臺灣博碩士論文加值系統

背景：近年來，空氣污染已經成為一個國際注目的嚴重問題，暴露空氣污染物會對於人類健康有許多負面的影響。許多文獻資料都顯示了PM2.5是造成男性精液質量惡化的危害因素，但有關的危害成分仍然不清楚，因此我們建立一個系統將PM2.5污染物分離為非水溶性PM2.5顆粒(ISP)及水溶性PM2.5萃取物(WSE)，探討PM2.5對於雄性生殖傷害之影響。
研究目的：探討雄性小鼠以氣管滴注模式，暴露ISP及WSE是否造成雄性小鼠的生殖危害。
方法：採集高雄地區之PM2.5，將其顆粒及水溶性萃取物做成分分析。再將C57BL/6雄性小鼠分成控制組及暴露組進行氣管滴注實驗。暴露組分別暴露ISP及WSE，暴露劑量又分為高劑量2 mg組及低劑量0.2 mg組，每次暴露50 L其中高劑量及低劑量組分別含有200 g與20 g的PM2.5，共暴露10次後進行犧牲。測量小鼠體重、器官重量、精子數量、活動力、型態、反應性氧化物質含量、粒線體膜電位功能、DNA結構完整性及睪丸組織內精子細胞週期分布與睪丸組織病理分析等變化量。
結果：在ISP當中主要的金屬元素為鈣元素、鐵元素、鎂元素、鉀元素及鋅元素。在WSE當中主要的離子為硫酸根離子、硝酸根離子、氯離子、鈉離子及銨離子。研究結果顯示，暴露ISP之小鼠的體重及四倍體比例會被影響，右副睪尾重量及正常型態精子比例會顯著下降，粒線體膜電位異常率及DNA斷裂指數會顯著上升。暴露WSE之小鼠的體重會被影響，右副睪尾重量、反應性氧化物含量、正常型態精子比例、瘦長型精細胞比例及雙倍體比例會顯著下降，圓形精細胞比例會顯著上升。
結論：雄性小鼠暴露高雄地區ISP及WSE可能影響體重以及精子功能。然而ISP與WSE之間的雄性生殖毒性之潛在機制尚未清楚，需要再進一步的研究。

Background：Recent years, air pollution has been a serious problem. Air pollutant exposure negatively affects human health. Studies have investigated that PM2.5 is a risky factor to the deterioration of semen quality in males. But, the related hazardous ingredients still unclear. Therefore, we developed a system separating PM2.5 pollutants to insoluble PM2.5 particles (ISP) and water-soluble PM2.5 extract (WSE) to investigate PM2.5-induced male reproductive damage.
Objectives：To explore the effects of exposure to ISP or WSE using intratracheal instillation on sperm functions in male mice.
Methods：PM2.5 from Kaohsiung, Taiwan, was collected, and its insoluble particles and water-soluble extract was subjected to component analysis. C57BL/6 mice were exposed to ISP and WSE (0.2 and 2 mg) through intratracheal instillation. The exposure was performed twice every week and continued for 5 weeks. On postnatal day 86 for all mice, epididymal sperm counts, motility, morphology, mitochondrial Membrane Potential (MMP) Functions, reactive oxygen species (ROS) generation, sperm chromatin DNA structure integrity, testicular DNA content in spermatogenesis and testicular histology were assessed.
Results：The most abundant metal elements in the ISP were Ca, Fe, Mg, K and Zn. There were considerable amounts of SO42-, NO3-, Cl-, Na+ and NH4+ in the WSE. Exposure to ISP disrupted body weight and tetraploid rate, decreased left cauda epididymis weight and normal morphology rate; and increased abnormal MMP rate and DNA fragmentation index. Exposure to WSE disrupted body weight, decreased left cauda epididymis weight, ROS content, normal morphology rate, elongating spermatids rate and diploid rate; and increased round spermatids rate.
Conclusion：Exposure to ISP and WSE from Kaohsiung might affect body weight and sperm functions. However, the underlying mechanisms of ISP and WSE-mediated male reproductive toxicity remain unclear and further investigation is needed.

摘要………………………………………………………………………………I
Abstract II
致謝……………………………………………………………………………IV
目錄…………………………………………………………………………….V
表目錄 VIII
圖目錄 IX
中英對照表 XI
壹、 緒論 1
1.1 前言 1
1.2 研究目的 3
貳、 文獻回顧 4
2.1 PM2.5介紹 4
2.1.1 PM2.5概論 4
2.1.2 PM2.5危害特性 5
2.2 生殖系統介紹 9
2.2.1 雄性生殖機能 9
2.2.2 精子的生成及結構 11
2.2.3 精子細胞週期及睪丸 DNA 含量 13
2.3 粒線體功能介紹 15
2.4 反應性氧化物質 17
2.4.1 何謂反應性氧化物 17
2.4.2 反應性氧化物質與精子功能影響 18
2.5 流式細胞儀 20
2.5.1 基本原理 20
2.5.2 儀器理論及應用 21
參、 材料與方法 26
3.1 實驗設計 26
3.1.1 PM2.5樣品的採樣與製備 26
3.1.2 PM2.5 金屬元素成分分析 28
3.1.3 PM2.5 離子成分分析 28
3.1.4 暴露方式 29
3.2 實驗動物來源 31
3.3 雄性生殖毒理評估 32
3.3.1 藥品與試劑 32
3.3.2 儀器與設備 36
3.3.3 懸浮液配製 37
3.3.4 精子型態評估 38
3.3.5 精子染色質結構分析 39
3.3.6 精子粒線體膜電位分析 40
3.3.7 精子細胞內過氧化氫含量分析 41
3.3.8 精子細胞內超氧陰離子含量分析 42
3.3.9 睪丸細胞DNA含量分析 43
3.3.10 睪丸組織病理分析 44
3.4 統計分析 45
肆、 結果 46
4.1 PM2.5化學成分分析結果 46
4.1.1 PM2.5金屬元素成分分析結果 46
4.1.2 PM2.5離子成分分析結果 49
4.2 雄性生殖功能影響評估 51
4.2.1 暴露期間體重分析 51
4.2.2 體重與臟器組織重量分析 53
4.2.3 精子數量及活動力分析 55
4.2.4 精子粒線體功能分析 55
4.2.5 精子細胞反應性氧化物分析 56
4.2.6 精子染色質DNA結構分析 56
4.2.7 精子型態 58
4.2.8 睪丸細胞DNA含量分析 60
4.2.9 睪丸組織病理學 62
伍、 討論 64
5.1 PM2.5化學成分分析結果之探討 64
5.1.1 PM2.5金屬元素成分分析結果之探討 64
5.1.2 PM2.5離子成分分析結果之探討 65
5.2 雄性小鼠生殖功能影響評估之探討 66
5.2.1 體重與臟器組織重量 66
5.2.2 雄性生殖功能影響 68
5.2.3 睪丸組織病理學影響 74
陸、 結論 77
柒、 參考文獻 78
捌、 研討會發表 83

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