠在注射 400 mg/kg APAP 1小時後，肝細胞中 Hepsin 蛋白表現量顯著下降，顯示 Hepsin 在 APAP 造成急性肝衰竭的早期即扮演角色。Hepsin 基因剔除小鼠在投予400 mg/kg APAP 的0.5~6小時間點，CYP2E1、CYP1A2 的表現量及肝組織 GSH 的消耗量均與野生型小鼠無差異，但肝組織中反應氧化壓力的硝基酪氨酸 (nitrotyrosine) 在2小時即顯著上升。由西方墨點法分析受 APAP肝毒性而活化的 MAPK 訊息傳遞路徑，發現 JNK 和 AKT 磷酸化程度在 Hepsin 基因剔除小鼠及野生型小鼠間並無差異，而 Hepsin 基因剔除小鼠在投予 APAP 2小時後 P38 磷酸化的比例和4小時後 ERK 磷酸化的比例皆顯著上升，而投予 P38 抑制劑，在施打 APAP 4小時後血清AST、ALT濃度明顯下降。初步推測，Hepsin 可能藉由抑制 P38 的活性，進而緩解 APAP 造成的肝臟損傷。 | zh_TW |
| dc.description.abstract | APAP overdose is the leading cause of acute liver failure in developed country. At high doses, cytochrome P-450 enzymes (CYP2E1, CYP1A2) convert APAP into a reactive quinone form, N-acetyl-p-benzoquinone imine (NAPQI). Although NAPQI is inactivated by conjugation with glutathione (GSH), once the pool of GSH is exhausted, remaining NAPQI covalently binds cellular proteins, causing the elevation of reactive oxygen species and nitrogen species. Finally, it results in oxidative stress, mitochondrial dysfunction, and DNA damage.
Hepsin, a type II transmembrane serine protease, is mainly expressed in the liver. Previous study in our lab found that Hepsin knockout (KO) mice treated with 400 mg/kg APAP resulted in severe liver injury and death of mice within 8 to 10 hours after APAP injection. Our data confirmed that the concentration of serum AST, ALT, and HMGB1 rises greatly in Hepsin knockout mice within 4 to 6 hours after 400 mg/kg APAP injection, indicated that Hepsin knockout mice had more serious liver injury. H&E stain of hepatocytes appeared vacuolization in both wild type and knockout mice 2 hour after APAP injection, and higher proportion of necrosis in Hepsin knockout mice compare to wild type mice 4 hour after APAP injection. Challenged wild type mice with 400 mg/kg APAP and found that the endogenous Hepsin in the liver of WT mice was significantly decreased within 1 hour after APAP injection, indicating that Hepsin played a role in APAP-induced acute liver failure at early time points. The protein level of P450 CYP2E1 and CYP1A2, and the GSH depletion level in liver tissue showed no difference between Hepsin knockout and WT mice under 400 mg/kg APAP treatment. However, the expression level of nitrotyrosine, which represents oxidative stress in cells, were elevated 2 hours after APAP treatment. The kinase of signaling pathway, which is activated under APAP-induced toxicity, were analysis by Western blots. We found phosphorylated JNK and AKT showed no difference between Hepsin knockout and WT mice, but p-P38/P38 and p-ERK/ERK ratios were significantly upregulated at 2 and 4 hour after APAP injection, respectively. Besides, the concentration of serum AST, ALT were attenuated in knockout mice pre-injection with P38 inhibitor 1 hour before 4 hour 400 mg/kg APAP been challenged. We proposed that Hepsin may be involved in APAP-induced liver injury by regulating the activity of P38 kinase. