温度敏感型信号轴HSF1/SOCS3调控自发性脊髓再生的机制研究

Several poikilothermic animals are able to regenerate their injured spinal cord completely, with the regenerative speed depending on environmental temperature. The endurable higher temperature has been found to be beneficial for eliminating the effects of inhibitory molecules on axonal regrowth, but the underlying mechanism is unclear. The negative regulator SOCS3 plays important dual roles in inhibition of axonal regrowth and inflammation. Our previous works have shown that SOCS3 expression is deficient in the neurons of regenerating spinal cord following gecko tail amputation, but it is significantly upregulated in the microglia to inhibit inflammation mediated by GM-CSF and IFN-γ; a DNA binding element of thermosensory factor HSF1 has been found to locate in the upstream promoter of SOCS3, and luciferase reporter assay displays that HSF1 is capable of inhibiting the activity of SOCS3; the expression of HSF1 is upregulated by rise in temperature, and is distributed in the neurons and microglia. These preliminary data suggest that thermosensory HSF1/SOCS3 pathway regulates spontaneous spinal cord regeneration. To confirm such hypothesis, we are going to clarify how temperature regulates HSF1/SOCS3 axis. Then we attempt to understand whether the regulation of HSF1 on SOCS3 is through a trimerization interaction with promoter or cooperation with SIRT1, and what the active sites of HSF1 are. At last we will unveil the cellular effects of HSF1/SOCS3 pathway on neurons and microglia, as well as relative mechanisms. Our studies will provide helpful clues for repair of injured mammalian spinal cord.

变温成体脊椎动物在适宜环境温度下可以完全再生脊髓，且再生速度呈温度依赖性变化。适宜的高温可以消除轴突再生抑制分子的影响，其中的分子机制尚未阐明。关键负调控分子SOCS3发挥抑制轴突生长和炎症反应的双重作用。课题组前期研究发现，在多疣壁虎断尾脊髓再生过程中SOCS3在神经元中表达缺失但在炎症细胞中表达上调，抑制GM-CSF、IFN-γ介导的炎症反应；SOCS3启动子区存在热敏感因子HSF1结合元件，HSF1显著抑制SOCS3表达；HSF1定位于脊髓神经元和小胶细胞中，其表达水平呈温度依赖性变化。该结果提示温度敏感型信号轴HSF1/SOCS3调控壁虎脊髓再生。课题组拟进一步揭示温度与HSF1/SOCS3信号轴之间的相互关系；阐明HSF1对SOCS3的调控模式及关键活性位点（域）；明确HSF1/SOCS3信号轴对神经元和小胶细胞的生物学效应及其机制。该研究为高等哺乳动物脊髓损伤修复提供重要参考。

多疣壁虎自发性脊髓再生过程中，神经元再生相关基因启动再生程序调控轴突生长，同时胶质细胞独有的细胞特性限制再生抑制微环境的形成。哺乳动物脊髓损伤后SOCS3抑制轴突生长，但也发挥抑制炎症反应的功能。课题组研究发现，多疣壁虎SOCS3启动子含有热休克因子HSF1结合元件。HSF1在壁虎脊髓损伤后的神经元和小胶质细胞中表达上调，而SOCS3仅在小胶质细胞中表达增加。HSF1调控SOCS3促进自发性脊髓再生的机制尚不清楚。为了揭示其中的分子机制，课题组首先建立了爬行动物中枢神经元原代培养方法，为开展后续研究提供了关键的细胞工具。荧光素酶报告基因检测表明，HSF1具有抑制SOCS3表达的作用，通过激活存活和生长相关基因，调控壁虎神经元轴突再生。在炎症细胞中，HSF1以不依赖于SOCS3的调控方式抑制NFκB激活，控制炎症细胞的炎症反应。在此基础上，课题组比较了多疣壁虎（再生）和大鼠（非再生）脊髓星形胶质细胞干细胞Marker的表达，结果表明，成体壁虎脊髓星形胶质细胞和E18大鼠脊髓星形胶质细胞具有类似的干细胞表达谱，并对炎症刺激不产生激活响应，提示其在壁虎脊髓损伤后几乎不参与再生抑制微环境的形成。课题组进一步研究显示，HSF1在大鼠脊髓损伤后的星形胶质细胞中表达上调。体内和体外实验证明，HSF1发挥抑制星形胶质细胞向A1型转变的作用，HSF1的激活有利于减少胶质疤痕的形成。上述结果充分说明，HSF1的激活对不同动物脊髓损伤修复都具有促进作用。为了减轻脊髓损伤后血-脊髓屏障破坏导致的二次损伤，课题组首次成功制备了多疣壁虎凝血酶重组蛋白。采用不同底物检测酶活性发现，壁虎凝血酶具有超强的促凝血活性，且对中枢神经细胞不产生任何毒副作用。该壁虎凝血酶独特功能的发现，对临床治疗肝慢性出血和脑溢血等具有潜在的药物开发价值。