【正文】 分類號(hào) S823 學(xué)校代碼 10129 U D C636 學(xué) 號(hào) 08202209 細(xì)胞因子對(duì)牛體外受精胚胎細(xì)胞凋亡的影響 Effect of Cytokines on Cell Apoptosis of Bovine In Vitro Fertilized(IVF) Embryos 申 請(qǐng) 人：蘇米雅 學(xué)科門類：農(nóng) 學(xué) 學(xué)科專業(yè)：動(dòng)物遺傳育種與繁殖 研究方向：動(dòng)物繁殖生物學(xué)與繁殖技術(shù) 指導(dǎo)教師：張家新 教 授 論文提交日期： 二〇一一年五月 0 要 本研究通過(guò)在牛卵母細(xì)胞成熟液和胚胎發(fā)育液中添加不同濃度的表皮生長(zhǎng)因子 （ EGF）和半胱胺（ CYS），比較其對(duì)牛卵母細(xì)胞成熟和胚胎發(fā)育的影響，利用 TUNEL 檢測(cè)法，檢測(cè)了不同處理?xiàng)l件下胚胎細(xì)胞的凋亡情況。為牛卵母細(xì)胞和胚胎體外發(fā) 育的提高提供理論基礎(chǔ)。研究結(jié)果如下： 試驗(yàn)一，研究了牛體外受精胚胎在不同發(fā)育階段的細(xì)胞凋亡情況。結(jié)果表明， 對(duì)于牛體外受精胚胎，凋亡信號(hào)最早出現(xiàn)于 16 細(xì)胞期，隨著胚胎的發(fā)育，凋亡發(fā)生 比率也增加。 16 細(xì) 胞凋亡率為 26%，桑葚胚凋亡率為 83%，囊胚為 97%,擴(kuò)展囊胚和 孵化囊胚為 100%。在囊胚階段，細(xì)胞凋亡指數(shù)也隨發(fā)育進(jìn)行呈現(xiàn)上升趨勢(shì)（囊胚凋 亡指數(shù)為 ，擴(kuò)展囊胚為 , 孵化囊胚為 ）。 試驗(yàn)二，比較不同濃度 EGF 或 CYS 對(duì)牛卵母細(xì)胞成熟的影響。結(jié)果表明 50ng/ml EGF 顯著提高卵母細(xì)胞的卵裂率。說(shuō)明 EGF 對(duì)卵母細(xì)胞成熟有促進(jìn)作用。在成熟液 中添加 CYS 對(duì)卵母細(xì)胞卵裂率和囊胚率沒(méi)有顯著影響，但 50ng/ml 濃度 EGF 和 100uM 濃度 CYS 處理組的囊胚凋亡指數(shù)都顯著低于對(duì)照組 (, v. )，說(shuō)明 成熟液添加適當(dāng)濃度 EGF 或 CYS 能夠顯著提高牛卵母細(xì)胞的質(zhì)量。 試驗(yàn)三，比較了在牛胚胎發(fā)育液中添加不同濃度 EGF 或 CYS 的效果。結(jié)果表明 EGF 對(duì)牛胚胎體外發(fā)育沒(méi)有明顯影響。在胚胎發(fā)育液中添加 100uMCYS 可以明顯提高 胚胎發(fā)育能力，囊胚率顯著高于對(duì)照組（ v. ,P），囊胚的凋亡指數(shù) 顯著低于其他組 ( v. , P)。說(shuō)明 CYS 對(duì)牛胚 胎體外發(fā)育有明顯促進(jìn)作用。 試驗(yàn)四，比較了在牛卵母細(xì)胞成熟液和胚胎發(fā)育液中同時(shí)添加 EGF 或 CYS 的效 果。結(jié)果表明，同時(shí)添加 EGF 或 CYS 對(duì)牛卵母細(xì)胞的成熟及胚胎的發(fā)育沒(méi)有產(chǎn)生疊 加效應(yīng)。 以上結(jié)果表明，（ 1）牛體外受精胚胎，凋亡信號(hào)最早出現(xiàn)于 16 細(xì)胞期，隨著胚 胎的發(fā)育，凋亡發(fā)生比率也增加。（ 2） EGF 和 CYS 可以促進(jìn)卵母細(xì)胞成熟。（ 3） EGF 對(duì)牛胚胎體外發(fā)育沒(méi)有明顯影響。 CYS 可以提高牛胚胎體外發(fā)育能力。（ 4）在成熟 液和發(fā)育液中同時(shí)添加 EGF 和 CYS 可以提高牛卵母細(xì)胞成熟和胚胎的發(fā)育，但不產(chǎn) 生疊加效應(yīng)。 關(guān)鍵詞： 牛；體外受精胚胎；細(xì)胞凋亡；細(xì)胞因子 Effect of Cytokines on Cell Apoptosis of Bovine In Vitro Fertilized(IVF) Embryos Abstract In this study, development of oocyte and embryo of bovine were examined after epidermal growth factor (EGF) and cysteamine (CYS) were supplemented in in vitro maturation medium and embryo culture medium, cell apoptosis of embryo under different treatment conditions were detected using TUNEL fluorescence method. The purpose of this study is to improve bovine oocyte maturation and embryo development. The results are as follows: In experiment one, cell apoptosis of bovine in vitro fertilized embryo at different developmental stages were detected. The results showed that apoptosis signal first appeared in the 16cell stage, and apoptosis rates was increased with embryo development. The apoptosis rate was 26% in 16cell embryo, 83% in morula , 97% in blastocysts and 100% in expanded blastocysts and hatching blastocysts. In blastocysts stage, cell apoptosis index were increased with embryo development(apoptosis index was in blastocysts, in expanded blastocysts, in hatched blastocysts ) In experiment two, the effects of EGF or CYS on bovine oocytes maturation were pared. The results showed that 50ng/ml EGF added in maturation medium could improve oocytes maturation which