SOST基因在淇河鲫肌间骨骨化过程中的表达研究

The mRNA and protein expression of gene SOST during ossification process of intermuscular bone in crucian carp(Carassius auratus) in Qihe River

为探索硬化蛋白(sclerostin,SOST)基因在淇河鲫肌间骨骨化过程中的调控作用,本研究以淇河鲫仔稚鱼为研究对象,利用整体骨骼染色法对肌间骨的形态发生进行观察,并采用q RT-PCR、Western Blot和免疫组织化学技术检测SOST基因在肌间骨不同骨化阶段的m RNA和蛋白表达变化情况。结果显示,淇河鲫发育至24 dpf(day post fertilization),肌膈中出现纤维束;25 dpf髓弓小骨出现,28 dpf脉弓小骨出现,33 dpf髓弓小骨出现分叉,45 dpf肌间小骨发育完全。SOST基因在肌间骨骨化发生各阶段均有表达,23 dpf的表达量最低(P<0.05),随着肌间小骨的骨化发育,SOST基因呈递增趋势,45 dpf表达量显著高于其他骨化阶段(P<0.05);SOST蛋白在45 dpf时显著高于23和27 dpf(P<0.05),与其他发育阶段无显著差异(P>0.05)。SOST m RNA和蛋白变化趋势基本相同,都在23dpf处于最低,肌间骨全部出现后表达量达到最高,与淇河鲫肌间骨的形态发生趋势一致。因此,推测SOST与淇河鲫肌间骨骨化具有一定的相关性,可调控肌间骨的分化和形成。

To explore the function of gene SOST in intermuscular bone ossification of Carassius auratus in Qihe River, a modified whole bone staining method was firstly used to investigate ossification development of intermuscular bone. Then, the q RT-PCR, Western Blot and immunohistochemistry methods were employed to detect the m RNA and protein expression of gene SOST in different ossification stages. The results showed that the ligaments were found in myoseptum at 24 dpf（day post fertilization）. The earliest epineural bones appeared at 25 dpf, and the epipleural bones were subsequently found at 28 dpf. At 33 dpf, the epineural bones began to bifurcate.Until 45 dpf, all intermuscular bones were fully developed in myoseptum. The gene SOST was expressed in various ossification stages of intermuscular bone and the lowest was at 23 dpf（P〈0.05）. The m RNA abundance of gene SOST was increased along with the ossification development and reached a maximum level at 45 dpf（P〈0.05）. The relative expression level of SOST protein at 45 dpf was significantly higher than at 23 and 27 dpf（P〈0.05）, but had no obvious differences with other ossification stages（P〉0.05）. The expression of gene SOST protein showed the similar trend with the m RNA and both of them varied along with the development process of intermuscular bone, which was at the lowest point at 23 dpf and climbed to the highest level when all the intermuscular bones appeared. Thus, we concluded that there were relationships between the gene SOST and ossification development of intermuscular bone, and the gene SOST may play an important role in morphology and ossification of intermuscular bone.