本文選題：激活素 + 抑制素��； 參考：《中國協(xié)和醫(yī)科大學》2007年博士論文

【摘要】： 目前人類尚無法完整揭示卵泡生長發(fā)育過程中的調控機理，主要原因在于對卵巢內部因子作用機制了解不清。激活素-抑制素-卵泡抑素(activin-inhibin-follistatin，ACT-INH-FS)系統(tǒng)是最重要的卵巢內部因子之一，其卵巢內部的旁自分泌作用機制有待闡明。 近年來人類未成熟卵母細胞的體外成熟(in vitro maturation，IVM)技術在不育癥治療中顯示出重要的臨床價值。但其妊娠率、種植率以及自然流產率尚不理想，主要原因在于尚未建立能夠使未成熟卵母細胞同步獲得細胞核和細胞漿成熟的培養(yǎng)體系。ACT-INH-FS作為卵泡體內成熟微環(huán)境中的重要因子，可能也是IVM培養(yǎng)體系中不可或缺的組分。 本研究目的是通過以下三方面內容，探討激活素-抑制素-卵泡抑素系統(tǒng)在卵母細胞成熟過程中的旁分泌作用機制及其在人類不成熟卵母細胞體外成熟技術中的應用價值。 一、激活素-抑制素-卵泡抑素系統(tǒng)對卵母細胞成熟及胚胎發(fā)育潛能的影響 二、激活素-抑制素-卵泡抑素系統(tǒng)卵巢局部生理作用的階段性與交互性 三、卵母細胞體外成熟培養(yǎng)體系的安全性相關研究 第一部分激活素-抑制素-卵泡抑素系統(tǒng)對卵母細胞成熟及胚胎發(fā)育潛能的影響 目的：探討激活素-抑制素-卵泡抑素系統(tǒng)在小鼠生發(fā)泡(GV)期卵母細胞體外成熟及早期胚胎發(fā)育中的的影響機制。方法：3-4周齡昆明系雌性小鼠腹腔注射PMSG后48小時，獲取GV期卵母細胞隨機分入以下八組。對照組采用HTF+10％SPS；試驗組分別為：100ng／ml ACTA；100ng／mlACTA+100ng／mlFS；100ng／mlINHA；100ng／mlINHA+100ng／mlFS；100ng／mlACTA+INHA；100ng／mlACTA+100ng／mlINHA+100ng／mlFS；100ng／ml FS。GV期卵母細胞體外培養(yǎng)16-18小時后，MⅡ期的卵母細胞進行體外受精，觀察ACTA-INHA-FS對GV期卵母細胞生發(fā)泡破裂(GVBD)、第一極體(PB1)排出及受精率、卵裂率、囊胚形成率的影響。使用MitoTracker RED熒光探針對ACTA組以及ACTA+INHA合用組MⅡ期卵母細胞內活性線粒體數目及分布進行檢測。結果：(1) ACTA、ACTA+INHA、單獨使用FS均可顯著促進GV期卵母細胞GVBD發(fā)生率(P＜0.05)。(2) ACTA、ACTA+INHA、INHA均可顯著促進MⅡ期卵母細胞發(fā)生率(P＜0.05)，(3) INHA組受精率低于對照組(P＜0.05)，其余各組與對照組相比，，受精率無明顯差異(P＞0.05)。(4) ACTA、INHA、ACTA+INHA組8細胞及囊胚形成率均顯著高于對照組(P＜0.05)。(5) FS可以特異性拮抗ACTA的促進作用，但不能中和ACTA與INHA的協(xié)同作用(P＞0.05)。(6)單獨使用FS組囊胚形成率高于對照組(40％vs 24.7％)，但差異尚不具有統(tǒng)計學意義(P=0.051)。(7)體外成熟后的MⅡ期卵母細胞內線粒體形態(tài)可以分為以下三類：周邊型、半周型、彌散型，ACTA組以及ACTA+INHA合用組半周型以及彌散型的比例明顯高于對照組，整體分布存在顯著性差異(P＜0.01)結論：(1) ACTA、INHA對GV期卵母細胞體外成熟及胚胎發(fā)育潛能具有顯著的促進作用，并可以被FS所拮抗。(2)在ACTA／INHA缺乏或不足的條件下，FS可出現ACTA樣作用，促進卵母細胞成熟。(3) ACTA-INHA對體外成熟卵母細胞核質成熟同步化具促進作用。 第二部分激活素-抑制素-卵泡抑素系統(tǒng)旁自分泌作用的階段性及交互性 目的：探討生理周期中LH峰前后ACTA／INHA對GV期卵母細胞體外成熟的不同影響；激活素-抑制素-卵泡抑素系統(tǒng)與EGF的相互作用方式。方法：3-4周性成熟前小鼠分為兩組，一組給予PMSG超排卵48小時后取卵，另一組予PMSG超排卵48小時后繼續(xù)給予HCG10U 2小時后取卵。