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系統識別號 U0026-3107201216194900
論文名稱(中文) 巨噬細胞移動抑制因子於順鉑引起急性腎損傷的角色
論文名稱(英文) The Role of Macrophage Migration Inhibitory Factor in Cisplatin-induced Acute Kidney Injury
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 100
學期 2
出版年 101
研究生(中文) 林玉慧
研究生(英文) Yu-Huei Lin
學號 s96994023
學位類別 碩士
語文別 英文
論文頁數 68頁
口試委員 口試委員-邱元佑
口試委員-邢中熹
指導教授-林秋烽
中文關鍵字 順鉑  急性腎臟損傷  腎發炎  腎毒性  細胞凋亡  腎小管壞死  肝醣合成酶激酶3  巨噬細胞移行抑制因子  皮質部腎小管細胞  硫胱氨酸蛋白酶  脫氧核醣核酸損傷  訊息傳遞  抗癌 
英文關鍵字 Cisplatin  AKI  Renal inflammation  Nephrotoxicity  Apoptosis  Tubular necrosis  GSK-3  MIF  Cortical tubular cell  Caspase  DNA damage  Signaling  Anticancer 
學科別分類
中文摘要 抗癌藥物順鉑誘導急性腎臟損傷 (AKI) 係經過腎發炎及腎毒性產生包括腎小管細胞凋亡伴隨壞死現象。在本研究中利用順鉑誘導急性腎臟損傷模式，我們探討肝醣合成酶激酶3 (GSK-3) 促使腎毒性產生繼而誘發巨噬細胞移行抑制因子 (MIF) 媒介腎發炎。體內動物模式證明藥物性抑制巨噬細胞移行抑制因子有效地減緩順鉑誘導急性腎臟損傷；然而，體外細胞模式證明巨噬細胞移行抑制因子的表現並不參與順鉑誘導腎小管細胞凋亡。免疫組織染色顯示腎臟器官內巨噬細胞移行抑制因子特定地表現在腎小管細胞，並且順鉑刺激會造成細胞內巨噬細胞移行抑制因子表現的缺失。無論在體內動物模式或體外細胞模式，實驗證實伴隨著順鉑誘導腎毒性產生會進而引起皮質部腎小管細胞內巨噬細胞移行抑制因子表現缺失或由胞內釋出至胞外。體外細胞模式證明包括硫胱氨酸蛋白酶的活化、脫氧核醣核酸損傷反應的誘導以及表皮生長因子接受器/磷酸激酶Src/蛋白質酶C-δ/活性氧化物/ p38有絲分裂原活化蛋白質激酶/ c-Jun胺基末端激酶的訊息傳遞都可調控於順鉑細胞毒性以及巨噬細胞移行抑制因子的釋出。重要地是順鉑刺激可活化肝醣合成酶激酶3伴隨著巨噬細胞移行抑制因子的釋出以及肝醣合成酶激酶3調控腎毒性與腎發炎。考量於肝醣合成酶激酶3對順鉑媒介抗癌活性的重要性，在以順鉑為基的抗癌模式下，抑制巨噬細胞移行抑制因子有效地減緩急性腎臟損傷的發生並且無損於順鉑的抗癌活性。本研究結果不只闡明肝醣合成酶激酶3調控之腎毒性與巨噬細胞移行抑制因子調控之腎發炎分子機制並且提供一個治療策略透過抑制細胞毒性相關的巨噬細胞移行抑制因子進而減緩順鉑誘導急性腎臟損傷。
英文摘要 Anticancer agent cisplatin causes acute kidney injury (AKI) following renal inflammation and nephrotoxicity including renal cell apoptosis and acute tubular necrosis. In this study, we investigated a novel role of glycogen synthase kinase (GSK)-3-facilitated nephrotoxicity followed by macrophage migration inhibitory factor (MIF)-mediated renal inflammation in cisplatin AKI. Pharmacologically inhibiting MIF abated cisplatin AKI in vivo; however, MIF expression was not required for cisplatin-induced tubular cell apoptosis in vitro. Immunohistochemistry showed a specific renal expression of MIF in cortical tubular cells and cisplatin caused MIF decrease from these cells. Accompanied by cisplatin nephrotoxicity in vivo and in vitro, cortical tubular cell MIF was lost and/or secreted from intracellular to extracellular sites. In vitro studies showed that activation of caspases, induction of DNA damage responses, and signaling pathways of epidermal growth factor receptor/Src/protein kinase C-δ/reactive oxygen species/p38 mitogen-activated proten kinase/c-Jun N-terminal kinase were all regulated for cisplatin cytotoxicity as well as MIF secretion. Notably, cisplatin activated GSK-3 followed by MIF secretion and induced GSK-3-regulated nephrotoxicity and renal inflammation. Once GSK-3 was also required for cisplatin-induced anticancer, as demonstrated in a cisplatin-based anticancer model, inhibiting MIF prevented AKI without interference on anticancer efficacy. These results not only prove the molecular mechanisms for GSK-3-regulated nephrotoxicity and MIF-regulated renal inflammation but also confer a therapeutic strategy by inhibiting cytotoxicity-associated MIF to ameliorate cisplatin AKI.
