神经粘附分子NB3和CHL1在高原低氧脑损伤中的作用和机制

Cerebral hypoxic damage is an important patho-physiological mechanism induced by special environmental factors such as plateau and high pressure. The damage mainly presents as cerebral edema, neuron degeneration and regeneration repair and so on. There are currently no effective intervention measures. NB3 and CHL1 is a pair of mutually regulated neural adhesion molecules, belonging to the immunoglobulin super-family. We reported for the first time the following findings: NB3 deficiency resulted in aggravation of cerebral hypoxia injury, while CHL1 deficiency caused mitigation of cerebral hypoxia injury; NB3 and CHL1 were distributed over different brain tissues in different cell types, but their expression was down regulated in the brain after hypoxia-treatment; CHL1 content in the human serum was increased after hypoxia-treatment. The current work focuses on the hypothesis that "NB3 / CHL1 have different regulating molecular and cellular mechanism in hypoxic brain damage". To investigate the mutual control of NB3/CHL1, we prepared in vivo NB3 / CHL1 double knockout animals. At the cellular level, their cellular and sub-cellular distributions were examined; the roles of their key target cells were assayed by using E - G - N 3D culture techniques. At the molecular level, the cell surface interaction between NB3 and CHL1 and their downstream signaling pathways in cells were studied. For practical purposes, we examined the level of NB3 and CHL1 in the serum from patients with cerebral brain injury. By recourse to the existing anti-hypoxia compound libraries, drug monomers with regulating NB3 / CHL1 expression were obtained. This initial study of the role of cell adhesion molecules and the mechanism involved in hypoxic brain damage is expected to be instrumental in developing new warning and intervention measures.

低氧造成的脑水肿和神经元变性是高原等特殊环境脑损伤的共性问题。我们研究发现NB3/CHL1这对相互调控的细胞粘附分子在低氧脑损伤中有重要作用： ①它们的表达均受低氧调控；②它们在神经-血管单元的不同类型细胞中分布不同；③NB3敲除加重而CHL1敲除减轻低氧脑损伤；④低氧后人血清中CHL1含量上升。本工作以“NB3/CHL1在低氧脑损伤中的作用与机制”为科学问题。主要研究内容：①以NB3/CHL1双敲除动物进一步验证它们的作用和相互调控；②利用E-G-N 3D培养技术等研究它们作用的关键靶细胞；③利用细胞共培养，研究它们的同嗜、异嗜作用，以及相互调节；④利用生物信息学预测和分子生物方法，研究NB3与Notch、CHL1与Nrp1等相互作用及下游信号通路；⑤验证高原病患者血清CHL1含量变化的临床意义；本工作首次研究细胞粘附分子对低氧脑损伤的作用与调控机制，有望发展新的预警与干预措施。

低氧造成的脑水肿和神经元病变是高原等特殊环境致脑损伤的共性问题。神经粘附分子NB3和CHL1 (Close Homologue of L1)属于免疫球蛋白超家族中的成员，具有相似的胞外结构，同源性大于50%。我们前期的研究发现NB3或CHL1缺失小鼠在急性低氧条件下的对脑损伤的作用不同：提示NB3可能是脑水肿和血脑屏障的内源性保护因子；而CHL1可能是脑损伤的负调控因子。因此，本项目以“神经粘附分子NB3/CHL1在低氧脑损伤中的不同作用及机制”为科学问题。在整体水平上制备NB3/CHL1敲除动物，进一步验证二者在高原低氧下的作用和功能上的相互调控；在细胞水平研究它们作用的关键靶细胞，并利用分子生物学等方法探讨其作用机制。主要结果如下：（1）建立了高原低氧脑损伤的小鼠动物模型；阐明了炎症协同低氧加重高原脑水肿的发生及其调节机制。（2）阐明了神经粘附分子NB3在高原低氧性脑损伤中作用和细胞分子机制。我们首次发现NB3在脑血管内皮细胞中表达，NB3的缺失通过调节血脑屏障的功能的完整性，增加低氧下BBB的通透性，而加重脑水肿的发生。（3）阐明了神经粘附分子CHL1通过调节星型胶质细胞减轻脑内的炎症反应而减轻高原低氧脑损伤。研究发现了LPS刺激明显上调星形胶质细胞CHL1的表达水平，CHL1通过亲同性作用调节星型胶质细胞炎症反应；CHL1缺失下调LPS刺激下星形胶质细胞炎症因子水平；CHL1敲除能够明显减少小鼠高原低氧模型下小胶质细胞的活化，以及脑海马组织中炎症分子如IL1、IL6、TNFα的表达；LPS以及高原低氧复合LPS 处理均能够上调血清中炎性相关因子，而CHL1敲除能够减轻高原低氧暴露后小鼠血清中的炎性反应。（4）同时拓展研究了CHL1在肠道炎症中的调节作用。研究发现神经粘附分子CHL1在肠道组织中表达，CHL1在炎症性肠病时表达明显升高；CHL1缺失后通过增强炎症细胞的浸润和肠道的通透性，加重炎症性肠病的发生；该研究为炎症性肠病的干预提供了新的思路。本工作首次研究细胞粘附分子对低氧脑损伤的作用与调控机制，有望发展新的预警与干预措施。

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