斑马鱼促甲状腺激素生物节律的研究

Thyroid hormones (T4 and T3) produced by the thyroid gland play a critical role in animal growth, development,and metabolism. The activity of the thyroid gland is regulated predominantly by thyroid stimulating hormone (TSH) secreted by thyrotrope cells in the anterior pituitary gland. TSH is a glycoprotein complex composed of subunits α and β. It is the β subunit (TSHβ) that determines the specificity of TSH to its receptor. Previously, we characterized the zebrafish tshβ gene promoter, which harbors circadian regulation-related E-Boxes. However, molecular mechanisms underlying circadian regulation of tshβ and the effects of tshβ on expression of circadian clock genes as well as locomotor activities remain elusive. In this study, qPCR, in situ hybridization, and tshβ:luc transgenic fish lines will be employed to examine circadian expression of tshβ; in vitro cell transfection assays and a zebrafish circadian clock gene null mutant (per1b-/- ) will be used to examine the regulatory roles of circadian clock genes in tshβ expression; and tshβ-/- zebrafish generated via TALEN (transcription activator-like effector nuclease) will be used to examine the effects of tshβ on expression of circadian clock genes and locomotor rhythmicity . This study should provide invaluable insights into circadian regulation of tshβ and also clinical diagnosis and treatment for various human thyroid diseases.

甲状腺素在维持生物体生长，发育和新陈代谢等方面起到重要的调节作用,而主导调控甲状腺活性的则是促甲状腺激素（TSH）。TSH 是由α和β亚基构成的糖蛋白复合体，其中β亚基决定TSH对其受体的特异性。申请者已克隆和鉴定了斑马鱼tshβ基因的启动子，并且发现其拥有生物钟调控元件E-Box，但是TSH节律调控的分子机制以及TSH可能对生物节律的影响尚需深入研究。本研究拟采用qPCR和整体原位杂交技术以及构建tshβ:luc转基因斑马鱼检测TSH表达的节律的特征；通过体外细胞转染手段和斑马鱼生物钟基因无效突变体(per1b-/- )检验不同生物钟基因对tshβ的调控作用；利用TALEN构建tshβ-/- 突变鱼研究tshβ对生物钟基因表达和运动节律的影响。该研究将有助于深入理解TSH的生理功能和节律调节的作用机制，在临床上对甲状腺类疾病的诊断和治疗具有重大。

甲状腺滤泡上皮细胞所分泌的甲状腺素，对于生命体的生长，发育和代谢具有十分重要的作用。之前我们克隆了斑马鱼tshβ基因的启动子，启动子上具有节律调节的E-box元件，但是节律如何调控tshβ以及tshβ是否对节律具有反馈作用，这些都未知。在本研究中，我们利用了ELISA，定量PCR以及tshβ:luc转基因的方法发现在斑马鱼的幼鱼和成鱼中Tshβ，T4 和 T3都呈现明显的节律表达并且在ZT16达到最高峰。细胞转染和ChIP显示tshβ可以被Clock1a 和Bmal1b激活但是被Cry1ba抑制，同时在clock1a-/- , per2-/- 和 rev-erbα-/- 突变体中tshβ的表达受到了改变，这些都表明tshβ直接受到生物钟的调控。我们通过TALEN技术构建了tshβ-/-纯合突变体，发现在完全黑暗的条件下突变体的振幅比野生型对照要高，在正常光暗条件下突变体游动的距离比野生型对照要短，同时钟基因clock1a,clock1b,clock2,bmal1a,bmal1b,bmal2, per1a,per1b,per2, cry1ab,cry1ba,cry1bb, rorαa, nr1d1在突变体的表达有影响，这些都表明tshβ对节律调节的影响。突变体中下丘脑-垂体-甲状腺（HPT）轴的下游基因tra, trb, slc5a5, deio1 and deio也有明显的变化。本研究证明了在斑马鱼中tshβ直接受到生物钟的调控同时也反作用于生物节律