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研究生:朱建安
研究生(外文):Jian-An Ju
論文名稱:注射c-mossiRNA對豬卵母細胞成熟、受精及發育的影響
論文名稱(外文):The effects of injecting c-mos siRNA on maturation, fertilization and development of porcine oocyte
指導教授:陳銘正陳銘正引用關係
指導教授(外文):Ming-Cheng Chen
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術研究所碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:90
中文關鍵詞:豬卵母細胞
外文關鍵詞:porcine oocyte
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本研究之目的在於藉由顯微注射c-mos 小干擾核醣核酸(small interference RNA, siRNA)到豬卵丘卵母細胞複合體(cumulus-oocyte complexes, COCs),觀察其對卵母細胞成熟形態學、c-mos mRNA表現量及隨後體外受精(in vitro fertilization, IVF)或ICSI(Intracytoplasmic sperm injection, ICSI)卵受精與發育的影響。為了解正常卵母細胞成熟過程形態學及c-mos mRNA表現量的變化,本試驗分別逢機選取成熟培養期間0, 12, 24, 36及44小時的卵母細胞,進行RT-realtime PCR檢驗c-mos RNA表現量,並用位相差及螢光顯微鏡術觀察其形態學變化。為避免顯微注射傷害卵母細胞,因此在注射前,NCSU-23成熟培養液分別添加1 μg/ml、10 μg/ml的cycloheximide,並培養8小時後進行卵母細胞的穿刺(penetration),再培養44小時,比較兩種濃度的cycloheximide是否可以增加豬卵母細胞穿刺後的存活率。構築c-mos siRNA後,將c-mos siRNA注射到未成熟卵母細胞細胞質,成熟培養44小時,去除卵丘細胞,繼續培養16 小時。在培養12 小時與24 小時分別檢驗卵母細胞核成熟及c-mos 基因表現量。最後將已注射c-mos siRNA的卵母細胞分別進行體外受精及精子顯微注射,IVF卵母細胞培養7日,ICSI的卵母細胞培養16 小時,分別觀察卵母細胞的受精與發育情形。結果顯示,生發泡的瓦解發生於成熟培養12-24小時期間,24小時之後成熟率開始逐漸上升,培養44小時達到最高;c-mos mRNA表現量在培養12小時達到最高,12小時後便逐漸下降。經由1 μg/ml cycloheximide處理的豬卵母細胞存活率為78.5 %,顯著高於對照組的36.9 %(p<0.05)。卵母細胞經c-mos siRNA注射後,不影響卵母細胞成熟的過程,但是培養至60 小時,有57.5 %卵母細胞致活,且c-mos基因表現量有下降的現象。而IVF之後siRNA注射組雄原核形成率為49.5 %,與僞注射對照組(28.6 %)比較有顯著差異(p < 0.05),且培養至7日,有5 %的胚,其分溝細胞數超過10細胞。ICSI之後,注射c-mos siRNA的卵母細胞致活率與雄原核形成率為80.97 %與 22.57 %,顯著高於僞注射組者(64.57 %與0 %)。綜合上述結果證明注射c-mos siRNA對於卵母細胞之成熟作用及隨後之受精與發育能力,確實有顯著之影響效果。
The objective was to study c-mos small interference RNA(siRNA) by microinjecting to porcine cumulus-oocyte complexes(COCs)which influenced maturation morphology, c-mos mRNA expression quantity, fertilization and development of porcine oocytes after in vitro maturation(IVM), fertilization(IVF) or intracytoplasmic sperm injection(ICSI) . In order to understand the variation of normal oocyte maturation morphology and c-mos mRNA expression quantity, ovarian oocytes randomly selected and subjected to maturation in vitro for 0, 12, 24, 36, and 44 h respectively. Subsequently, these oocytes were subjected to examination of c-mos mRNA expression by RT- realtime PCR and their morphology by phase-contrast and fluorescence microscopy. Microinjection could damage oocyte, so we added 1 μg/ml or 10 μg/ml cycloheximide to NCSU-23 maturation medium respectively and cultured for 8 h before the oocytes were punctured. After 44 h of maturation culture in vitro, these oocytes were compared whether the two concentration cycloheximide treatment could increase the proportion of survival oocyte. The c-mos siRNA was injected directly into the cytoplasm of an oocyte. After 44 h of maturation culture, these oocytes were removed cumulus cell and cultured for another 16 h. These oocytes were examined the variation of nucleus maturation and c-mos mRNA quantity during the culture period(12 h and 24 h). At last, the matured oocytes which had been injected c-mos siRNA were fertilized in vitro (IVF) or injected sperm (Intracytoplasmic sperm injection, ICSI) respectively. After fertilization, the oocytes were cultured for 7 day. After ICSI, the oocytes were cultured for 16 h. I examined the variation of morphology during the culture period. The results showed that germinal vesicle breakdown occurred during the 12 to 24 h of culture, then the oocyte maturation rate were increased gradually after 24 h of culture, and reached the highest at 44 h. The highest quantity of c-mos mRNA could attain at 12 h of IVM culture, then decrease gradually. The survival rate of oocytes via 1 μg/ml cycloheximide treatment(78.5 %)was significantly(p<0.05) higher than that of the control group(36.9 %). After injecting c-mos siRNA to oocytes, the process of maturation oocyte was not influenced, but c-mos gene expression declined, and 57.5 % oocytes was activated at 60 h of culture. After in vitro fertilization, the percentage of male pronucleus formation in oocytes that were injected c-mos siRNA(49.5 %)was significantly(p<0.05)higher than that in the punctured oocytes(28.6 %). Five percentage of fertilized oocytes that were injected c-mos siRNA development to the 10 cells morula stage. After ICSI, The rates of activation(80.97 %) and male pronucleus formation(22.57 %) of oocytes that were injected c-mos siRNA were higher than those of punctured oocytes(64.57 %, 0 %). These results suggest that injecting c-mos siRNA to porcine oocytes can influence oocyte maturation, fertilization and development.
