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博碩士論文 etd-0627116-094848 詳細資訊
Title page for etd-0627116-094848
論文名稱
Title
過氧化氫在胚胎發育期促進肌細胞釋放IGF-1機制之研究
Studies on the role of hydrogen peroxide in the myocyte secretion of IGF-1 at developing neuromuscular synapse
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
102
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2016-06-21
繳交日期
Date of Submission
2016-07-27
關鍵字
Keywords
ryanodine receptor、IGF-1、分泌、鈣離子儲存池、H2O2
IGF-1, secretion, H2O2, sarcoplasmic reticulum, ryanodine receptor
統計
Statistics
本論文已被瀏覽 5710 次,被下載 368
The thesis/dissertation has been browsed 5710 times, has been downloaded 368 times.
中文摘要
突觸形成的過程中,需要突觸前和突觸後一些訊息分子之間的交換,進而促使突觸的成熟。雖然ROS過量已被證實會影響體內的氧化還原恆定(redox homestasis)、毒殺正常細胞、改變分子結構等,導致老化、突變、神經退化性疾病,但越來越多研究證實ROS也扮演生理上的角色。本實驗室之前的實驗已證實,H2O2能促進胚胎發育時期的神經自發性傳遞物質乙醯膽鹼 (ACh) 的釋放,而這種刺激作用與H2O2間接造成肌細胞釋出IGF-1有關。另外,預實驗中利用western blot試驗方式我們已知H2O2可以促進肌細胞釋放IGF-1使得培養液中IGF-1蛋白的量增加,然而H2O2如何對於IGF-1的詳細釋放機制卻不是很清楚,因此本論文研究先構築爪蟾IGF-1基因表現在紅螢光蛋白載體pDsRed-C1上,再利用螢光顯微鏡拍攝觀察IGF-1紅色螢光的即時變化來進行探討H2O2造成DsRed_IGF-1釋放的分子機制。
100μM H2O2能促使肌細胞內DsRed_IGF-1螢光融合蛋白強度的下降,鈣離子螢光探針Fluo-4亮度的增加,當處理鈣離子螯合劑BAPTA-AM時可以顯著抑制H2O2促進myocyte釋放DsRed_IGF-1螢光融合蛋白的作用,結果顯示鈣離子扮演著H2O2能促使肌細胞內DsRed_IGF-1螢光融合蛋白釋放的重要的角色。
進一步我們設計了一系列實驗釐清鈣離子的來源,實驗結果顯示將細胞外溶液置換成不含鈣離子的Ca2+ free Ringer 生理緩衝液,細胞外鈣離子缺乏的情況下觀察發現H2O2依舊能促使Fluo-4螢光亮度的增加,細胞鈣離子濃度的上升,並且myocyte釋放DsRed_IGF-1螢光融合蛋白,因此我們認為H2O2造成肌細胞釋出IGF-1與細胞內鈣離子儲存池有關。此外,為更一步證實H2O2造成細胞內鈣離子濃度上升為鈣離子儲存池所提供,我們利用鈣離子儲存池上的鈣離子通道阻斷劑TMB-8、Ruthenium red (Ryanodine receptor inhibitor),實驗結果顯示TMB-8、Ruthenium red能夠顯著抑制H2O2促進myocyte釋放DsRed_IGF-1螢光融合蛋白的作用。
Abstract
The successful synaptogenesis at neuromuscular junction is an intricate process relies on dynamic interaction of nerve- and muscle-derived molecules. We have previously demonstrated that muscle-derived insulin-like growth factor-1 (IGF-1) facilitate spontaneous neurotransmitter relese and is important in the development of neuromuscular synapse.
Furthermore, the IGF-1 content in the conditioned medium of Xenopus myocyte culture is significantly increase after hydrogen peroxide (H2O2) application suggesting H2O2 plays an important role in the secretion of IGF-1 from myocyte. Here we dig further to explore the molecular mechanism of H2O2 in myocyte-derived IGF-1 secretion by measure the florescence intensity of DsRed_IGF-1 in cultured Xenopus myocyte. The red fluorescent protein (DsRed) is used as a reporter and DsRed_IGF-1 intensity in a myocyte is monitored by using fluorescence microscope. Bath application of H2O2 significantly attenuates the intensity of DsRed_IGF-1 overexpressed in myocyte.
The H2O2-induced IGF-1 secretion is followed by an increase of cyotosolic calcium while Fluo-4 is used to as a probe. Pretreatment of the culture with Ca2+ chelator BAPTA-AM significantly hampered the H2O2-induced IGF-1 secretion. Moreover, exclusion of Ca2+ from culture medium abolished the H2O2-induced IGF-1 secretion, suggesting Ca2+ influx play a crucial role in this process. Bath application of ryanodine receptor inhibitors either antagonist 8-(dethylamino) octyl 3, 4, 5-trimethoxybenzoate (TMB-8) or Ruthenium red significantly occluded H2O2-induced IGF-1 secretion.
Overall, results from our current studies have shed some lights on signaling mechanism underlying the H2O2-induced secretion of IGF-1 from myocyte in the early development of neuromuscular synapse.
目次 Table of Contents
論文審定書 i
致謝 ii
中文摘要 iii
英文摘要 v
目錄 vii
圖目錄 viii
附圖目錄 x
縮寫表 xi
緒論 1
實驗目的 11
實驗材料 12
實驗方法 14
實驗結果 23
討論 34
參考文獻 41
Figure 1. H2O2 facilitates IGF-1 release from developing Xenopus myocyte. 53
Figure 2. H2O2 induce a sequential change in Hyper and DsRed-IGF-1 fluorescence intensity in the developing Xenopus myocyte. 55
Figure 3. Depolarization of the membrane potential induced by potassium chloride facilitates IGF-1 release. 57
Figure 4. H2O2 induced a rise in intracellular calcium concentration at developing Xenopus myocyte. 59
Figure 5. Effect of N-acetylcysteine in H2O2-induce IGF-1 release. 61
Figure 6. Effect of N-acetylcysteine in KCl-induce IGF-1 release. 63
Figure 7. Effects of N-Acetylcysteine in KCl- and H2O2-incuded rise of cytosolic calcium. 65
Figure 8. Calcium is responsible for H2O2-induced IGF-1 release. 67
Figure 9. Role of calcium influx in H2O2-induced IGF-1 release. 69
Figure 10. Calcium released from internal store is responsible for H2O2-induced IGF-1 release. 71
Figure 11. IP3 receptor activation is not involved in H2O2-induced IGF-1 release. 73
Figure 12. Activation of ryanodine receptor is required for H2O2-induced IGF-1 release. 75
Figure 13. Ryanodine receptor is involvement in H2O2-induced calcium rise at developing Xenopus myocyte. 77
Figure 14. Response to ionomycin of myocytes loaded with Fluo-4. 79
Figure 15. Change of IGF-1 punctate induced by H2O2 in high resolution confocal microscope. 81
Figure 16. Effect of H2O2 on properties of single acetylcholine (ACh) channel in Xenopus cultured myocyte. 83
Figure 17. Proposed model of IGF-1 secretion requires oxidation. 85
附圖 一 87
附圖 二 88
附圖 三 89
附圖 四 90
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