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
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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。
Objective:
To comparis the gene profiles in the MII eggs from blocking and non-blocking strain mice,and investigate the effects of FGF7 on the in vitro development of mouse preimplantation embryo.
Methods:
1. Mouse cDNA microarray (Affymetric GeneChip Mouse Genome 430 2.0) was applied to analyse the gene expression profiles of KM and B6C3F1 mouse eggs, and then Real-time PCR was used to validate some different genes.
2. Reverse transcription polymerase (RT-PCR), Immunofluorescence staining and confocal lascer scanning microscope were used to detect the expression and distribution of FGFR1, FGFR2 and FGFR3.
3. KM 1-cell embryos were cultured in M16 media which were added different concentrations of FGF7 protein. The effects of FGF7 on the development of preimplantation embryo in vitro were observed, and the percentage of mouse embryos developing from 2- to 4-cell stage was used as evaluation criteria.
Results:
1. Of 45037 genes in the mouse cDNA microarray, 3144 genes were differentially expressed between KM and B6C3F1 mouse MII oocytes, including 1818 up-regulated genes and 1326 down-regulated genes for B6C3F1 mice. We retain the gene which signal intensity exceed 200, containing 793 up-regulated genes and 202 down-regulated genes for B6C3F1 mice MII oocytes. These genes involved in regulation of gene transcription, oxidation reduction, protein biosynthesis, protein transport, protein amino acid phosphorylation, development processes were up-regulated in B6C3F1 mouse MII oocytes .
2. FGFR1, FGFR2 and FGFR3 mRNA were detectable in both the oocytes and the preimplantation embryos of KM mice by using RT-PCR. Immunofluorescence staining analysis showed that FGFR1 and FGFR3 were mainly distrubuted in the peripheral cytoplasm of the oocytes and preimplantation embryos. However, FGFR2 were located uniformly in the cytoplasm of the oocytes and preimplantation embryos.
3. KM 1-cell embryos were cultured in M16 media which were added by different concentrations of FGF7 protein. The percentages of mouse embryos developing from 2- to 4-cell stage had significant differences comparing to the control. In vitro, most of KM 1-cell embryos were arrested in 2-cell stages when cultured in M16 media, while most of KM 1-cell embryos could develop beyond 2-cell stage and to 4-cell in experimental group.
Conclusion:
These maternal genes involved in regulation of gene transcription, protein biosynthesis and transport, oxidation reduction, protein amino acid phosphorylation, development processes were up-regulated in B6C3F1 mouse MII oocytes, which might have an effect on the in vitro development of mouse preimplantation embryo. The expression of FGFR1, FGFR2, FGFR3 in oocytes and preimplantation embryo and the promotive effects of FGF7 on the in vitro development of KM mouse preimplantation embryo confirmed the effects of maternal genes on the development of mouse preimplantation embryo further.
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。
Objective:
To comparis the gene profiles in the MII eggs from blocking and non-blocking strain mice,and investigate the effects of FGF7 on the in vitro development of mouse preimplantation embryo.
Methods:
1. Mouse cDNA microarray (Affymetric GeneChip Mouse Genome 430 2.0) was applied to analyse the gene expression profiles of KM and B6C3F1 mouse eggs, and then Real-time PCR was used to validate some different genes.
2. Reverse transcription polymerase (RT-PCR), Immunofluorescence staining and confocal lascer scanning microscope were used to detect the expression and distribution of FGFR1, FGFR2 and FGFR3.
3. KM 1-cell embryos were cultured in M16 media which were added different concentrations of FGF7 protein. The effects of FGF7 on the development of preimplantation embryo in vitro were observed, and the percentage of mouse embryos developing from 2- to 4-cell stage was used as evaluation criteria.
Results:
1. Of 45037 genes in the mouse cDNA microarray, 3144 genes were differentially expressed between KM and B6C3F1 mouse MII oocytes, including 1818 up-regulated genes and 1326 down-regulated genes for B6C3F1 mice. We retain the gene which signal intensity exceed 200, containing 793 up-regulated genes and 202 down-regulated genes for B6C3F1 mice MII oocytes. These genes involved in regulation of gene transcription, oxidation reduction, protein biosynthesis, protein transport, protein amino acid phosphorylation, development processes were up-regulated in B6C3F1 mouse MII oocytes .
2. FGFR1, FGFR2 and FGFR3 mRNA were detectable in both the oocytes and the preimplantation embryos of KM mice by using RT-PCR. Immunofluorescence staining analysis showed that FGFR1 and FGFR3 were mainly distrubuted in the peripheral cytoplasm of the oocytes and preimplantation embryos. However, FGFR2 were located uniformly in the cytoplasm of the oocytes and preimplantation embryos.
3. KM 1-cell embryos were cultured in M16 media which were added by different concentrations of FGF7 protein. The percentages of mouse embryos developing from 2- to 4-cell stage had significant differences comparing to the control. In vitro, most of KM 1-cell embryos were arrested in 2-cell stages when cultured in M16 media, while most of KM 1-cell embryos could develop beyond 2-cell stage and to 4-cell in experimental group.
Conclusion:
These maternal genes involved in regulation of gene transcription, protein biosynthesis and transport, oxidation reduction, protein amino acid phosphorylation, development processes were up-regulated in B6C3F1 mouse MII oocytes, which might have an effect on the in vitro development of mouse preimplantation embryo. The expression of FGFR1, FGFR2, FGFR3 in oocytes and preimplantation embryo and the promotive effects of FGF7 on the in vitro development of KM mouse preimplantation embryo confirmed the effects of maternal genes on the development of mouse preimplantation embryo further.
引文
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