HCG刺激(HCG+)組和HCG未刺激(HCG-)組的GV期卵丘-卵母細胞復合物(COC)，在含有100ng／mlACTA+100ng／mlINHA培養(yǎng)液中體外培養(yǎng)14-18小時，MⅡ期的卵母細胞進行體外受精，觀察ACTA-INHA-FS對GV期卵母細胞生發(fā)泡破裂(GVBD)、第一極體(PB1)排出及受精率、卵裂率、囊胚形成率的影響。GV期卵母細胞在含10ng／ml EGF及10ng／ml EGF+100ng／mlFS的培養(yǎng)液中培養(yǎng)16-18小時后，MⅡ期的卵母細胞進行體外受精，比較生發(fā)泡破裂(GVBD)、第一極體(PB1)排出及受精率、卵裂率、囊胚形成率的差別。結果：(1) HCG(+)組COC經體外成熟培養(yǎng)14小時后，卵丘細胞團擴散比例明顯高于HCG(-)組(81.5％vs5.1％)。(2) HCG(+)組與HCG(-)桑椹胚形成率以及囊胚形成率存在非常明顯差異(P＜0.01)。(3) FS可以顯著抑制EGF對PB1排出率的促進作用(P＜0.01)但不能抑制EGF對胚胎發(fā)育的促進作用(P＞0.05)結論：(1) LH峰后的數小時內是卵母細胞成熟發(fā)育、維持核漿成熟同步化的關鍵時期。(2) LH峰值前后ACTA／INHA對GV期卵母細胞體外成熟的影響不同，LH峰后ACTA／INHA對早期胚胎發(fā)育的促進作用消失。(3)胞核成熟與胞漿成熟存在不同的調控途徑；在核成熟的調控機理中，ACT系統(tǒng)是EGF的下游作用通路。。 第三部分卵母細胞體外成熟培養(yǎng)體系的安全性相關研究 目的：通過對MⅠ期卵母細胞骨架的激光共聚焦研究探討體外成熟卵母細胞紡錘體、染色體結構異常與非整倍體發(fā)生率的關系；通過對MⅡ期卵母細胞染色體核型的檢測評價激活素A-抑制素A用于卵母細胞體外成熟培養(yǎng)體系的安全性。方法：(1) GV期卵母細胞體外培養(yǎng)7小時，獲取MⅠ期卵母細胞作為體外成熟組，體內發(fā)育的MⅠ期卵母細胞作為對照組。經免疫熒光染色，共聚焦熒光顯微鏡觀察紡錘體形態(tài)、染色體分布。(2) GV期卵母細胞隨機分入以下兩組：對照組采用HTF+10％SPS；試驗組采用100ng／mlACTA+INHA；體外培養(yǎng)16-18小時后，選擇MⅡ期卵母細胞進行染色體核型分析。結果：(1)體外成熟組紡錘體結構異常以及染色體分布異常的比例均明顯增高(34.6％vs 7.1％；26.9％vs 0；P＜0.01)。但成熟后的MⅡ期卵母細胞非整倍體發(fā)生率(0／31)并無增高。(2) ACTA+INHA組非整倍體發(fā)生率為7.1％，與對照組相比非整倍體發(fā)生率無顯著增高(P＞0.05)。結論：(1)不成熟卵體外培養(yǎng)過程中存在紡錘體結構以及染色體分布的異常；卵母細胞可通過自檢方式阻止結構異常的細胞進一步分裂。(2) ACTA-INHA不增加體外成熟卵母細胞的非整倍體發(fā)生率。 激活素-抑制素-卵泡抑素系統(tǒng)在卵母細胞成熟過程中的影響機制相關研究在國內尚無先例。國外的相關研究大多數限于對核成熟的調控作用方面，3篇關于牛的研究分別觀察了ACTA或INHA對胚胎發(fā)育潛能的影響，但結果不甚一致。本研究首次將ACT-INH-FS作為一個系統(tǒng)，較以往研究更為全面地探討其對卵母細胞成熟的調節(jié)機理，首次借助對卵母細胞超微結構的觀察證實了其對胞漿成熟的促進作用。并探討了激活素系統(tǒng)生理作用的階段性和交互性，及其用于IVM培養(yǎng)體系的安全性，這些內容及結論亦未見相關報道。
[Abstract]:At present , the mechanism of regulation and regulation in the growth and development of follicles is not fully revealed . The main reason is that the mechanism of the internal factors of the ovary is not clear . The activin - statin - follistatin ( ACT - INH - FS ) system is one of the most important internal factors of the ovary , and the mechanism of paratual self - secretion in the ovary needs to be clarified .


