垂体-甲状腺轴起源探索：文昌鱼促甲状腺素β亚基及其受体的相互作用和功能鉴定

The pituitary-thyroid axis, or thyroid-stimulating hormone (TSH)/TSH receptor (TSHR) system, has been identified in all vertebrate species including lamprey, however, its origin remains a mystery. Given that amphioxus has the Hatschek's pit, a pituitary-like organ, and the endostyle, a thyroid gland-like organ, we thus propose that a TSH/TSHR system might have emerged in this basal chordate. In accordance with our hypothesis above, TSHR gene has been identified in the genome database of Florida amphioxus. In addition, we have also identified a gene encoding β subunit of TSH (TSHβ) in the genome databases of Florida amphioxus and Xiamen amphioxus. However, functional characterization of both TSHβ and TSHR remains open in amphioxus. We have successfully cloned both TSHβ and TSHR genes from the amphioxus Branchiostoma japonicum. Next, we plan to perform the following experiments: (1) RT-PCR analysis and whole mount hybridization of TSHβ and TSHR genes in adult and embryos/larvae; (2) Recombinant expression and purification of TSHβ and TSHR proteins, and preparation of antibodies; (3) Immunohistochemical localization of TSHβ with TSHR; (4) Binding analysis of recombinant TSHβ with TSHR; (5) Activation of TSHR by recombinant TSHβ or/and fusion hormone GpA2/TSHβ; and (6) Examination in vivo of TSHβ and/or GpA2/TSHβ. Based on the experiments above, we will conclude if the TSH/TSHR system or pituitary-thyroid axis already emerged in amphioxus. This study will certainly enrich and deepen our understanding of the origin and evolution of endocrine system in vertebrates.

垂体-甲状腺轴即TSH（促甲状腺素）/TSHR（促甲状腺素受体）信号通路存在于包括七鳃鳗在内的所有脊椎动物中，但其起源和演化迄今仍是一个未解之谜。鉴于文昌鱼存在类似垂体的同源器官哈氏窝和类似甲状腺的同源器官内柱，因此我们推测TSH/TSHR信号通路可能已出现于文昌鱼中。有报道表明文昌鱼存在类似TSHR基因。新近，我们发现文昌鱼也有TSHβ亚基。但是，关于文昌鱼TSHβ和TSHR的功能一直缺乏研究。我们拟（1）采用原位杂交和免疫组化技术，研究TSHβ和TSHR组织分布和定位；（2）采用ELISA以及配体、受体和报告基因共转染技术，分析TSHβ和TSHR的互作及TSHβ和融合激素GpA2/TSHβTSHR的活化作用；（3）采用体内实验确立TSHβ和GpA2/TSHβ的生理功能。由此，确定文昌鱼是否存在类似脊椎动物的TSH/TSHR信号通路，进而描绘出脊椎动物垂体-甲状腺轴起源与演化图景。

垂体-甲状腺轴即TSH（促甲状腺素）/TSHR（促甲状腺素受体）信号通路存在于包括七鳃鳗在内的所有脊椎动物中，但其起源和演化迄今仍是一个未解之谜。鉴于文昌鱼存在类似垂体的同源器官哈氏窝和类似甲状腺的同源器官内柱，因此我们推测TSH/TSHR信号通路可能已出现于文昌鱼中。有报道表明文昌鱼存在类似TSHR基因。但是，关于文昌鱼TSHβ和TSHR的功能一直缺乏研究。我们首先克隆了文昌鱼thyrostimulin（GpA2/GpB5）以及TSHR基因全长，明确文昌鱼的gpa2/gpb5和tshr基因在脑泡、哈式窝和内柱等重要的神经内分泌器官有表达。体外重组表达蛋白thyrostimulin（GpA2/GpB5）和BjTSHR，发现二者能相互作用，且thyrostimulin在表达TSHR的细胞中能激活下游的PKA和PKC信号通路。thyrostimulin能诱导血液中T4水平的升高，提示其能刺激鱼类甲状腺激素的释放，具有类似脊椎动物TSH的功能效果。这些结果提示文昌鱼体内有功能性的thyrostimulin（GpA2/GpB5）-TSHR系统，其发挥着类似脊椎动物垂体-甲状腺轴的功能，并推测文昌鱼中存在着脑泡-哈式窝-内柱组成的原始甲状腺内分泌调节轴。并证明了文昌鱼中存在着功能性的Orexin-OrexinR系统，其发挥着类似脊椎动物调节能量代谢的功能，并推测文昌鱼的脑泡与脊椎动物的脑存在着进化上的同源关系。明确文昌鱼Miple是脊椎动物MK/PTN的祖先；还发现Miple具有广谱的抗菌活性。以上结果均提示文昌鱼中存在脑泡-哈式窝-内柱组成的原始的垂体-甲状腺内分泌调节轴。