硼对雏鸵鸟骨和免疫器官生长发育的影响及其调控机制

Ostrich chicks are the little ostrich birds from hatching to 3 months old.They grow very fast with imperfect immune system and are very sensitive to the environmental change. Consequently, the incidence of leg-bone disease among ostrich chicks is up to 50% and the death rate is 30%, leading to big loss. At present, it is very urgent for the orstrich farming industry to find a way to improve ostrich chicks' immunity and boost the growth and development of their bones. According to our preliminary study, boron is very beneficial to bones and immune organs. Taking ostrich chicks as the test object, we adopt technologies such as immunohistochemical SABC, flow cytometry, ELISA, Radioimmunoassay(RI), In situ hybridization and colloidal gold immunological electron microscope to carry out a comprehensive study on the development and regulation of ostrich chicks' bones and immune organs from multiple aspects like hormone levels, nucleic acid levels, microscopic and ultrastructural levels. During the breeding practice of ostriches, the appropriate supplemental amount of boron and the appropriate time to add boron are found out, providing an effective way in auxology and immunology to improve ostrich chicks' immunity and reduce leg-bone disease. As a result, it not only offers theoretical basis for both the disease prevention and control of ostrich chicks and their production practice, but also enriches and develops scientific theories of morphology, immunology and developmental biology. Besides, it is very referential for the prevention and control of osteoporosis among the middle and old-aged human beings. There is no doubt that it has important academic and practical value.

雏鸵鸟是指出壳到3月龄的幼鸟，因生长速度快、免疫系统发育不完善，对环境变化等应激反应敏感，所以雏鸟腿骨病发病率高达50%，死亡率高达30%，损失惨重。因此提高雏鸵鸟免疫力，改善骨的生长发育，已成为目前鸵鸟养殖业急需攻克的难题。我们前期的研究结果发现添加硼对骨和免疫器官有重要作用。因此以雏鸵鸟为试验动物，采用免疫组化SABC、流式细胞术、ELISA、放射免疫、原位杂交和胶体金免疫电镜等多种技术，从激素水平、核酸水平、显微和超微结构水平，对雏鸵鸟骨和免疫器官的生长发育及调控进行多角度、多层次的综合研究，结合鸵鸟生产实践，探明硼的适宜添加剂量和添加时间，从发育学、免疫学角度探明提高雏鸵鸟免疫力和降低腿骨病的有效途径，为雏鸵鸟的疾病防治和生产实践提供理论依据，丰富和发展形态学、免疫学、发育生物学等科学理论，并为人类中老年骨质疏松症的防治与研究提供参考依据，因而具有重要的学术价值和实际应用价值。

雏鸵鸟生长快，免疫功能不完善，对环境变化敏感，腿骨病发病率高，死亡率高，损失严重。提高雏鸵鸟免疫力，改善骨的生长有重要意义。选刚出壳非洲雏鸵鸟分6组，饮水中添加不同剂量的硼酸，A组0mg/L、B组40mg/L、C组80mg/L、D组160mg/L、E组320mg/L、F组640mg/L，在45和90天取腿骨、胸腺、脾和法式囊，采用组织学、免疫组化和分子生物学综合技术做多层面的研究。结果如下：. 与对照组A相比，添加硼的B组、C组、D组的雏鸵鸟腿骨小梁区域的宽度明显增加，骨髓腔增大，骨小梁周长、骨小梁面积、骨小梁相对体积显著增大，其中D组达到最大值，骨小梁分离度最小，显著低于A、B组，与C组差异不显著。D组骨小梁的表面成骨细胞和破骨细胞数量最多；而E组和F组在钙化的软骨基质区域有些软骨细胞逐渐变成空泡状，出现核缩现象，最后退化死亡。TUNEL技术检测腿骨中细胞凋亡显示，45d后随硼添加剂量的加大，细胞凋亡渐增，大于80mg/L时，随硼剂量增加而促进凋亡细胞增多，高剂量320-640mg/L时细胞凋亡量最大。用ELISA检测血清内骨钙素和糖皮质激素的变化，结果显示随硼添加剂量的加大，在雏鸵鸟血清中的BGP水平呈先升高再降低的趋势。. 采用RNA-Seq技术检测硼在鸵鸟胸腺免疫应答反应中的作用，选A组、C组和F组做转录组学分析，结果显示：①三个文库的Clean Data占reads总数的比例分别为98.61%，98.60%和98.68%。②A组和F组，C组和F组，A和C组差异基因比较总计分别有2044个，1085个和902个。③所有的差异基因共分为7种表达趋势，其中趋势0，趋势1和趋势3具有差异显著性。④KEGG信号通路富集分析结果显示：细胞因子-受体互作通路，癌症通路，钙离子信号通路，肌动蛋白细胞骨架调节通路和MAPK信号通路显著富集，这些通路主要参与机体的免疫应答；除上述通路外，硼还能参与TLR信号通路，B/T细胞受体通路和凋亡通路的调节。综上所述，硼对鸵鸟机体免疫机能调控的相关通路，主要有MAPK信号通路，钙离子通路，B细胞受体通路，T细胞受体通路，肌动蛋白调节信号通路，TLR信号通路，凋亡信号通路及癌症信号通路。