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研究生:蔡玲惠
研究生(外文):Ling-Hui Tsai
論文名稱:不同溫度暴露影響大鼠與人類精子染色質DNA異常之研究
論文名稱(外文):Effects of different temperature exposure on chromatin DNA integrity in rats and human spermatozoa
指導教授:許昺奇許昺奇引用關係
指導教授(外文):Ping-Chi Hsu
學位類別:碩士
校院名稱:國立高雄第一科技大學
系所名稱:環境與安全衛生工程所
學門:工程學門
學類:環境工程學類
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:42
中文關鍵詞:流式細胞儀DNA人類精子大鼠精子
外文關鍵詞:flow cytometryheatDNAhuman spermatozoarat spermatozoa
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高溫作業是人類產業活動中,屬於傳統而且具有危險性的工作之一。雖然目前的科技進步,大部分的工業製程已自動化,但仍有許多工廠因部分製程需要,使勞工須於熱環境下作業。而大部分的哺乳動物,特別是人類,精子的生成乃是倚賴著溫度的變化,正常睪丸功能須在低於身體內部溫度2~4℃的環境,若以人為方式增加正常生育男性陰囊或睪丸的溫度,則所排出的精子量與精液品質皆有降低的現象。在精子的形成時期,精子細胞核型態與染色質凝結和其他細胞核的現象有關。畸形的精子細胞核DNA染色質結構發生改變,而對位DNA對於受熱變性的抵抗力與其本身的功能影響有關,因此精子DNA染色質結構對溫度改變具有高度敏感性,也是決定生育力的重要因素。本研究目的是利用流式細胞儀(flow cytometry, FCM)測量以Acridine Orange染色受到不同溫度暴露的大鼠和人類精子DNA綠色及紅色螢光強度,以研究精子DNA因溫度變化而產生變異。
本研究將實驗分為大鼠精子與人類精子兩部分:(1)大鼠精子-控制組設置在23 ℃,加熱組分別在37 ℃、50 ℃、80 ℃與98 ℃的恆溫水浴槽加熱10分鐘;(2)人類精子-控制組設置在23 ℃,加熱組分別在37 ℃、50 ℃、60 ℃、80 ℃與98 ℃的恆溫水浴槽加熱10分鐘。取200 µl精液樣本加入400 µl的lysis solution混合30秒,再加入1.2 ml的Acridine Orange(AO)染劑混合3分鐘後,將樣本置於FCM分析,每個樣本約有10,000顆精子進行綠色(FL1-正常DNA)及紅色(FL3-受損DNA)螢光強度的記錄。
研究結果發現大鼠與人類精子DNA受熱壓迫時會受損,隨著溫度增高,DNA受損程度增加。大鼠精子實驗中,加熱98 ℃的COMP aT(%)明顯異於控制組(P=0.0026),而aT(aT=red/red+green)部分,雖然有隨溫度升高而增加的情形,但在ANOVA的統計上並無顯著地差異。人類精子DNA部分,加熱50 ℃、60 ℃、80 ℃和98 ℃的COMP aT(%)均與其他組有差異(P<0.0001),而aT部分,98 ℃的加熱組有較明顯的差異(P=0.0130)。因此,我們認為精子受熱暴露後會引起DNA完整性方面劑量反應的損害。


Occupational heat exposure has been already known to be dangerous to workers. To date, although more advanced techniques have been widely adopted by industries, however, workers might still suffer to heat exposure in some specific environments. Normal testicular function requires a temperature 2-4℃ below body temperature. Artificial increases in scrotum or testicle temperature in fertile men reduce both sperm output and quality. Thermal denaturation of DNA in situ depends on chromatin conformation may be related to the diminished fertility. However, studies addressing the direct effect of heat exposure on DNA integrity in rat and human spermatozoa lacking.
In this study we used acridine orange (AO) staining to follow changes in DNA packaging exposed to different temperature in rat and human spermatozoa, respectively. The rat sperm head was separated by sonicator and exposed to increasing heat at 23, 37, 50, 80 and 98℃ for 10 min, respectively. The human sperm was exposed to increasing heat at 23, 37, 50, 60, 80 and 98℃ for 10 min, respectively. Sperm DNA integrity was assessed by the sperm chromatin structure assay using flow cytometry. The monomeric AO bound to native DNA fluoresces green, whereas the aggregated AO on denatured DNA fluoresces red.
Human sperm heated to 98℃ resulted in significantly higher aT(aT=red/green+red), the proportion between red and green fluorescent distribution and red fluorescence levels than control groups(P=0.0130). Rat sperm heated to 98℃ resulted in significantly higher COMP aT(%), the cells outside the main population, than at 23, 37, 50 and 80℃(P=0.0026). Human sperm heated to 50, 60, 80 and 98℃ resulted in significantly higher COMP aT(%) than at 23 and 37℃(P<0.0001). In conclusion, spermatozoa with increasing heat exposure may induce the dose-response damage of DNA integrity.


中文摘要i
英文摘要iii
目錄v
表目錄viii
圖目錄ix
第一章 緒論
1.1 前言1
1.2 研究緣起1
1.3 研究目的2
第二章文獻回顧
2.1 去氧核醣核酸3
2.1.1 DNA的構造3
2.1.2 DNA的性質5
2.1.3 DNA與染色質(Chromatin)5
2.2 熱對精子DNA的影響7
2.3 Acridine Orange螢光染劑的應用11
2.3.1 螢光染色技術11
2.3.2 Acridine Orange螢光染劑11
2.4 精子DNA結構分析技術12
2.4.1 精蟲分析(Sperm Analysis)12
2.4.2 精子染色質結構分析(SCSA)13
第三章 材料與方法
3.1 樣品處理14
3.1.1 大鼠精液14
3.1.2 人類精液14
3.1.3 Acridine Orange染色15
3.2 分析方法15
3.2.1 分析儀器15
3.2.2 FCM SCSA16
3.2.3 Cell Quest套裝軟體17
3.2.4 WinMDI流式數據分析軟體17
第四章 結果
4.1 大鼠精子熱暴露分析21
4.2 人類精子熱暴露分析28
第五章 討論
5.1 溫度的影響35
5.2 精子DNA對熱的抵抗力35
5.3 熱暴露的風險評估36
第六章 結論與建議
6.1 結論….38
6.2 建議…38
參考文獻39


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