本文選題：基因芯片 + 母源基因�。� 參考：《福建醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的: 檢測阻滯品系(昆明)小鼠與非阻滯品系(B6C3F1)小鼠次級卵母細(xì)胞基因表達(dá)水平的差異;觀察成纖維細(xì)胞生長因子7(fibroblast growth factor 7,FGF7)對KM小鼠植入前胚發(fā)育和克服2-細(xì)胞阻滯的作用。 方法: 1.Affymetrix表達(dá)譜芯片檢測比較KM與B6C3F1小鼠次級卵母細(xì)胞基因表達(dá)差異,并運用Real time PCR對部分差異基因進(jìn)行驗證; 2.RT-PCR和免疫熒光細(xì)胞化學(xué)顯色方法檢測成纖維細(xì)胞生長因子受體(fibroblast growth factor receptor,FGFR)-1、2和3在KM小鼠卵母細(xì)胞和植入前胚中的表達(dá)與分布; 3.收集KM小鼠1-細(xì)胞胚,采用微滴培養(yǎng)法連續(xù)培養(yǎng),觀察在M16培養(yǎng)液中添加FGF7蛋白對植入前胚發(fā)育的影響,觀察其是否能克服2-細(xì)胞阻滯。 結(jié)果: 1.基因芯片檢測顯示KM與B6C3F1小鼠卵母細(xì)胞內(nèi)許多與基因轉(zhuǎn)錄、轉(zhuǎn)錄調(diào)節(jié)、蛋白質(zhì)合成等功能相關(guān)的基因表達(dá)存在差異;Real time PCR驗證結(jié)果與芯片檢測結(jié)果較為相符,證明本次芯片檢測結(jié)果較為可靠。 2.RT-PCR檢測顯示,FGFR1、FGFR2和FGFR3 mRNA在KM小鼠卵母細(xì)胞和植入前胚均有表達(dá);免疫熒光細(xì)胞化學(xué)結(jié)合共聚焦掃描顯微鏡觀察顯示FGFR1、FGFR3免疫陽性反應(yīng)見于卵母細(xì)胞和植入前胚胞質(zhì)周邊,靠近細(xì)胞膜;FGFR2陽性反應(yīng)較均勻分布于卵母細(xì)胞和植入前胚的胞質(zhì)。 3.KM小鼠1-細(xì)胞胚在添加FGF7蛋白的M16培養(yǎng)液中培養(yǎng),其4-細(xì)胞胚發(fā)育率明顯高于空白M16培養(yǎng)液(對照組),差異有顯著性意義。 結(jié)論: B6C3F1小鼠卵母細(xì)胞表達(dá)上調(diào)基因主要與基因轉(zhuǎn)錄調(diào)節(jié)、抗氧化應(yīng)激、蛋白質(zhì)合成與轉(zhuǎn)運、氨基酸磷酸化修飾、細(xì)胞周期、發(fā)育等功能相關(guān),提示B6C3F1小鼠卵母細(xì)胞內(nèi)與自身基因轉(zhuǎn)錄、蛋白質(zhì)合成與轉(zhuǎn)運、抗氧化等功能相關(guān)的調(diào)節(jié)更為完善,阻滯與非阻滯品系小鼠卵母細(xì)胞內(nèi)這些母源基因表達(dá)差異可能影響小鼠植入前胚體外發(fā)育能力;KM小鼠卵母細(xì)胞和植入前胚內(nèi)有成纖維細(xì)胞生長因子受體1、2和3的表達(dá);FGF7可促進(jìn)KM小鼠植入前胚體外發(fā)育,顯著提高2-細(xì)胞至4-細(xì)胞的發(fā)育比率。
[Abstract]:Objective: To investigate the difference of gene expression in secondary oocytes between Kunming and non-blocking strain B6C3F1, and to observe the effects of fibroblast growth factor (7(fibroblast growth factor 7) on preimplantation embryo development and overcoming 2-cell block in km mice. Methods: The difference of gene expression between km and B6C3F1 secondary oocytes was detected by 1.Affymetrix microarray, and some differentially expressed genes were verified by Real time PCR. The expression and distribution of fibroblast growth factor receptor FGFRC-1 and 3 in km mouse oocytes and preimplantation embryos were detected by 2.RT-PCR and immunofluorescence cytochemical method. 3. The 1-cell embryos of km mice were collected and cultured continuously by microdrop culture. The effects of FGF7 protein on the development of preimplantation embryos in M16 medium were observed, and whether they could overcome the 2-cell block was observed. Results: 1. Gene chip analysis showed that there were differences in gene expression related to gene transcription, transcription regulation and protein synthesis between km and B6C3F1 mouse oocytes. The results of Real time PCR verification were consistent with those of microarray analysis. It is proved that the chip test result is reliable. 2.RT-PCR analysis showed that FGFR1FGFR2 and FGFR3 mRNA were expressed in oocytes and preimplantation embryos of km mice, and immunofluorescence cytochemistry combined with confocal scanning microscopy showed that FGFR1 and FGFR3 immunoreactive reaction were observed in oocytes and the peripheral cytoplasm of preimplantation embryos. The FGFR2 positive reaction near cell membrane was more homogeneously distributed in oocytes and cytoplasm of preimplantation embryos. When 3.KM mouse 1-cell embryos were cultured in M16 medium supplemented with FGF7 protein, the development rate of 4-cell embryos was significantly higher than that in blank M16 medium (control group, the difference was significant. Conclusion: The up-regulation of oocyte expression in B6C3F1 mice is mainly related to gene transcription regulation, antioxidant stress, protein synthesis and transport, amino acid phosphorylation modification, cell cycle, development and other functions. The results suggest that the regulation of autogene transcription, protein synthesis and transport, antioxidation and other functions in B6C3F1 mouse oocytes is more perfect. The difference in the expression of these genes in oocytes of mice with and without blocking may affect the developmental ability of mouse preimplantation oocytes and fibroblast growth factor receptors 1 and 3 in mouse oocytes and preimplantation embryos. The expression of FGF7 could promote the development of km mouse preimplantation embryos in vitro. The development ratio of 2-cell to 4-cell was significantly increased.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R321

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相關(guān)期刊論文 前2條

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本文編號：1810253