臺灣博碩士論文加值系統

三氧化二砷 (As2O3) 為砒霜之主要成份，傳統醫學中利用少量三氧化二砷做為治療疾病的藥物。三氧化二砷可做為肝癌、大腸癌、乳癌等多種癌症的治療藥物。目前並不了解三氧化二砷做為治療藥物是否對病患的生殖系統造成影響，故本實驗探討動物施用三氧化二砷後其生殖系統是否受到傷害。利用雄性ICR小白鼠為實驗動物進行皮下注射不同濃度之三氧化二砷 (0, 0.15, 0.3, 1.5及3.0 mg/g體重)，以施打5天休息2天為一周期。三周期後，觀察雄性小白鼠生殖系統的各項變化，以期進一步了解三氧化二砷對人類生殖系統可能產生的影響。由實驗結果得知，注射三氧化二砷後小鼠體重、攝食量、睪丸及副睪的器官重量之變化均無統計上差異。利用Hematoxylin & Eosin 染色法觀察睪丸組織中精子細胞的發育，發現實驗組的精原母細胞 (spermatogonia) 數量減少，同時可觀察到精蟲之分化也受到抑制。此外，在肝組織切片中觀察到細胞核濃縮 (nuclear condensation) 的現象。透過電腦輔助精子分析系統 (computer-aided sperm analysis, CASA) 分析副睪的精子活動，顯示精子活動力下降，且精蟲數目會隨著注射三氧化二砷劑量的增加而減少。利用螢光顯微鏡以SYBR14/PI判別精子細胞存活率時，亦呈現精子存活率降低，同時測得副睪中精子細胞內抗氧化酵素superoxide dismutase (SOD)， glutathione peroxidase (GP)，glutathione S-transferase (GST) 和 catalase 之活性皆低於控制組，顯示精子的抗氧化能力降低。另一方面利用酵素免疫法 (Enzyme-linked immunosorbent assay, ELISA) 檢測血清中荷爾蒙濃度，結果顯示濾泡刺激激素 (follicle stimulating hormone, FSH) 濃度無明顯變化，黃體刺激激素 (luteinizing hormone, LH) 及血清中的睪固酮 (testosterone) 濃度明顯降低，但睪丸組織中的睪固酮濃度卻無明顯變化。以 Real-Time PCR 分析三氧化二砷處理小白鼠睪丸組織中睪固酮生合成中主要酵素的mRNA變化，得知cytochrome P450 side chain cleavage (P450scc)、3b-hydroxysteroid dehydrogenase (3b-HSD)、cytochrome P450 17-a hydroxylase/C17-20 lyase (Cyp17) 等三種酵素的基因表現均低於控制組。以RT-PCR分析與調控睪丸睪固酮濃度相關的基因表現，結果顯示ABP (androgen binding protein)、AR (androgen receptor) 和5-reductase基因表現均無顯著差異。利用免疫螢光染色觀察睪固酮在睪丸組織中分佈情形，顯示睪固酮累積在間質細胞。此一發現合理解釋睪固酮運往支持細胞之過程受到干擾，而影響生精作用，而所觀察到的各種現象是否會使小白鼠生殖功能受到影響，則有待進一步實驗探討。

Arsenic trioxide (As2O3) is an effective therapeutic agent for the treatment of acute promyelocytic leukemia (APL), liver cancer, breast cancer and colorectal cancer. The effect of arsenic trioxide on male reproductive system is unclear. Using male mice as an animal model, we attempt to illustrate the effects of arsenic trioxide on male reproductive system. The mice were administrated with arsenic trioxide in various dosages subcutaneously. The treatments were sustained for 5 days in a week and continued for 3 weeks. Our data showed that no difference in the food consumption, body weight, liver weight, testis weight and epididymis weight. Using histological observation, we showed the decrease of spermatogonia number and inhibition of spermatogenesis. The DNA condensation was found in liver cells. A significant decrease in sperm motility and viability were found in computer assised spermatozoa analysis (CASA) and SYBR14/PI staining assay. The activities of antioxidative enzyme including superoxide dismutase (SOD), glutathione peroxidase (GP), glutathione S-transferase (GST) and catalase (CAT), were significantly decreased in epididymal spermatozoa. Using enzyme-linked immunosorbent assay, we found the significant decrease of luteinizing hormone (LH) and testosterone levels in plasma but no difference in testicular testosterone level. Moreover, the mRNA of enzymes which involved in testicular testosterone synthesis, cytochrome P450 side chain cleavage (P450scc), 3-hydroxysteroid dehydrogenase (3-HSD) and cytochrome P450 17- hydroxylase/C17-20 lyase (Cyp17) were significantly decreased. Using immunohistological observation, we showed the accumulation of testosterone in leydig cell. These results suggest that testosterone transportation should be interfered by arsenic trioxide treatment and then disrupt spermatogenesis. Further investigation should be continued to elucidate the fertility of male mice of arsenic trioxide treatment.

