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研究生:黃中佑
研究生(外文):Chong Yu Huang
論文名稱:低壓氧環境減低光損傷時視網膜感光細胞凋亡現象
論文名稱(外文):Hypobaric Hypoxia Decreased Photoreceptor Apoptosis In the Retina After Photic Injury
指導教授:周崇龍張正忠張正忠引用關係
指導教授(外文):Chung Long ChouCheng Jong Chang
學位類別:碩士
校院名稱:國防醫學院
系所名稱:航太醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:83
中文關鍵詞:光損傷共軛顯微鏡流式細胞分析儀TUNEL分析annexin V 分析低壓艙
外文關鍵詞:photic injuryconfocalflow cytometryTUNELannexin Vhypobaric chamber
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視覺功能在現今飛航安全操控中扮演著非常重要的角色,對於飛航器空間中的定位、飛航訊息的判讀、操控等均可以提供一個即時的糾正。但相對的,視覺也是飛航中最容易受到干擾和傷害的生理器官;例如在高空中所可能遭遇到日光直射、雲層強烈反光而引起的暫時性眩光或是失明、因為高空缺氧導致的視覺功能障礙…等,對於視覺功能都有機會造成某些程度上不可逆的傷害。因為視覺中,視網膜感光細胞為主要的光線能量接收、傳遞訊息部位,對光線的刺激敏感性相當高。所以本實驗針對模擬一萬英呎高空時所可能遭遇到因大氣壓力降低而導致缺氧問題,在不同光照時程下配合急性光損傷引發動物之視網膜感光細胞凋亡結果,利用最新之共軛顯微分析技術(LSCM)與流式細胞分析技術(FACS)觀察低壓氧對於動物視網膜感光細胞凋亡現象之影響。其目的在於評估在高空缺氧環境下,連續的光照刺激對於視網膜感光細胞是否會造成更嚴重的傷害。由實驗結果顯示,在急性光損傷動物模式中,低壓氧環境對於視網膜凋亡現象可能具有延緩作用。

To observe the effects of hypobaric hypoxia on photoreceptor apoptosis in an animal model of photic injury. Methods:Six to eight weeks old, Lewis albino rats were reared, for 2 weeks, in cyclic light and dark adapted for 24 hr before light exposure. Rats were placed in the low pressure cabin with controlled inner pressure down to 520~530 mm Hg. After exposed to continuous light for 1 to 24hr in 1,000-1,200 Lux of light intensity, light-exposed rats were repressurlized and followed by 24 hr of dark recovery. Compared to controlled light-exposed retina under normoxia condition, retinal specimens after photic injury under hypobaric hypoxic environment were evaluated by flow cytometry using Annexin V-FITC labeling. The total amount of apoptotic photoreceptor nuclei were further calculated by terminal transferase-mediated biotin dUTP nick end labelling (TUNEL)technique under confocal microscopy. Results:After 1, 6, 12,and 24 hr light exposure, fluorescent was significant higher in the normoxic (21% oxygen) group than the hypobaric hypoxic (14% oxygen) group with Annexin V-FITC assay. TUNEL-positive photoreceptor nuclei were first observed at 6hr in normoxic conditions and increased in numbers through the period of 24hr. Compared to normoxic condition, hypobaric hypoxic environment induced less TUNEL-positive in photoreceptors in the retina. Conclusions:The present study demonstrates that hypobaric hypoxia may play a role for inhibition of photoreceptor apoptosis after photic injury.