increased the cleavage rate of oocyte significantly. CYS supplemented in maturation medium did not influence oocyte cleavage rate and blastocyst rate ,but apoptosis index in 50ng/ml EGF or 100uM CYS treatment groups were significantly lower than that of control group(, ,P), which indicate supplementation with appropriate concentrations of EGF or CYS could improve the development of bovine oocyte significantly. In experiment three, the effect of EGF or CYS on bovine embryo development were pared. The results showed that EGF did not effect bovine embryo development. Supplement with 100uM CYS in culture medium could increase blastocyst rate significantly to pare to control group( v. ， P).Apoptosis index of blastocyst in 100uM CYS were significantly lower than that of other groups ( V. ,P). This result indicate CYS can promote the bovine embryo development. In experiment four, the effects of EGF bined with CYS on bovine oocyte maturation and embryo development were result showed that supplementation with EGF and CYS together did not produce additive effects on bovine oocyte maturation and embryo development. These results suggest that (1)in bovine IVF embryo, the apoptosis signal first appeared in the 16cell stage. Apoptosis rate was increased with embryo development. (2) EGF and CYS could promote oocyte maturation. (3)EGF had no significant effect on embryo development。 CYS could improve development of bovine embryo. (4)Supplement with EGF and CYS together could improve bovine oocyte maturation and embryo developmet, but without accumulated effect. Key words： Bovine。 In Vitro Fertilized Embryo。 Cell Apoptosis。 Cytokine Directed by： prof. ZHANG Jiaxin () Applicant for Master degree： Sumiya (Animal Science College. Inner Mongolia Agricultural ) （ Animal Geics Breeding amp。 Reproduction） 目 錄 1 引言 ................................................................ 1 卵母細(xì)胞凋亡現(xiàn)象 ................................................ 1 卵母細(xì)胞凋亡假說(shuō) ................................................ 2 卵母細(xì)胞凋亡分子機(jī)理 ............................................ 2 細(xì)胞凋亡相關(guān)基因 ................................................ 3 胚胎的細(xì)胞凋亡 .................................................. 4 細(xì)胞凋亡檢測(cè)方法 ................................................ 4 形態(tài)學(xué)檢測(cè) .................................................... 4 生化檢測(cè) ...................................................... 5 2 實(shí)驗(yàn)研究 ............................................................ 8 實(shí)驗(yàn)方法 ........................................................ 8 口吸撿卵針的準(zhǔn)備 .............................................. 8 卵母細(xì)胞的準(zhǔn)備 ................................................ 8 撿卵及成熟培養(yǎng) ................................................ 8 精子獲能 ...................................................... 9 體外受精 ...................................................... 9 胚胎培養(yǎng)及發(fā)育 ................................................ 9 TUNEL 試劑盒工作原理 ........................................... 9 TUNEL 試劑盒處理前準(zhǔn)備