初步探索孕酮促进创伤性脑损伤血管生成的分子调控机制

There is no effective drug for the traumatic brain injury(TBI) even after world wide hard exploration for more than five decades. Progesterone is the first drug approved by NIH to be applied in the III phase clinical trial for TBI in more than 30 years research. Thus the research of progesterone on TBI becomes one of the hot issues in TBI field. In our previous study, we have conifirmed that progesterone is able to improve TBI by stimulating angiogenesis in the injured brain tissue.Based on our TBI rat model, we plan to detect the correlationship between progesterone and both of the angiogenesis related VEGF/Notch signal pathway and miRNAs(miRNA-120, miRNA-200 and miRNA-320). Whether the porgesterone is combined with Notch receptors will be also explored. An in vitro endothelial progenitor cells(EPCs) culture model will be established and the cDNA transfection, siRNA turning down, miRNA mimics and inhibitors are all adopted to modulate the gain and loss function of VEGF/Notch signals and angiogenesis related miRNAs in the EPCs.Both the matril gel based in vitro angiogenesis assay system and hopping probe ion conductance microscopy- patch-clamp system are established to assay the effect of the progesterone and the expression level change of VEGF/Notch signals and angiogenesis related miRNAs on EPCs functional phenotype and surface potential. A 30 cases of clinical volunteer trial will be processed to confirm the therapeutic effect of progesterone on both of the severe TBI patients and their angiogenesis related moleculars. This grant proposal is designed to provied the more comprehensive molecular basis materials than before in order to modify and improve the progesterone treatment in TBI.

多年来研究者一直潜心寻找治疗颅脑创伤（TBI）的有效药物，但一直未获理想结果，而孕酮是30年来首个获得NIH巨资资助进行TBI临床实验的药物，成为当今TBI研究新热点。本项目在前期率先证实孕酮可促进损伤脑组织血管生成从而促进神经修复基础上，拟建立TBI大鼠模型，检测孕酮与血管生成相关VEGF/Notch信号通路、miRNA-120，miRNA-200和miRNA320等分子群的相关关系、是否与Notch受体结合？建立大鼠内皮祖细胞体外模型，人工调控VEGF/Notch信号和miRNA表达增强和减弱后与孕酮孵育，以基质血管形成模型、离子电导显微镜-膜片钳系统检测孕酮以及血管生成相关信号分子群调控对内皮祖细胞功能形态和表面电位的影响，确定孕酮与血管生成相关分子群变化在血管生成中的作用。最后以临床志愿者实验验证孕酮对重型TBI的疗效和对血管生成相关分子群的影响，为优化孕酮的TBI应用提供依据。

孕酮是一种雌性体内主要激素，具有脂溶性，能透过血脑屏障，在颅内发挥作用。体内实验证实了孕酮不仅能明显促进大鼠脑外伤后神经功能恢复，还能明显减轻脑外伤后大鼠血脑屏障的破坏及显著减轻脑水肿，同时促进大鼠TBI后血脑屏障的修复和完整性。在体外实验中，孕酮能增加DLL4蛋白和Notch1蛋白的表达，短期使用还能促进EPC的增殖能力及表达CXCL12和pAkt的能力；使用相应受体阻滞剂则能抵消孕酮的效果；孕酮还能调控EPC细胞的成管能力、迁移能力、粘附能力及分泌VEGF的能力，在孕酮剂量为10-9M时效果最佳。在探索脑外伤新型治疗策略的过程中，我们发现Fingolimod作为一种新型的免疫调控剂，能透过血脑屏障在颅内发挥免疫调控作用。应用Fingolimod干预能显著提高TBI小鼠的学习及空间记忆能力，同时改善TBI引起的小鼠运动功能缺陷。TBI 小鼠外伤后3天，使用Fingolimod干预能减少脑组织过多的淋巴细胞浸润，同时增加抗炎的Treg细胞含量；高通量细胞因子芯片结果显示TBI后1天损伤灶周围即有多达20种细胞因子以及趋化因子积聚，并在伤后3天达到高峰，14天时逐渐减少，使用Fingolimod干预则可以减少多种细胞因子以及趋化因子积聚，并在3天达到最大效果。因此，Fingolimod可以广泛调节TBI后多种免疫炎性反应，进而改善TBI后神经功能缺陷，提示Fingolimod可能成为TBI治疗的新策略。