硼对雏鸵鸟免疫器官细胞凋亡信号通路的调节机制

Ostrich chicks represent a critical period of growth and development of the ostrich, but they often suffer a high mortality rate in livestock farm, which usually results from the trace elements. The immune function of ostrich chicks is closely related to the growth of immune organs. As thymus and spleen anf bursa of Fabricius are important immune organs, their development is closely related with the immunity. Our study has found that appropriate dose of boron could promote the development of multi-cellular organism and regulate the body’s immune function. Randomly divided into different groups, healthy ostriches were supplemented with different doses of boron concentration respectively. To investigate the mechanism of boron on the ostrich immune organ, studies in three aspects are carried out as follow: firstly, to study the regulation of boron on the genes (Foxn1, BMP2, BMP4) related to the immune organs development; secondly, to explore how boron participates in the inflammatory responses, mainly by means of MAPK signal path, stress-related proteins: HSP70/ HSP40 and PI3K/AKT signal path; thirdly, to investigate the mechanism of different doses of boron on the cell apoptosis, the RNA-seq of the ostrich chick thymus and spleen and bursa of Fabricius, from the result to found boron could regulate the cell apoptosis through the exogenous cell apoptosis signaling pathway. The present study mainly focused on the RNA-seq sequencing regulated signal path and Ca2+ regulated signal path. With molecular biological and other techniques above investigation, we would like to explore the regulating mechanism of boron on the ostrich chick thymus and spleen, and to provide the theoretical basis of boron involved in regulating the body’s immune function.

雏鸵鸟是鸵鸟生长发育的关键期，而微量元素缺乏等的影响，使育雏期鸵鸟大量死亡。鸵鸟免疫机能的强弱与育雏期免疫器官的发育有关。胸腺、脾和法氏囊是重要的免疫器官，其发育和功能与机体免疫力密切相关，而免疫器官机能的完善与雏鸵鸟的生长发育密切相关。我们研究发现适量的硼能促进动物机体的正常发育，调控机体的免疫。本课题选初生鸵鸟随机分组，饮水中添加不同剂量硼；饲喂至90日龄宰杀取胸腺、脾和法氏囊，从三方面研究硼对免疫器官的作用机制：①研究不同浓度的硼对免疫器官发育相关基因Foxn1、BMP2/BMP4的调节作用；②从MAPK信号通路，应激相关蛋白HSP70/HSP40和PI3K-AKT信号通路探讨硼参与免疫器官炎症反应的机制；③根据RNA转录组结果，探明硼参与免疫器官细胞凋亡机制。用分子生物学等多种技术探讨硼在鸵鸟胸腺、脾和法氏囊中的调节机制和作用机理，为深化硼参与调节机体免疫机能提供充分的依据。

采用形态学和分子生物学技术手段，系统研究了硼对雏鸵鸟免疫器官细胞凋亡信号通路的调节机制。研究结果如下：①饮水加适量的硼促进鸵鸟胸腺、脾和法氏囊的正常发育；过量的硼破坏鸵鸟免疫器官组织结构，诱发细胞凋亡，活化体caspase-3表达增强，抑制鸵鸟免疫器官发育。②鸵鸟Foxn1基因是高度保守基因。Foxn1阳性信号主要在胸腺髓质上皮细胞表达。80 mg/L硼促进Foxn1在胸腺内的表达和胸腺上皮细胞的分化，从而促进T细胞的发育，增强机体免疫力。高剂量硼显著抑制Foxn1蛋白的表达，导致胸腺结构的破坏和退化，抑制T细胞的生长发育，降低机体免疫力。③适量硼促进BMP2和BMP4在鸵鸟免疫器官的表达，高剂量硼抑制BMP2和BMP4的表达，造成T细胞的分化受阻，免疫器官发育异常，免疫机能下降。④用RNA-Seq首次对硼在鸵鸟免疫器官的作用做了高通量测序分析。硼对鸵鸟炎症及免疫机能相关信号通路的影响，通过MAPK信号通路，钙离子通路，B细胞受体通路，肌动蛋白调节信号通路，凋亡信号通路及癌症信号通路而实现。⑤硼对鸵鸟MAPK信号通路的调节，优先调控Ras-ERK通路和JNK通路，对p38MAPK通路的影响小。通过对MAPK信号通路活性的影响，调控T细胞的生长发育和机体免疫应答。硼对钙离子信号通路的影响集中在对钙-钙调磷酸酶-NFAT通路的调节，影响T细胞的生长发育。T、B细胞受体信号通路与MAPK信号通路，钙离子信号通路和肌动蛋白细胞骨架调节通路相关，硼参与调节T、B细胞受体信号通路的活性。高硼抑制Toll样受体信号通路的活性，使其功能下降，鸵鸟应对外界刺激的炎症应答及免疫应答降低。硼影响鸵鸟免疫器官热休克蛋白的表达，调节细胞凋亡，参与机体免疫及炎症应答。⑥硼对鸵鸟细胞凋亡通路的影响，主要调控TRAIL诱导的外源性细胞凋亡通路的活性，且硼参与凋亡抑制蛋白FLIP和IAP的活性调节。