In recent years , the in vitro maturation ( IVM ) technique of human immature oocytes has shown an important clinical value in the treatment of infertility , but its pregnancy rate , implantation rate and spontaneous abortion rate are not ideal .


The purpose of this study was to explore the mechanism of the parasecretory action of the activin - statin - follicle - inhibiting system in the maturation of oocytes and its application in the in vitro maturation of human immature oocytes by the following three aspects :


Effects of Activin - Inhibin - follicle - statin on the maturation and embryonic development potential of oocytes


2 . Phase and Interactivity of the Local Physiological Function of Activin - Inhibin - follicle Suppressor System in the Ovary


Study on the safety of mature culture system of oocytes in vitro


Effects of the first part of activin - statin - follicle - inhibiting system on oocyte maturation and embryonic development potential


Objective : To investigate the mechanism of activin - statin - follicle - inhibiting system in vitro maturation and early embryonic development of mouse oocytes . Methods : Forty - eight hours after intraperitoneal injection of PMSG into Kunming female mice , the following eight groups were randomly divided into eight groups : HTF + 10 % SPS ;
The experimental groups were 100 ng / ml ACTA , respectively .
100 ng / ml ACTA + 100ng / ml FS ;
100ng / ml INHA ;
100ng / ml INHA + 100ng / ml FS ;
100 ng / ml ACTA + INHA ;
100 ng / ml ACTA + 100ng / ml INHA + 100ng / ml FS ;
Results : ( 1 ) ACTA , ACTA + INHA and INHA could significantly promote the incidence of GVBD in GV phase ( P < 0.05 ) . ( 1 ) ACTA and INHA promoted the maturation and development potential of oocytes in vitro and could be antagonized by FS . ( 2 ) The ACTA - INHA promoted the maturation of oocytes and promoted the maturation of oocytes .


Phase and Interactivity of the Second Part of Activin - Inhibin - follicle Suppressor System in the Presence of Self - Secreting


Objective : To investigate the effect of ACTA / INHA on the maturation of GV oocytes in vitro before and after LH peak .
( 2 ) The effect of ACTA - INHA - FS on the growth of oocytes in GV stage was significantly higher than that in HCG ( - ) group ( 81.5 % v5.1 % ) .
In the mechanism of nucleus maturation , the ACT system is the downstream pathway of EGF .


Study on the safety of the third part oocyte in vitro maturation culture system


Objective : To investigate the relationship between chromosome structural abnormalities and the incidence of non - euploid in vitro maturation of oocytes in vitro .
Methods : ( 1 ) The oocytes were cultured in vitro for 7 hours in vitro . The oocytes were cultured in vitro for 7 hours in vitro .
100 ng / ml ACTA + INHA was used in the test group .
Results : ( 1 ) The abnormal structure of spindle structure and the abnormal distribution of chromosome distribution in vitro were significantly higher than that in the mature group ( 34 . 6 % vs 7.1 % ) .
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本文編號：1731660