ATP经P2X4受体介导在全脑缺血再灌注损伤中的作用及机制的研究

The ischemic cerebral paralysis is the most common disease, which threatens human health severely.The important treatment is the restoration of local blood supply as soon as possible. But the neurons will further be injured in the ischemic region after restroration of blood supply, which is designated as cerebral ischemia reperfusion injury(CIRI). The mechanisms of CIRI are very complicated. The previous data showed that accumulation of extracellular toxins such as excitable amino acid, ATP, neuronal calcium overloading and radical generation etc are involved in the processes of CIRI. The previous data demonstrated that CIRI are often observed in CA1 region of hippocampus, cerebral cortex and striatum. Our preliminary data showed that the pyramidal neurons being apoptosis expressed high level of P2X4 receptor in the hippocampal CA1 region after global ischemia and reperfusion; the blockage of P2X4 receptor by 5BDBD(a selective antagonist for P2X4 ) could partly inhibited the apoptosis in this region; some of the neurons over-expressing P2X4 receptor via virus vectors in the CA1 region are also observed to be apoptosis. Those preliminary data of ours clearly indicated that up-regulation of P2X4 receptor after global ischemia and reperfusion is involved in the CIRI of hippocampal CA1 region. This research project will further investigate the mechanisms of CIRI induced by extracelluar ATP via P2X4 receptor by the following techniques such as tissue microanalysis, western blot, calcium assay and immunohistochemistry etc. The data from this reseach project will be useful for the treatment of the ischemic cerebral paralysis.

缺血性脑中风是目前严重威胁人类健康的常见疾病。治疗原则是尽快恢复缺血区血供。然而缺血后再恢复血供往往会造成神经元的进一步损伤即脑缺血再灌注损伤（CIRI)。CIRI形成机制极复杂，文献提示可能与细胞外毒性物质(兴奋性氨基酸、细胞外ATP)聚集、细胞内钙离子超载、自由基生成等因素有关。动物实验发现全脑缺血再灌注后会诱发海马、大脑皮质和纹状体等区域发生CIRI发生。我们发现缺血再灌注后48小时正在凋亡的海马CA1区神经元高表达P2X4受体；P2X4受体拮抗剂可部分抑制神经元凋亡并改善损伤后动物的学习记忆功能；病毒载体使海马CA1神经元过表达P2X4受体后神经元出现凋亡。上述预实验清楚地说明海马CA1的CIRI与P2X4受体上调相关。本实验将通过组织微透析、蛋白印迹、钙离子测定、免疫组织化学等技术进一步研究细胞外ATP通过P2X4受体诱发CIRI的分子机制,为缺血性脑中风治疗提供有益的理论依据

脑中风的发病率、死亡率和致残率都相当高，是全球人口死亡和致残的首要原因，是一类严重危害人类健康的疾病。缺血性中风的治疗原则一般是及时恢复缺血区的血液供应。但是缺血区再次血流灌注后，却会使缺血区的脑功能和神经元遭受进一步损坏，这种损伤机制复杂。本课题的主要研究进展，重要结果和关键数据：1、建立四血管结扎全脑缺血再灌注模型。我们实验室已经建立了稳定的四血管结扎全脑缺血再灌注模型，成功率在90%以上。建立了IRI后微透ATP分析技术，发现ATP浓度在缺血再灌注后逐渐升高，48小时到达高峰，此后下降4天后基本与对照相似，这一重要结果提示ATP在脑IRI中起一定作用。2、用不同的技术显示缺血后大鼠海马区锥体细胞P2X4 受体表达上调，这些P2X4受体上调的神经元 正是凋亡的神经元。 3，海马注射P2X4受体拮抗剂（5BDBD）可将少海马区神经元死亡， 凋亡的神经元数目减少。4，应用AAV病毒，建立了稳定P2X4受体在海马锥体细胞过表达动物，发现海马区过表达P2X4受体可促进海马区锥体神经元的凋亡，并使其神经元死亡时间推前至IRI后24小时。 5，体外实验证明ATP可通过P2X4受体使神经元内钙信号升高，P2X4的拮抗剂可明显抑制这一钙信号。6， 脑IRI后5BDBD 可明显改善动物的学习记忆行为。 7，高通量基因表达芯片结果显示炎性反应因子，钙信通路分子参与了脑IRI的发生发展过程。此外，我们还继续了嘌呤受体的相关研究，发现：ATP通过P2X7受体参与了脑外伤后由小胶质细胞的激活，微泡的释放参与局部炎症和继发性神经细胞损伤过程；ATP通过P2X7受体参与了周围神经损伤修复过程中施万细胞的增殖过程， 这一过程是通过MAPK / ERK细胞内信号传导途径。在基金资助下还建立了一套多色荧光原位杂交技术，可同时显示三种不同的mRNAs，为研究者提供了快速高效mRNA原位检测技术。本科题这些重要结果和关键数据表明ATP通过P2X4受体参与了脑缺血后神经组织继发性损伤过程，为临床治疗提供了靶点，所以P2X4受体拮抗剂的开发应用具有很好的临床应用前景。