臺灣博碩士論文加值系統

本文的目的在探討類卵巢中濾泡液內胰島素生長因子(IGF-II及IGF-II)及其結合蛋白(IGF binding proteins)與IGFBP4之蛋白酶(pregnancy-associated plasma protein-A)、及取卵後卵子之後的發育情況間的關係。
總共有162位不孕婦女進行人工生殖(in vitro fertilization)，共收集了241管的濾泡液(FF)檢體且沒有血液污染；此外，這些濾泡液檢體也都有與其相對應之發育成熟的卵細胞，並詳細紀錄卵細胞發育成胚胎的詳細資料。而濾泡液中IGFs、IGFBPs及PAPP-A的濃度則是利用酵素結合免疫吸附法(ELISA)測量而得。
我們發現濾泡液中IGF-II，IGFBP3及IGFBP4的濃度與胚胎發育第三天的分級分數(embryo score)成正相關(其P值依序為：P < 0.02, P < 0.02 and P < 0.005)；意即胚胎之第三天的分級分數(embryo score)越高，其濾泡液中IGF-II，IGFBP3及IGFBP4的濃度越高。此外，測量濾泡液中PAPP-A的濃度卻發現與IGFBP4的濃度呈現負相關(P < 0.01)。此外，卵細胞成功受精且發育到第三天的濾泡(Day3 embryo score ³ 5)其濾泡液中IGFBP3及IGFBP4的濃度，比卵細胞沒有受精及胚胎最後沒有成功發育到第三天者(Day3 embryo score = 0)來的高(P< 0.00001及P< 0.00003)。
比較那些沒有成功受精或在受精後72小時後，胚胎停止發育的濾泡(Day3 embryo score =0)其濾泡液中PAPP-A的濃度，與成功受精且胚胎發育至第三天的濾泡(Day3 embryo score ³ 5)之PAPP-A；前者的PAPP-A濃度比後者的高(P< 0.0003)。
以上的實驗結果，可以假設濾泡液中之第二型類胰島素生長因子(insulin-like growth factor II，IGF-II)，及其第三、第四結合蛋白(IGFBP3，IGFBP4)的濃度，也許能反應出卵子的成熟度及之後胚胎發育的情況。

The objective of the present study was to investigate the association between levels of insulin-like growth factors (IGF-I and IGTF-II), their binding protein (IGFBP1, IGFBP3 and IGFBP4) and a protease of IGFBP4, pregnancy-associated plasma protein-A (PAPP-A), in follicular fluid (FF) and the quality of subsequent embryo development of the in vitro fertilized oocytes aspirated form the corresponding follicle.
162 infertile women undergoing controlled ovarian hyperstimulation from in vitro fertilization and embryo transfer were recruited. 241 pairs of FF samples and corresponding mature oocytes were studied. Concentration of IGFs, IGFBPs and PAPP-A were determined by enzyme-linked immunosorbent assay (ELISA).
We found that levels of IGF-II, IGFBP3 and IGFBP4 in follicular fluid (FF) were significantly correlated with embryo scores on Day3 (72 hours after oocyte retrieval; embryo score ³ 5) (P < 0.02, P < 0.02 and P < 0.005, respectively). Additionally, concentrations of PAPP-A in FF on the day of oocyte retrieval showed a significantly reverse association with of IGFBP4 (P < 0.01). FF levels of IGFBP3 and IGFBP4 from follicles in which oocytes were fertilized and developed into embryos on Day3 were significantly higher than those follicles both in which oocytes were unfertilized and in which fertilized oocytes fail to develop into Day3 embryos (Day3 embryo score = 0) (P < 0.00001 and P < 0.00003, respectively). Lower levels of PAPP-A in FF were noticed from follicles in which retrieved eggs were fertilized and developed into Day3 embryos as compared to those from follicles in which oocytes were unfertilized and in which fertilized eggs became arrested by 72 hours after oocyte retrieval. Results of this study suggest that insulin-like growth factor-II (IGF-II), IGF-biding protein-3 (IGFBP3) and IGFBP4 in FF at the time of oocyte retrieval may reflect the oocyte maturation and later embryo development.