中文摘要..............................................................................................5
英文摘要..............................................................................................6
縮寫表..................................................................................................7
中英對照表..........................................................................................9
第一章 緒論
前言......................................................................................................11
第二章 材料與方法
(一) 實驗藥品.....................................................................................19
(二) 實驗材料.....................................................................................21
(三) 實驗儀器.....................................................................................21
(四) 試劑配方.....................................................................................22
(五) 實驗方法.....................................................................................24
一、 動物飼養條件.................................................................24
二、 實驗設計.........................................................................24
1、 體內實驗 (in vivo)
2、 體外實驗 (in vitro)
三、 組織切片染色.................................................................24
1、 組織固定 (fixation)
2、 組織包埋 (paraffin embedded)
3、 石臘塊切片法 (paraffin block section)
4、 蘇木紫與伊紅染色 (Hematoxylin &Eosin
staining,H&E)
5、 封片
6、 組織學量化
(1) 計算肝組織切片中核濃縮數量
(2) 計算睪丸組織切片中精原母細胞數量
(3) 估算睪丸內可產生精蟲的上皮細胞之表面積
四、 精蟲數目決定..................................................................27
五、 精蟲活動力評估..............................................................27
1、 體內實驗
2、 體外實驗
六、 精蟲存活率 (viability) 評估.........................................29
七、 精蟲內ROS含量測定....................................................29
八、 精蟲抗氧化酵素活性測量..............................................30
1、 樣品置備
2、 Superoxide dismutase活性測定
3、 Catalase活性測定
4、 Glutathione peroxidase活性測定
5、 Glutathione S-transferase活性測定
九、 細胞內GSH與GSSG含量測定..................................31
1、 樣品置備
2、 GSH含量測量
3、 GSSG含量測量
十、 蛋白質定量分析.............................................................32
十一、 荷爾蒙濃度測量.............................................................32
1、 血清收集
2、 血清中LH濃度測量
3、 血清中FSH濃度測量
4、 血清中testosterone濃度測量
5、 睪丸內睪固酮濃度測量
十二、 睪丸組織內相關基因之mRNA表現...........................35
1、 RNA萃取
2、 RNA定量
3、 DNase處理
4、 反轉錄(Reverse transcription)置備cDNA
5、 即時定量PCR (Quantitiative Real Time PCR)
6、 反轉錄聚合酶連鎖反應 (Reverse-Transcription Polymerase Chain PCR)
7、 洋菜凝膠電泳 (Agarose gel electrophoresis)
8、 各基因引子 (primer) 序列
十三、 睪丸內睪固酮分布情形.................................................39
十四、 統計分析.........................................................................39
第三章 實驗結果
(一) 三氧化二砷對於小鼠體重、飼料消耗量和組織器官
重量影響....................................................................................40
(二) 三氧化二砷影響小鼠肝細胞分化……………………………40
(三) 三氧化二砷抑制生精作用……………………………………40
(四) 三氧化二砷對性荷爾蒙的影響………………………………41
(五) 睪固酮生合成過程中p450scc、CYP17和3-HSD
基因表現………………………………………………………42
(六) 維持睪丸睪固酮濃度之相關基因表現………………………43
(七) 睪丸內睪固酮分布情形………………………………………43
(八) 三氧化二砷對精蟲數量、游動力和存活率影響……………43
(九) 精蟲抗氧化酵素能力評估……………………………………44
(十) 三氧化二砷對精蟲活動力之直接影響………………………45
(十一) 三氧化二砷對精蟲細胞內ROS含量之直接影響……….45
第四章 討論.......................................................................................47
表
表一、三氧化二砷對小鼠體重、飼料消耗量、肝臟、睪丸
和副睪重量之影響………………………………………….51
表二、可以產生精蟲之上皮細胞表面積之估算………………….52
圖
圖一、三氧化二砷對肝細胞影響………………………………….53
圖二、三氧化二砷對生精作用的影響…………………………….55
圖三、三氧化二砷對荷爾蒙的影響……………………………….57
圖四、睪固酮合成酵素的基因表現……………………………….58
圖五、睪丸內ABP、AR及5-reductase基因表現…………….59
圖六、睪丸內睪固酮分布情形…………………………………….61
圖七、三氧化二砷對精蟲數目、精蟲游動力與存活率之影響….63
圖八、三氧化二砷對副睪精蟲GSH含量之影響………………...65
圖九、三氧化二砷對副睪精蟲抗氧化酵素活性之影響………….66
圖十、三氧化二砷對精蟲活動力直接影響……………………….67
圖十一、三氧化二砷對精蟲細胞內ROS含量之直接影響……...68
參考文獻.............................................................................................69
附錄.....................................................................................................75

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