Study of Annual Fluctuations of Fecal Reproductive Steroids in Formosan Black Bears (Ursus thibetanus formosanus)

本研究應用非侵入性之酵素免疫分析法，對6隻圈養在特有生物研究保育中心低海拔試驗站之台灣黑熊糞中性類固醇之濃度變化做一為期3年多觀察與研究。結果顯示，台灣黑熊為季節性發情的動物，繁殖季主要在3至6月間。公熊睪固酮濃度具有季節性變化，睪固酮於冬季開始有增加的趨勢，而在春季達到最高的濃度。有分娩記錄的台灣黑熊，雌二醇及孕酮濃度變化較無配種或有配種無分娩的母熊有規則性且孕酮呈現出階段性增加。懷孕期約為6-7.5個月，但因懷孕期差異頗大，推測可能包含有胚延遲埋植期。母熊於分娩前後會出現不飲、不食、不排泄及行動減緩等類似冬眠的行為。此外，於繁殖季睪固酮及雌二醇濃度高低並非唯一決定交配與否的因素，而圈養條件下台灣黑熊會因環境、氣候、年齡、健康、哺乳等因素，造成個體上頗大繁殖差異。本研究可推論出台灣黑熊於類似自然環境下的年活動模式，主要分為活動期及似冬眠期兩個模式且可能因氣候適宜及食物來源較不匱乏，因此不包括冬眠期。本研究的結果可作為台灣黑熊野外的調查及研究之參考，並為台灣生物多樣性的維護略盡綿薄。The present study utilized non-invasive enzyme-linked immunosorbent assays(ELISA) to measure the changes of fecal reproductive steroids in 6 Formosan black bears (Ursus thibetanus formosanus)at the Low Altitude Experimental Station of Endemic Species Research Institute. The results indicated Formosan black bears were seasonal breeder and the major breeding season was between March and June. Testosterone concentrations were fluctuated according to seasonal changes. The concentrations increased in late winter and reached to the highest peak in spring. Relatively regular patterns of estradiol and progesterone concentrations in pregnancy were observed in parturient females, and the gestation periods were about 6 to 7.5 months. Because the gestation periods were different, it might include a special reproductive phenomenon, delayed implantation of embryo. Moreover, female Formosan black bears have hibernated behaviors, such as, fasting, non-excretion and sluggish activity at about a period before and after parturition. In non-mated females, progesterone concentrations were irregular and the peaks were lower than the mated bears. Health condition and environmental factors influences caused different reproductive behaviors in each individual. Thus, testosterone and estradiol concentrations were not the only factor that had influenced on mating behavior. The annual pattern could be divided into an active and like-hibernate period. Because of well climate and abundant food, the annual pattern did not include hibernation. This study provides useful information for the wildlife research and management of the Formosan black bears. Furthermore, the present study could also contribute to the conservation of Taiwanese biodiversity.一、緒言 二、文獻探討 第一節 台灣黑熊簡介 第二節 熊科動物年活動週期繁殖模式 第三節 熊科動物繁殖行為 第四節 熊科動物繁殖生理 一、雄性熊科動物 (一) 睪丸解剖學與細胞學 (二) 精子生成 (三) 性類固醇生成酵素 (四) 公熊繁殖內分泌 二、雌性熊科動物 (一) 母熊繁殖器官解剖學與細胞學特徵 (二) 性類固醇生成酵素 (三) 特殊繁殖現象 (四) 母熊繁殖內分泌 第五節 性類固醇之研究與應用 一、類固醇的合成、生物效應及臨床應用 (一) 類固醇的合成 (二) 睪固酮的生物效應及臨床應用 (三) 動情素的生物效應及臨床應用 (四) 孕酮的生物效應及臨床應用 二、類固醇的代謝及分析 (一) 類固醇的代謝 (二) 類固醇的分析 三、糞材中性類固醇之研究與應用 三、雄性台灣黑熊糞中睪固酮分析 第一節 前言 第二節 材料與方法 一、實驗動物 二、糞材收集及萃取 三、酵素免疫分析法 (一) 試藥之製備 (二) 抗體及睪固酮-酵素聯結體之製備與濃度選擇 (三) 標準溶液之配置與標準曲線之建立 (四) 靈敏度、分析內與分析間變異係數 (五) 糞中睪固酮酵素免疫分析法之步驟 第三節 結果 第四節 討論 四、雌性台灣黑熊糞中雌二醇和孕酮的分析 第一節 前言 第二節 材料與方法 一、實驗動物 二、糞材收集及萃取 三、酵素免疫分析法 (一) 試藥之製備 (二) 抗體及類固醇-酵素聯結體之製備與濃度選擇 (三) 標準溶液之配置與標準曲線之建立 (四) 靈敏度、分析內與分析間變異係數 （五）糞中雌二醇和孕酮酵素免疫分析法之步驟 第三節 結果 第四節 討論 五、結