壹、 前言.1
貳、 文獻檢討.2
一、 卵母細胞的成熟受精與發育.2
(一) 卵母細胞的成熟.2
(二) 受精作用.5
(三) 胚的發育.6
二、 訊息分子對於卵母細胞成熟、受精與發育的調控.7
(一) 濾泡環境中的訊息分子對卵母細胞成熟、受精與發育的調控.7
(二) 卵母細胞內訊息分子對成熟與受精的調控.8
1. 細胞內訊息分子對卵母細胞成熟的調控.8
2. 細胞內訊息分子對卵母細胞受精的調控.10
(三) 細胞內影響卵母細胞的重要分子.10
1. MPF.10
2. MAPK.13
3. c-Mos.15
(一) c-Mos、MAPK 對卵母細胞成熟的影響.15
1. 影響卵母細胞第一次減數分裂的恢復.16
2. 影響卵母細胞停頓在第二次減數分裂中期.16
三、 RNA干擾用於卵母細胞之研究.17
(一) RNAi pathway.19
(一) 干擾RNA 的類型及對卵母細胞的應用性.21
1. 反義股RNA.21
2. 長片段雙股RNA.21
3. miRNA.22
4. shRNA.23
5. siRNA.23
參、 材料與方法.26
一、 siRNA的設計與製備.26
(一) 轉錄模板的製備.27
(二) 雙股RNA的合成.27
(三) siRNA的純化.29
二、 豬卵母細胞體外成熟、受精與發育的培養.29
(一) 體外成熟.29
(二) 體外受精.30
(三) 體外發育.31
三、 顯微注射卵存活率提升試驗.31
四、 c-mos siRNA顯微注射.32
五、 精子顯微注射.34
六、 形態學觀察.34
(一) Hoechst 33258 螢光染色.34
(二) Lacmoid 染色.35
七、 RNA萃取.36
(一) 藥品配製.36
1. Denature solution.36
2. 2M Sodium acetate pH4.0(2M NaOAC).36
(二) RNA 萃取流程.36
八、 RT與realtime PCR.37
(一) RT合成cDNA.37
(二) 即時定量PCR(realtime PCR).37
九、 試驗設計.39
(一) 豬卵母細胞成熟期間形態學及c-mos mRNA 表現量的變化.39
(二) cycloheximide 處理是否會增加被穿刺卵母細胞的存活率.40
(三) 注射c-mos siRNA 對豬卵母細胞成熟的影響.40
(四) 注射c-mos siRNA 對豬卵母細胞受精與發育的影響.41
(五) 注射c-mos siRNA對豬卵母細胞精子顯微注射的影響.41
肆、 結果.42
一、 豬卵母細胞成熟期間形態學及c-mos mRNA表現量的變化.42
(一) 豬卵母細胞在成熟期間之形態學變化.42
(二) 豬卵母細胞成熟期間c-mos mRNA 表現量的變化.47
二、 被穿刺卵母細胞存活率提升試驗.52
三、 注射c-mos siRNA對豬卵母細胞成熟的影響.56
四、 注射c-mos siRNA對豬卵母細胞受精與發育的影響.60
五、 注射c-mos siRNA對豬卵母細胞精子顯微注射的影響.68
伍、 討論.70
陸、 結論.76
柒、 參考文獻.77
捌、 附錄.83
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