【摘要】 目的:检测生长分化因子5在小鼠肢芽组织的表达,分析该基因在肢体骨骼发育中的表达变化,从而进一步认识生长分化因子5基因在肢体发育过程中的作用。方法:实验于2005-05/07在华中科技大学同济医学院附属协和医院骨科实验室完成。选取清洁级2～3月龄昆明种小鼠30只,雌性20只,雄性10只,于每天下午6时按雌雄2∶1合笼,次日晨8时取出,查见阴道栓者定为孕期第0.5天。分别在胚胎11.5,12.5,13.5,14.5,15.5d颈椎脱臼处死孕鼠,无菌条件下分离出胚胎,解剖显微镜下用眼科剪取肢芽组织备用。分别采用反转录聚合酶链反应和Westernblotting免疫印迹法检测生长分化因子5在小鼠胚胎发育过程中肢芽组织mRNA和蛋白表达的变化。结果:①小鼠胚胎肢芽组织生长分化因子5mRNA的表达:孕11.5,12.5,13.5,14.5,15.5d胚胎肢芽组织均可见一条219bp的生长分化因子5特异扩增条带,其与β-actin吸光度的比值分别为0.521±0.023,1.210±0.014,1.221±0.008,0.556±0.014,0.510±0.019。孕12.5d和孕13.5d生长分化因子5mRNA的相对含量与孕11.5,14.5,15.5d相比有极显著性差异(P<0.01)。②小鼠胚胎肢芽组织生长分化因子5蛋白的表达规律:孕11.5,12.5,13.5,14.5,15.5d胚胎肢芽组织均观察到相对分子质量为13700的蛋白条带,其与β-actin蛋白吸光度的比值分别为0.589±0.021,1.103±0.124,1.112±0.041,0.601±0.015,0.552±0.006。孕12.5d和孕13.5d生长分化因子5mRNA的相对含量与孕11.5,14.5,15.5d相比有极显著性差异(P<0.01)。结论:生长分化因子5mRNA及其蛋白表达的变化趋势一致,在小鼠肢体骨骼发育早期阶段持续表达,孕12.5d和孕13.5d呈高表达,继而减弱。表明生长分化因子5是肢体骨骼发育早期的一个关键因子,在小鼠肢体骨骼发育和关节形成中具有重要作用。更多还原

【Abstract】 AIM To detect the expression of growth differentiation factor 5 GDF5 in limb bud tissues analyze the expression trend of GDF5 in limb skeletogenesis so as to recognize effect of GDF5 during limb growth of mice.METHODS The experiment was conducted at the orthopedic lab Union Hospital Tongji Medical College Huazhong University of Science and Technology from May to July in 2005. Thirty clean-grade Kunming mice aged 2-3 months were selected composed of 20 females and 10 males and put into cages at 6 00 PM every afternoon according to the proportion of 2 to 1 and taken out of the cages at 8 00 in the following morning. The day a vaginal plug was found was considered as embryonic day 0.5 E 0.5. At E 11.5 E 12.5 E 13.5 E 14.5 and E 15.5 cervical vertebra luxation the pregnant mice were killed respectively. Embryos were removed and kept in reserve for using with ophthalmic scissors under anatomic microscope. The changes of the expression of GDF-5 mRNA and protein of mouse fetal limb buds were detected with RT-PCR and Western blotting respectively. RESULTS ①Expression of GDF-5 mRNA in mouse embryonic limb buds A 219bp of specific electrophoretic strip was observed in every limb bud at pregnant for E11.5 E 12.5 E 13.5 E 14.5 and E 15.5 and absorbance A ratio of GDF5/β-actin was 0.521±0.023 1.210±0.014 1.221±0.008 0.556±0.014 0.510±0.019 respectively. The expression of GDF-5mRNA at pregnant for E12.5 and E 13.5 had extremely significant difference as compared with that at pregnant for E 11. 5 E 14.5 and E 15.5 (P < 0.01). ②Expressive rule of GDF5 protein in the embryonic limb bud tissue of mice Protein strip with relative molecule mass of 13 700 was found in E 11.5 E 12.5 E 13.5 E 14.5 and E 15.5 limb bud tissue and the ratio with β-actin protein A was 0.589±0.021,1.103±0.124,1.112±0.041,0.601±0.015,0.552±0.006 respectively. The relative content at E 12.5 and E 13.5 GDF5 mRNA had extremely significant difference as compared with the E 11.5 E 14.5 and E 15.5 (P < 0.01=. CONCLUSION During early stage of limb skeletogenesis of mice the expression of GDF-5mRNA and protein has the same tendency and is constant highlights at embryos E 12.5 and E 13.5 subsequently dropped. It suggests that GDF5 is a key factor in early bone growth and plays an important role in limb skeletal development and joint formation.更多还原