論文目次 Abstract in Chinese II
Abstract in English III
Contents VIII
Chapter 1 Introduction 1
1-1. Cisplatin 1
1-2. Cisplatin-induced Nephrotoxicity 1
1-3. Pathophysiological events in cisplatin-induced nephrotoxicity 2
1-4. Macrophage migration inhibitory factor (MIF) 3
1-5. MIF in kidney disease 3
1-6. MIF and inflammation 4
1-7. MIF and Tumorigenesis 4
1-8. Glycogen synthase kinase-3 (GSK-3) 5
1-9. GSK3β in kidney disease 6
Chapter 2 Objective and Specific aims 7
2-1 Objective 7
2-2 Specific Aims 7
2-3 Experimental flowchart: 8
Chapter 3 Materials and Methods 9
3-1. Animal treatment 9
3-2. Computer-assisted quantification of immunohistological information 9
3-3. Immunohistochemical staing 10
3-4. Histology 10
3-5. Apoptosis Assay 11
3-6. Serological Examination 11
3-7. Cell Culture 11
3-8. Cytotoxicity 12
3-9. Enzyme-linked Immunosorbent assay (ELISA) 12
3-10. Reagent 12
3-11. Analysis of cell apoptosis 13
3-12. Immunostaing 13
3-13. Western blotting analysis 13
3-14. ROS Detection 14
3-15. Lentiviral-based shRNA transfection 14
3-16. Immune cell infiltration 15
3-17. Tumor Model 15
3-18. Statistical analysis 16
Chapter 4 Results 17
4-1 Pharmacologically inhibiting MIF attenuates cisplatin AKI 17
4-2 Expression of MIF in mouse tissues and organs 17
4-3 MIF is highly expressed in cortical tubules 18
4-4 Cisplatin causes cytotoxicity accompanied by MIF loss in vivo and in vitro 18
4-5 Caspase signaling determines MIF secretion 20
4-6 Cisplatin induces MIF secretion through DNA damage responses 20
4-7 Cisplatin induces MIF secretion following EGFR/Src/PKC-δ activation 21
4-8 Activation of p38 MAPK and JNK by ROS is required for cisplatin cytotoxicity and MIF secretion 21
4-9 GSK-3 mediates cisplatin-induced MIF secretion and AKI 22
4-10 Cisplatin induces GSK-3 activation and apoptosis in head and neck cancer cells 23
4-11 Inhibiting MIF abates cisplatin-induced AKI without inhibition on the therapeutic effects in human head and neck tumor xenografts 24
Chapter 5 Discussion 25
Chapter 6 Conclusion and Implication 30
Chapter 7 References 31
Chapter 8 Figure and Figure Legends 38
Appendix 55
A. Materials 55
A-1 Chemicals 55
A-2 Antibodies 58
A-3 Kits 59
A-4 Consumables 59
A-5 Apparatus 60
B. Methods 61
B-1 Cell culture 61
B-2 Western blot 63
B-3 Lentiviral-based shRNA knockdown 66
B-4 PI staining 68

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