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研究生:林立德
研究生(外文):Li-te Lin
論文名稱:探討脫氫表雄酮對卵巢反應不良者之卵丘細胞細胞凋亡和粒腺體功能之影響
論文名稱(外文):Effect of dehydroepiandrosterone on apoptosis and mitochondrial function of cumulus cells in poor ovarian responders
指導教授:崔冠濠陳錦翠
指導教授(外文):Kuan-Hao TsuiJiin-Tsuey Cheng
學位類別:博士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:124
中文關鍵詞:脫氫表雄酮卵丘細胞粒線體細胞凋亡卵巢反應不良者
外文關鍵詞:apoptosisdehydroepiandrosteronemitochondriacumulus cellspoor ovarian responders
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  • 下載下載:26
  • 收藏至我的研究室書目清單書目收藏:0
卵巢反應不良患者一直以來都是人工生殖技術的一大挑戰,過去的研究指出,補充脫氫表雄酮可能對於卵巢反應不良患者之試管嬰兒預後有助益,不過,確切的作用機轉尚不清楚。本研究旨在探討脫氫表雄酮對於卵巢反應不良患者在臨床上的助益及其對卵丘細胞細胞凋亡及粒線體功能的影響,並以顆粒細胞株進一步驗證。本研究共納入131位準備接受試管嬰兒療程的不孕婦女,其中的59位為正常卵巢反應者,其餘的72位為卵巢反應不良患者;卵巢反應不良患者再分成進入試管嬰兒療程前有補充和沒有補充脫氫表雄酮二組。比較三組試管嬰兒的預後及收集三組受試者之卵丘細胞進行研究,探討卵丘細胞中細胞凋亡相關基因的表現、分析細胞凋亡狀況、粒線體脫氫酶活性、粒線體質量及粒線體功能相關TFAM基因的表現。另外,並以顆粒細胞株作為模式進行西方點墨法、免疫螢光標記法、流式細胞儀分析及海馬生物能量測定等實驗進一步提出佐證。臨床結果顯示,和未補充者相較,卵巢反應不良患者補充脫氫表雄酮明顯增加受精率、高品質胚胎數及植入胚胎數,雖然並未具有顯著統計上之差異,但在懷孕率及活產率上皆有增加的趨勢。在卵丘細胞模式研究結果顯示,脫氫表雄酮能降低卵丘細胞DNA之損壞及細胞凋亡,且能增加粒線體質量、粒線體脫氫酶活性及粒線體TFAM基因的表現。顆粒細胞株的研究發現脫氫表雄酮可抑制細胞凋亡、增加粒線體之質量及耗氧量、減少粒線體之膜電位流失及活性氧化物產生。總結而言,本研究結果顯示卵巢反應不良患者補充脫氫表雄酮對試管嬰兒預後具有正面效果,並為脫氫表雄酮之功效可能是藉由改善卵丘細胞粒線體功能及抑制細胞凋亡來達成提供佐證。
Poor ovarian responders (PORs) pose a great challenge for in vitro fertilization (IVF). Previous studies have suggested that dehydroepiandrosterone (DHEA) may improve IVF outcomes in PORs; however, the precise mechanisms were unclear. The current study aimed to investigate the clinical benefits of DHEA in PORs and the possible effects of DHEA on cumulus cells (CCs) regarding apoptosis and mitochondrial function. HO23 granulosa cell line was used for further confirmation of DHEA effects. A total of 131 women who underwent IVF treatment participated, including 59 normal ovarian responders (NORs) and 72 PORs. PORs were assigned to receive DHEA pretreatment or not before the IVF cycle. IVF outcomes were compared among the three groups and CCs were obtained after oocyte retrieval in all patients. In the CCs, mRNA expression of apoptosis-related genes and mitochondrial transcription factor A (TFAM) gene, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, mitochondrial dehydrogenase activity and mitochondrial mass were measured. Additionally, we performed immunoblotting, immunofluorescence labeling, flow cytometry and seahorse analyzer in HO23 cells to verify the effects of DHEA. The results showed that PORs with DHEA supplementation produces a great number of top-quality embryos at day 3 and increased the number of transferred embryos and fertilization rate compared with those without DHEA supplementation. Moreover, PORs with DHEA pretreatment displayed a tendency of higher clinical pregnancy rate, ongoing pregnancy rate and live birth rate than PORs without DHEA pretreatment. In the CCs, supplementation with DHEA in PORs decreased DNA damage and apoptosis while enhancing the mitochondrial mass, mitochondrial dehydrogenase activity and TFAM expression. In the HO23 cells, treatment with DHEA improved mitochondrial capacity to defend against apoptosis through the elimination of mitochondrial ROS, increasing mitochondrial biogenesis levels, and the enhancement of mitochondrial mass. In conclusion, our results showed that the benefits of DHEA supplementation on IVF outcomes in PORs were significant, and the effects may be partially mediated by improving mitochondrial function and reducing apoptosis in CCs.
前 言…………………………………………………………….... 1
卵巢反應不良患者…………………………………………....... 2
脫氫表雄酮(dehydroepiandrosterone, DHEA)…………… 5
卵丘細胞或顆粒細胞………………………………………….. 10
細胞凋亡……………………………………………………….. 13
粒腺體………………………………………………………….. 16
研究動機與假設……………………………………………….. 19
材 料 與 方 法………………………………………………….. 20
研究設計與分組……………………………………………….. 21
試管嬰兒療程………………………………………………….. 23
卵丘細胞之分級與收集……………………………………….. 25
總RNA萃取及即時定量聚合酶連鎖反應…………………..... 26
細胞凋亡分析 (TUNEL assay) …………………................. 27
粒線體脫氫酶活性分析………………………….................. 27
粒線體質量測量………………………………….................. 27
HO23人類顆粒細胞株之培養與治療………………………... 28
細胞存活分析………………………………………………….. 29
免疫螢光標記法……………………………………………….. 29
粒線體膜電位、活性氧化物、質量之測量………………….. 29
西方墨點法(Western blot)………………………………… 30
粒線體耗氧量之測量………………………………………….. 31
統計方法……………………………………………………….. 32
結 果……………………………………………………………... 33
臨床病人資料與試管嬰兒預後……………………………….. 34
脫氫表雄酮對於卵子-卵丘細胞複合體之影響…....………… 37
脫氫表雄酮對於卵丘細胞細胞凋亡之影響………………….. 38
脫氫表雄酮對於卵丘細胞粒線體之影響…………………….. 39
脫氫表雄酮對於HO23顆粒細胞株細胞凋亡之影響………... 40
脫氫表雄酮對於HO23顆粒細胞株粒線體功能之影響……... 43
討 論……………………………………………………………... 45
結論及未來研究方向……………………………………........... 52
參考文獻…………………………………………………………. 53
附表………………………………………………………………. 75
附圖………………………………………………………………. 80
縮寫表…………………………………………………………... 103
附錄……………………………………………………………... 105
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