阻滞与非阻滞品系小鼠次级卵母细胞基因表达水平的比较及FGF7对小鼠植入前胚发育的影响

发布时间：2018-04-27 09:54

本文选题：基因芯片 + 母源基因　；参考：《福建医科大学》2010年硕士论文

【摘要】： 目的: 检测阻滞品系(昆明)小鼠与非阻滞品系(B6C3F1)小鼠次级卵母细胞基因表达水平的差异;观察成纤维细胞生长因子7(fibroblast growth factor 7,FGF7)对KM小鼠植入前胚发育和克服2-细胞阻滞的作用。方法: 1.Affymetrix表达谱芯片检测比较KM与B6C3F1小鼠次级卵母细胞基因表达差异,并运用Real time PCR对部分差异基因进行验证; 2.RT-PCR和免疫荧光细胞化学显色方法检测成纤维细胞生长因子受体(fibroblast growth factor receptor,FGFR)-1、2和3在KM小鼠卵母细胞和植入前胚中的表达与分布; 3.收集KM小鼠1-细胞胚,采用微滴培养法连续培养,观察在M16培养液中添加FGF7蛋白对植入前胚发育的影响,观察其是否能克服2-细胞阻滞。结果: 1.基因芯片检测显示KM与B6C3F1小鼠卵母细胞内许多与基因转录、转录调节、蛋白质合成等功能相关的基因表达存在差异;Real time PCR验证结果与芯片检测结果较为相符,证明本次芯片检测结果较为可靠。 2.RT-PCR检测显示,FGFR1、FGFR2和FGFR3 mRNA在KM小鼠卵母细胞和植入前胚均有表达;免疫荧光细胞化学结合共聚焦扫描显微镜观察显示FGFR1、FGFR3免疫阳性反应见于卵母细胞和植入前胚胞质周边,靠近细胞膜;FGFR2阳性反应较均匀分布于卵母细胞和植入前胚的胞质。 3.KM小鼠1-细胞胚在添加FGF7蛋白的M16培养液中培养,其4-细胞胚发育率明显高于空白M16培养液(对照组),差异有显著性意义。结论: B6C3F1小鼠卵母细胞表达上调基因主要与基因转录调节、抗氧化应激、蛋白质合成与转运、氨基酸磷酸化修饰、细胞周期、发育等功能相关,提示B6C3F1小鼠卵母细胞内与自身基因转录、蛋白质合成与转运、抗氧化等功能相关的调节更为完善,阻滞与非阻滞品系小鼠卵母细胞内这些母源基因表达差异可能影响小鼠植入前胚体外发育能力;KM小鼠卵母细胞和植入前胚内有成纤维细胞生长因子受体1、2和3的表达;FGF7可促进KM小鼠植入前胚体外发育,显著提高2-细胞至4-细胞的发育比率。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R321

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