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:30:17Z (GMT). No. of bitstreams: 1 ntu-106-R04424001-1.pdf: 38694325 bytes, checksum: b533b066b05845463b3994c619cc99ad (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書 I 誌謝 II 中文摘要 III 英文摘要 V 圖目錄 X 表目錄 XI 第一章 緒論 1 1.1 Hepsin 1 1.1.1 Hepsin 的蛋白質結構 1 1.1.2 Hepsin 組織分佈與功能探討 2 1.1.3 Hepsin的病理角色 2 1.2乙醯胺酚 (Acetaminophen, APAP) 造成的急性肝衰竭 3 1.2.1 APAP 臨床現況與案例 3 1.2.2 APAP 的代謝與造成肝損傷的分子機制 4 1.2.3 APAP與細胞壞死 6 1.2.4 APAP 與影響其毒性的基因 7 1.2.5 APAP 動物模型 8 1.3 研究目的 10 第二章 實驗材料與方法 11 2.1 實驗動物 11 2.2 動物實驗設計、血清收集及犧牲 11 2.2.1 APAP 注射 11 2.2.2 心臟採血與血清收集 11 2.3 小鼠血清中 AST、ALT 及 HMGB1 之測量 12 2.3.1 小鼠血清天冬胺酸轉胺酶 (Aspartate transaminase, AST)、丙胺酸轉胺酶 (Alanine transaminase, ALT) 濃度測量 12 2.3.2 小鼠血清 HMGB1 (high mobility group box 1) 濃度測量 12 2.4 小鼠肝臟穀胱甘肽 (glutathione, GSH) 之測量 12 2.5 石臘切片的製作、組織染色 (H&E stain) 與肝臟壞死區域的觀察與定量 12 2.5.1 石蠟組織檢體處理及切片 13 2.5.2組織染色 13 2.5.3 肝臟壞死區域之定量 13 2.6 小鼠肝細胞中訊息傳遞路徑各蛋白質之定量 13 2.6.1 小鼠肝臟總蛋白質萃取 13 2.6.2 蛋白質電泳及西方墨點法 14 2.7 P38 抑制劑處理小鼠體內 P38 之激酶功能實驗 15 2.8 數據分析與統計 15 第三章 實驗結果 16 3.1 Hepsin與乙醯胺酚 (APAP) 造成的急性肝損傷 16 3.1.1 血清 AST, ALT, HMGB1 的變化 16 3.1.2 基因剔除小鼠在 APAP 處理後肝臟壞死面積顯著增加 16 3.1.3 野生型小鼠肝臟 Hepsin 蛋白表現量在投予 APAP 後顯著下降 17 3.2 探討基因剔除小鼠在 APAP 處理下肝損傷較嚴重的可能原因 18 3.2.1 野生型及基因剔除小鼠對 APAP 代謝及解毒能力並無差異 18 3.2.2 基因剔除小鼠以 APAP 處理後氧化壓力顯著上升 18 3.2.3 小鼠以 APAP 處理後細胞壞死性凋亡調節蛋白表現量無差異 19 3.2.4 小鼠以 APAP 處理後訊息傳遞路徑激酶的蛋白表現量變化 19 3.3 基因剔除小鼠在 APAP 處理前給予 P38 抑制劑對肝損傷的影響 20 3.3.1 小鼠在 APAP 處理前給予 DMSO 會使肝損傷降低，但不影響 P38 活化 20 3.3.2 P38 抑制劑會使 APAP 造成的基因剔除小鼠的肝損傷降低 21 第四章 討論 22 4.1 Hepsin 蛋白與 APAP 造成的肝損傷的關係 22 4.2 Hepsin 基因剔除小鼠動物模型與 APAP 造成的急性肝衰竭的病理變化 22 4.3 Hepsin 基因剔除小鼠與 APAP 造成的急性肝衰竭的分子機制 23 4.4 P38 抑制劑與 APAP的實驗與疑慮 24 4.5 P38 激酶與 Hepsin 可能的調控關係 26 第五章 結論與展望 27 參考文獻 28 圖一、小鼠施打400mg/kg APAP 後產生肝損傷的血清生化值 36 圖二、小鼠施打400mg/kg APAP 後肝損傷的病理切片圖 38 圖三、小鼠施打400mg/kg APAP 後產生肝損傷的病理切片圖 40 圖四、小鼠施打400mg/kg APAP 後肝臟 Hepsin 蛋白量的變化 42 圖五、小鼠施打400mg/kg APAP 後肝臟 P450 酵素 CYP2E1 和 CYP1A2 表現量之變化 45 圖六、小鼠施打400mg/kg APAP 後 glutathione (GSH) 表現量之變化 46 圖七、小鼠施打400mg/kg APAP 後肝細胞中被硝化 (nitration) 之蛋白量的變化 48 圖八、APAP 處理後 RIP1 和 RIP3 蛋白表現量之變化 50 圖九、APAP 相關 mitogen-activated protein kinases (MAPK) 訊息傳遞路徑激酶 p-JNK/JNK之蛋白量的變化 52 圖十、APAP 處理後相關 MAPK 訊息傳遞路徑激酶 p-ERK/ERK 之蛋白量的變化 54 圖十一、APAP 處理後 MAPK 訊息傳遞路徑激酶 p-P38/P38 之蛋白量的變化 56 圖十二、APAP 處理後訊息傳遞路徑激酶 p-AKT/AKT 之蛋白量的變化 58 圖十三、APAP 處理後 MAPK 與 AKT 訊息傳遞路徑激酶之定量 59 圖十四、小鼠給予 SB203580 後 APAP 對肝損傷及 pP38/P38 表現量的影響 62 表一、西方墨點法使用之抗體 63 附錄一、Hepsin 活化示意圖 64 附錄二、APAP 造成的肝毒性的臨床表現 65 附錄三、APAP 的代謝與可能影響其毒性的因子 66 附錄四、Hepsin 野生型及基因剔除小鼠以 APAP 處理後存活率 67 | |
| dc.language.iso | zh-TW | |
| dc.subject | P38 激? | zh_TW |
| dc.subject | 訊息傳遞路徑 | zh_TW |
| dc.subject | 急性肝衰竭 | zh_TW |
| dc.subject | 乙醯胺酚 | zh_TW |
| dc.subject | Hepsin 基因剔除小鼠 | zh_TW |
| dc.subject | Hepsin knockout mice | en |
| dc.subject | acetaminophen | en |
| dc.subject | acute liver failure | en |
| dc.subject | signaling pathway | en |
| dc.subject | P38 kinase | en |
| dc.title | 探討 Hepsin 在 acetaminophen 導致的肝毒性中扮演的角色 | zh_TW |
| dc.title | Investigate the role of Hepsin in acetaminophen-induced hepatotoxicity | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林淑容,郭錦樺,莊雅惠 | |
| dc.subject.keyword | Hepsin 基因剔除小鼠,乙醯胺酚,急性肝衰竭,訊息傳遞路徑,P38 激?, | zh_TW |
| dc.subject.keyword | Hepsin knockout mice,acetaminophen,acute liver failure,signaling pathway,P38 kinase, | en |
| dc.relation.page | 67 | |
| dc.identifier.doi | 10.6342/NTU201703929 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-18 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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