正文目錄
頁次
正文目錄……………………………………………………………….Ⅰ
圖目錄…………………………………………………………….……Ⅴ
縮寫表……………………………………………………………….…Ⅵ
中文摘要…………………………………………………………….…Ⅶ
英文摘要…………………………………………………………….…Ⅷ
第一章 緒 言……………………………………………….………...1
第一節 視 覺……………………………………………..………..1
壹、前言………………………….……….…….………………1
貳、眼球壁結構與功能…………….…….……….……………1
參、視網膜結構與功能………………….…….……..…………2
肆、視覺與照明……………………….………..……..…………4
伍、視覺與飛行……………………….………..………..………4
陸、結語……………………………….………..…..……………5
第二節 視網膜光損傷模式…………………...…….…...………..6
壹、前言...……………………………….……..…...……………6
貳、視網膜光損傷理論……………….………...…....…………6
參、視網膜光損傷結果……………….……….….…..…………8
肆、細胞凋亡及壞死………………….……….….…..…………8
伍、視網膜光損傷測定……………….……….….……..…..…11
陸、結語……………………………….……….….….….…..…20
第三節 大氣環境與缺氧……………….………...…..….………21
壹、前言...……………………………….……………….……..21
貳、大氣壓力………………………….………….…………….21
參、高空缺氧………………………….………….…………….22
肆、缺氧對視覺之影響……………….……………………….25
伍、結語……………………………….………...….………..…26
第四節 顯微鏡的發展……………………...…………..………..27
壹、前言...………………………………….…….……………..27
貳、顯微鏡的起源…...………………….……….……………..27
參、共軛顯微鏡的發展……………...….………….…………..28
肆、共軛顯微鏡的運用……………...….…………….………..30
伍、結語………………………………….……….………….…31
第五節 研究背景及目的…………….…….….…………………32
第六節 資料之統計與分析……………………….……………..33
第二章 材料與方法……………...….……………….….…………..34
第一節 實驗材料與儀器……...….……………….….…………..34
壹、實驗動物與試劑……...….……………….…...………….34
貳、實驗設備與儀器……...….……………….…...………….35
第二節 實驗步驟與方法……...….……………….….……...…..37
壹、動物實驗步驟……...….………..………….…..…………..37
貳、視網膜組織之處理..….………..………….…...…………..38
參、視網膜細胞胞膜免疫螢光染色.………….…...…………..39
肆、視網膜細胞胞核免疫螢光染色.………….…...…………..39
伍、視網膜組織切片之處理.………….…………...…………..40
陸、視網膜組織切片之胞核免疫螢光染色…….…..…..……..41
第三章結果…….……………………………………………...……..43
第一節 視網膜細胞之形態變化………………….……...…..….43
第二節 視網膜細胞胞膜Annexin V/PI 免疫螢光染色分析…44
第三節 視網膜細胞胞核TUNEL免疫螢光染色分析....……...46
第四節 共軛焦顯微鏡觀察網膜凋亡細胞……….……...….…..49
第四章 討論.………………….……………………………...……...67
第一節 前言……………….……………………………....……...67
第二節 視網膜光損傷模式.……………………………....……...67
第三節 大氣環境與低壓艙.……………………………....……...68
第四節 利用Annexin V/PI 胞膜染色法偵測凋亡細胞..……....69
第五節 利用TUNEL 胞核染色法偵測凋亡細胞……...………71
第六節 缺氧對於光損傷的保護機轉…….………….…...……...72
第五章 結論……....…………………………………………….…...74
第六章 參考文獻....…………………………………………….…...75
附錄一、各種溶液配方
圖 目 錄
圖一、 光損傷實驗步驟…………………………………………………… 52
圖二、 照光箱裝置(正面及側面)……………………………………… 53
圖三、 低壓艙裝備………………………………………………………… 54
圖四、 凋亡細胞與壞死細胞之形態圖…………………………………… 55
圖五、 一大氣壓下胞膜Annexin V/PI螢光染色分析結果圖………….. 56
圖六、 低壓艙中胞膜Annexin V/PI螢光染色分析結果圖…………….. 57
圖七、 不同壓力下利用胞膜螢光染色之凋亡細胞數量統計圖………… 58
圖八、 一大氣壓下胞核TUNEL螢光染色分析結果圖………………… 59
圖九、 低壓艙中胞核TUNEL螢光染色分析結果圖…………………… 60
圖十、 不同壓力下利用胞核螢光染色之凋亡細胞數量統計圖………… 61
圖十一、 一大氣壓下H&E切片染色圖……………………………………. 62
圖十二、 一大氣壓下TUNEL螢光染色切片圖…………………………… 63
圖十三、 低壓艙中H&E切片染色圖………………………………………. 64
圖十四、 低壓艙中TUNEL螢光染色切片圖……………………………… 65
圖十五、 不同壓力下TUNEL螢光染色切片之凋亡細胞統計圖………… 66

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