扎里奴思方调控血脑屏障开放对脑缺血骨髓间充质干细胞移植由血管迁移脑内的影响

Zha li nu si recipe（ZLNS）han been recorded in the book of Hui Hui Medicine and Recipe, which could perform the effect of orifice-opening with aroma on stroke. In prior study, it has been confirmed that ZLNS recipe could perform the significant role in the treatment for cerebral infarct. Being in the view of the effect that medicines of orifice-opening with aroma could regulate the opening of brain blood barrier (BBB), rats were used to explore the influence of ZLNS recipe on the regulation of BBB opening and then to promote the grafting of bone marrow mesenchymal stem cells (BMSCs) from vascular to brain tissue after transplantation through artery in this study，as well to explore the role of BMSCs in migration to cerebral ischemic brain tissue and the differentiation of BMSCs to neurones. The ways of BMSCs migration and the mechanisms of differentiation would also be explored in this study. By this rearch, the questions of the insufficience of BMSCs getting into brain and the limitation of it' therapy effect on stroke all might be solved in some degree. Rats BMSCs were acquired through cultivation and labelled by Brdu before transplantation, and then to be transplanted through rats internal carotid artery. Rats' cerebral ischemic model of middle cerebral artery occlusion (MCAO) was duplicated and the brain were taken at different time after BMSCs transplantation. As the dye of material, EB contents in rats' brain were tested in the study, as well to test the expressions of P-Glycoprotein (P-gP) and aquaporin 4 (AQP4), by which the opening of BBB could be observed. The immunohistochemical double markng method was used to explore BMSCs being labelled by Brdu, and then to observe the grafting and attribution of BMSCs in the brain blood vessel and brain tissue. The expressions of Occludin and Claudin-5 which were the tight proteins of brain blood vessel, the signal of Integrinα4 and vascular cell adhesion molecule 1 (VCAM-1) were tested by the method of RT-PCR in order to clarify the ways of regulation effect on BBB opening. The neuronalnu-doantigen (NeuN) and growth association protein (GAP-43) mRNA would be tested in the study in order to explore the synapse remodeling of neuronalcyte. The actives of neural growth factor (NGF) and glial cell line derived neurotrophic factor (GDNF), Nogo-A and myelin associated glycoprotein (MAG ) were tested, and then to explore the mechanisms of the effects of ZLNS recipe being combined with BMSCs transplantation on regenaration of neurone after cerebral ischemia. The results of this study could provide the grounds for the use of the combination of two methods in the treatment for cerebral infarct.

扎里奴思方出自《回回药方》，具有芳香开窍功效，用于治疗中风。前期研究证实，其治疗脑梗死的作用明显。基于芳香开窍药对血脑屏障开放（BBB）的作用，课题拟以大鼠为实验对象，研究本方调控BBB开放以促进骨髓间充质干细胞（BMSCs）由动脉移植经BBB进入脑内，并向缺血组织迁徙和向神经元分化的作用和机制，以解决BMSCs进入脑内数量不足和疗效受限问题。体外培养和标记BMSCs；颈动脉移植BMSCs；分时间取脑组织，测EB含量、P-gP和AQP4表达，以观察BBB开放；测脑内Brdu标记细胞，以观察BMSCs迁徙和归巢；测Occludin和Claudin-5、Integrinα4和 VCAM-1信号表达，以明确调控BBB开放的途径；测NeuN、GAP-43，以观察神经元分化；测神经营养因子（NGF、GDNF）和再生抑制因子（Nogo-A、MAG）活性，以明确其作用机制，为二者联合治疗脑梗死提供依据。

骨髓间充质干细胞移植(BMSCs)是治疗脑缺血的重要方法，但存在移植后干细胞通过血脑屏障(BBB)通透性迁徙和成活的问题，芳香开窍类是否可通过对BBB开放的调控而增强其脑保护作用，有待开展研究。本研究以大鼠为研究对象，以脑缺血后BBB通透性改变为切入点，联合应用扎里奴思方（扎方）和BMSCs移植，从脑缺血后不同时间大鼠神经功能、脑水肿、脑梗死及脑组织病理变化等探讨对脑缺血损伤的保护作用；从对BBB通透性变化的观察及其机制分析，明确扎方对脑缺血后BMSCs移植的促进作用；从对移植后的BMSCs向神经细胞分化及分泌神经营养因子的观察研究联合对促进脑缺血后神经再生修复的影响。研究发现，扎方联合BMSCs移植对脑缺血保护作用明显，大鼠神经功能改善，以14d的评测分值增高显著（15.80±0.79）。扎方可通过调控BBB通透性而促进BMSCs由血管迁移脑内，检测脑内EB含量，各组均呈先增高后减低趋势，其中联合组14d的EB降低尤为显著（11.73±0.79）；联合14d组AQP4降低显著（89.17±1.62）；脑组织P-gp含量测定，联合组14d降低显著（120.41±2.13）。扎方可增强脑缺血再灌注损伤移植的BMSCs向神经样细胞的分化，移植及联合各组Brdu/NSE（162.49±2.21）、Brdu/GFAP（136.10±4.00）、Brdu/NGF（62.5±12.7）×102表达增高。研究认为，扎里奴思方能有效调控脑缺血损伤BBB通透性，促进骨BMSCs经由血管移植脑内以发挥神经保护作用，其机制可能与干预P-gp、AQP4、VCAM-1、Integrin α4、Occludin和Claudin-5动态表达有关；扎方可促进脑缺血骨BMSCs移植后向神经样细胞分化，并分泌神经营养因子，促进脑缺血后神经再生修复。