Study of the blockage effect of rapamycin in high fat diet-induced obesity mice

雷帕黴素是由土壤中細菌- Streptomyces hygroscopicus分離出的一種抗黴菌藥，它同時也是mTOR的專一性抑制劑。由於雷帕黴素具有免疫抑制的特性，因此，近年來，雷帕黴素常廣泛使用於器官移植後，做為抗排斥藥物。雖然，已有文獻發現雷帕黴素具有抑制脂肪細胞分化的作用，然而，雷帕黴素對於是否具抗肥胖的效果仍不是十分清楚。本論文分別利用C57BL/6J及KK/HlJ 等2種不同品係的肥胖動物模式小鼠，探討雷帕黴素對於代謝的影響。肥胖C57BL/6J小鼠以雷帕黴素每週2 mg/kg劑量連續16週腹腔注射後，發現有體重、副睪脂肪重量減輕，減少每日飼料效率，血中瘦體素及胰島素濃度下降之情形。然而，雷帕黴素卻會增加C57BL/6J小鼠的食物攝取量，並且對於禁食血糖沒有顯著的影響。組織形態學分析每週一次腹腔注射雷帕黴素的C57BL/6J小鼠，其脂肪肝程度、脂肪細胞形態、大型後腹腔脂肪細胞和大型副睪脂肪細胞的比例皆顯著減小。於第二型糖尿病特徵KK/HlJ小鼠則改以雷帕黴素每天2 mg/kg劑量連續42天腹腔注射，結果發現雷帕黴素在KK/HlJ小鼠同樣具有抗肥胖效果。然而，注射雷帕黴素KK/HlJ小鼠即使有較高的胰島素濃度，但雷帕黴素卻會增加活性氧自由基的濃度，同時並造成KK/HlJ小鼠葡萄糖耐受性不良。綜合上述，雷帕黴素藉由減少脂肪蓄積而對抗高脂飲食會增加體重的效果，因此，雷帕黴素可用於體重控制和防止肥胖的發生，但長期、高頻率地使用雷帕黴素，仍需考慮其影響血糖恆定的副作用，特別是當用於高胰島素血症和葡萄糖耐受性不良之情形。Rapamycin (RAPA), an anti-fungal macrolide, is an unique and specific mTOR kinase inhibitor isolated from soil bacterium, Streptomyces hygroscopicus. Subsequently, RAPA was shown to have potent immunosuppressive and antiproliferative effects. As an immunosuppressant drug, it is used extensively to prevent graft rejection in transplant patients. Although, it has been reported to inhibit adipogenesis in vitro, however, the full extent of the role of RAPA in treating human/animal obesity has not been fully explored. In this study, we investigated the metabolic effects of RAPA in two different obese animal models, C57BL/6J and KK/HlJ mice. The high fat diet-fed (HFD) C57BL/6J mice were injected intraperitoneally with RAPA (2 mg/kg/week for 16 weeks) had reduced body weight and epididymal fat pads/body weight, reduced daily food efficiency, and lower serum leptin and insulin levels compared with the HFD control mice. However, RAPA-treated mice were hyperphagic, demonstrating an increase in food intake and exhibited similar blood glucose levels relative to the control group. Dissection of RAPA-treated mice revealed a marked reduction in fatty liver scores, average fat cell size and percentage of large adipocytes of retroperitoneal and epididymal white adipose tissue (RWAT and EWAT), compared to the HFD control mice. On the other hand, obese KK/HlJ mice were treated with a daily intraperitoneal injection of RAPA at 2 mg/kg or vehicle for 42 days on a HFD. Treated KK/HlJ mice also exhibited the pronounced anti-obesity effects of RAPA in metabolic parameters based on histological and image analysis. However, RAPA-treated animals showed a marked decline in glucose tolerance as judged by the 180-minute area under the curve for plasma glucose levels, paralleled by increased generation of plasma reactive oxygen species (ROS), despite greater serum insulin levels. Tthese results suggest that RAPA preventsthe effect of a high fat diet on the rate of accretion in body weight via reducing lipid accumulation. It is likely that RAPA may serve as a potential strategy for body weight control and/or anti-obesity, while prolonged and continual use of RAPA can cause adverse effects such as the deterioration of blood glucose homeostasis, especially in conditions with hyperinsulinemia and glucose intolerance.中文摘要 ……………………………………………………………4 英文摘要 ……………………………………………………………5 目錄.................................................. 7 圖次.................................................. 9 表次.................................................. 10 第一章 緒言………………………………....................11 第一節 前言…………………………………………………......11 第二節 文獻回顧………………………………………………....14 一、肥胖 …………………………………………………… 14 （一）肥胖成因及定義 ………….………………….... 14 （二）肥胖對健康的危害 ………………...………… 15 二、mTOR ………………………………….……………… 21 （一）mTOR途徑訊息傳遞 ………………………... 22 （二）mTOR與代謝性疾病、癌症、炎症關係 …… 26 三、免疫抑制劑 ……………………….………………….. 29 四、雷帕黴素臨床應用之未來性…………………….…… 34 第三節 研究假說與設計…..………..………………….……… 38 第二章 雷帕黴素改善高脂飲食誘導C57BL/6J小鼠的肥胖......40 第一節 前言…………………………………………………... 40 第二節 材料和方法 ……………………………………………. 41 第三節 結果…………………..………………………………. 43 第四節 討論………………………………………………….. 46 第三章 長期使用雷帕黴素可減少高脂餵食KK/HlJ小鼠的 肥胖，但會造成小鼠葡萄糖耐受性不良 ………………. 56 第一節 前言…………………………………………………... 56 第二節 材料和方法 ……………………………………………. 57 第三節 結果…………………..………………………………. 61 第四節 討論………………………………………………….. 65 第四章 結論……..……………………………………………. 76 參考文獻 ……………………………………………………… 79 附錄………………………………………………...... 10