目錄
指導教授推薦書
口試委員審定書
目錄(Table of content)………………………………………………….I
圖目錄(List of figures)…………………………………………………V
表目錄(List of tables)………………………………………………….VI
附錄(List of appendix)
中文摘要………………………………………………………………VII
英文摘要………………………………………………………………IX
第一章：序論
1.1 濾泡生成過程 (Folliculogenesis)……………………………….1
1.2 累計的胚胎發育分數 (The cumulative embryo score)………. .3
1.3 概觀TGF-b superfamily (Overview of TGF-b superfamily)…....3
1.4 TGF-b superfamily：the bone morphogenetic proteins………….4
1.5 TGF-b superfamily之訊息傳遞路徑……………………………5
1.6 BMP蛋白質在調節濾泡生長及發育所扮演之角色…………..6
1.7 概觀IGFs/IGFBPs系統(Overview of IGF/ IGFBPs systems)…8
1.8 IGFs與IGFBPs所扮演之角色(Role of IGFs/ IGFBPs)……….9

第二章：研究目的與假說……………………………………………..17
2.1 Rationale…………………………………………………………18
2.2 Specific aims……………………………………………………..18
第三章：實驗材料與方法
3.1 誘發排卵的步驟(Protocols of ovulation induction)……………19
3.2 人工生殖與胚胎的評估………………………………………..20
3.3濾泡液的收集(Collection of follicular fluid)…………………...20
3.4 Enzyme-linked immunosorbent assay (ELISA) for IGF-I, IGF-II, IGFBP-1, IGFBP-3, IGFBP-4, PAPP-A………………………………..21
3.5西方墨點分析IGFBP4及Beta-actin之分子量……………….25
3.6收集並培養濾泡顆粒細胞……………………………………...26
3.7 免疫組織化學染色法(Immunohistochemical staining)………..27
3.8 Total RNA的粹取………………………………………………28
3.9 反轉錄聚合鏈反應 (Reverse transcription PCR)……………..29
3.10 Real-time quantitative polymerase chain reaction…………….29
3.11 Primers and Probes………………………………………….…30
3.12 Electroporation…………………………………………………31
3.13 Microinjection of granulosa cells……………………………..32
3.14 轉染 (Transfection)…………………………………………..32
3.15 Reporter vector preparation……………………………………34
3.16 Luciferase assay……………………………………………….36
3.17 cDNA microarray analysis…………………………………….38
3.18 數據分析……………………………………………………...38
3.19 傳統聚合酶鏈反應 (Polymerase Chain Reaction)………….40
3.21 濾泡顆粒細胞之藥物影響…………………………………...41
3.22 Radio immunoassay (RIA) for progesterone………………….42
第四章：實驗結果……………………………………………………44
4.1 收集濾泡中之濾泡液(FF)與該濾泡內卵子並紀錄早期胚胎發育情況…………………………………………………………………44
4.2 濾泡液中各種蛋白質與該濾泡中卵子發育之預後關係……44
4.3 利用免疫組織化學染色法來確認所收集之細胞……………46
4.4 確立real time quantitative PCR的正確性(efficiency)……….47
4.5 顆粒細胞內各種基因表現與該濾泡中卵子發育之預後關係.47
4.6 以微注射或轉染(transfection)將外來基因引導入顆粒細胞…48
4.7 以cDNA microarray來篩選與卵子發育有關之基因………..49
4.8 以傳統聚合鏈反應(PCR)及DNA電泳分析來確認cDNA microarray所篩選出之基因之正確性………………………………..49
4.9 濾泡顆粒細胞的In vitro treatment來看IGF-II、IGFBP3及IGFBP4間對顆粒細胞的影響………………………………………..50
第五章：討論…………………………………………………………52
5.1 由濾泡液著眼IGFs, IGFBPs及PAPP-A於胚胎預後所扮演角色之結果…………………………………………………………………52
5.2 由濾泡顆粒細胞著眼BMPs系統於胚胎預後扮演之角色的結果………………………………………………………………………57
5.3 由DNA微陣列分析篩選出與胚胎預後可能有關之基因…59
5.4 在high embryo score (Day2 embryo score ³ 10)這組，其基因表現被活化之基因……………………………………………………...60
5.5 由In vitro treatment著眼探討IGF-II、IGFBF3及IGFBP4間的交互作用對濾泡顆粒細胞的影響…………………………………..65
第六章：結論………………………………………………………..67
參考文獻…………………………………………………………….68

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