2015-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/685152摘要：由我們過去的研究發現，多囊性卵巢症後群中有較高比率的非酒精性脂肪肝病變的發生是獨立於肥胖的影響(Human Reproduction, 2011)。MCDdiet 餵食是誘發非酒精性脂肪肝病變(Non-alcoholic Fatty Liver Disease)常用的動物模式，此模式會造成動物的肝功能異常。肝功能異常是多囊性卵巢症後群的臨床觀察之一，但彼此的相關性和致病機轉，除了雄性賀爾蒙外，其餘並非很清楚。我們由MCD 餵食的母鼠卵巢觀察到多囊性卵巢的現象﹔這樣的發現過去並沒有文獻探討過。在本研究中我們將以動物模式探討MCD 影響卵巢濾泡成熟過程的生物效應與作用機制，並進一步與我們已經建立之雄性素誘導PCOS 動物模式進行分子機轉的比較:期望能釐清MCD干擾卵巢濾泡成熟與排卵的機制。第一年的研究目標是離探討 MCD diet 餵食導致之脂肪肝纖維化與PCOS 的關係。研究策略與方法為:(1)探討單純的脂肪肝與脂肪肝纖維化病變對卵巢多囊化的效應：分別以高油脂飼料與MCD diet 餵食4 週；進行肝藏與卵巢組織病理切片分析；量化脂肪肝程度、纖維化病變與卵巢濾泡大小及數量的關係。(2)建立 MCD diet 餵食導致肝纖維化與PCOS 之時序關係。比較餵食不同週數的成熟母鼠之體重變化、血中肝功能指數(GGT, ALT,AST,ALP)與生殖相關荷爾蒙濃度(FSH, LH, E2, P4)，並進行肝藏與卵巢組織病理切片分析；量化脂肪肝纖維化病變與卵巢多囊化的程度。(3)探討 MCD diet 餵食導致的多囊性卵巢是否仍具有生殖週期性:以促濾泡成熟激素作用後；測試不同時間點之血中生殖相關荷爾蒙濃度變化與運用卵巢組織切片分析不同期別之濾泡。第二年的研究目標是釐清 MCD diet 餵食導致PCOS 的分子機轉。研究策略與方法為:(1) 探討 MCD diet 餵食是否造成卵巢組織細胞凋亡:以TUNEL assay 偵測顆粒細胞之凋亡情況,並以免疫組織染色偵測凋亡相關分子caspase3,Bcl-2,Mcl-1 的表現。(2) 探討 MCD diet 餵食是否改變卵巢組織顆粒細胞增生與相關訊息傳遞:以免疫組織染色偵測顆粒細胞之PCNA 以證明細胞增生狀態, 並偵測FSHR, ER, PR, p-Akt, cyclinD1, PTEN 等訊息傳遞分子的表現。(3) 探討 MCD diet 餵食是否改變卵巢組織顆粒細胞產生血管通透相關因子的能力：先以卵巢過度刺激的方式促進濾泡成熟，再以血管新生相關因子蛋白質晶片分析濾泡中血管通透相關蛋白質的表現模式。本研究成果將能釐清 MCD diet 餵食導致動物多囊性卵巢現象之分子機制並比較其與雄性素誘導PCOS 之機轉差異。進一步解釋本模式能否合理運用於探討臨床之非酒精性脂肪肝導致多囊性卵巢症候群。<br> Abstract: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women during reproductiveage and characterized with hyperandrogenism, chronic anovulation, and higher prevalence of obesity andinsulin resistance disorders. According to our previous study, women with PCOS were reported to havehigher prevalence of NAFLD than those without PCOS and independent from obesity (HumanReproduction, 2011). Methionine-and choline- deficient (MCD) diet mice model is a conventional anduseful model of nonalcoholic fatty liver disease in rodents. Mice treated with MCD show macrovesicularsteatosis, hepatitis, and enhancement of proinflammatory cytokines and oxidative stress in the liver likethose found in human non-alcoholic fatty liver disease (NAFLD). In our preliminary study, we found thatthe MCD diet mice revealed the polycystic ovaries and anovulation similar to those found in women withPCOS. Such phenomenon has never been reported after reviewing the literatures. In this project, we aimedto investigate the potential biological effect and its mechanism on the ovarian follicle development in MCDdiet NAFLD mice model. Besides, we will investigate the difference between MCD diet mice and ourestablished androgenized mice model in anovulation at molecular and hormonal level in order to clarify themechanism of MCD diet related reproductive dysfunction.The aim of the first year is to clarify the relationship between the MCD diet mediated fibrotic fatty liver andovulatory dysfunction. The study strategies and methods are:1. Investigate the effect of fatty liver on follicle development by feeding the mice with high fat diet andMCD diet respectively for 4 weeks. Detection and quantify the fatty liver and fibrotic liver by oil redand Masson's trichrome staining. Quantify the follicle numbers and stages by series section/HE staining.The quantitative results of pathological data will be used to calculate the correlation between fatty liverand follicular development.2. Investigate the time course effect of MCD diet feeding on fatty liver fibrosis and polycystic ovaries.Female mice with different feeding durations will be used to detection the changes of body weight, liverfunction (GGT, ALT, AST, ALP), reproductive related hormones (FSH, LH, E2,P4). Furthermore, thepathological change of liver and ovary will be analyzed.3. Investigate the bioactivity of MCD diet mediated polycystic ovaries. Female mice with MCD dietmediated polycystic ovaries and anovulation will be treated with PMSG to test the capability of folliclematuration. Serum level reproductive related hormones, the pathological change of liver and ovarywill be analyzed during natural cycle.The aim of the second year is to clarify the molecular mechanism of MCD diet feeding caused PCOS-likephenotypes as polycystic ovaries and anovulation. The study strategies and methods are:1. To evaluate weather apoptosis promoting effect be in ovarian tissue. Tissue sections will be processedwith TUNEL assay for apoptosis determination and with immunological analysis of caspase3, Bcl-1 andMcl-1 for apoptosis related protein detection.2. To evaluate weather cell proliferation related signaling were changed in ovarian tissue. Tissuesections will be processed with immunological analysis of PCNA for proliferation determinationand analysis of FSHR, ER, PR, p-Akt, cyclinD1 and PTEN for cell proliferation related signalingdetection.3. To evaluate weather vascular permeability factors producing capability was changed in ovariangranulosa cells. PCOS–like female mice with MCD diet will be treated with PMSG to stimulatefollicle maturation. The expression of vascular permeability factors in follicular fluid will bedetermined by angiogenesis protein array.The results of this project may help to clarify the molecular mechanism of MCD diet caused PCOS-likephenotypes, and to realize its difference or similarity with androgen caused PCOS-like phenotypes.Furthermore, the results might also clarify whether the NAFLD related PCOS could be represented byMCD diet feeding animal model.Klebsiella pneumoniaeliver abscesscapsulecarrier proteinsbacteriophageendoglycosidasevaccine