ALK1参与神经干细胞治疗缺血性脑卒中的相关机制研究

Stroke has high mortality and morbidity in human disease. But until now still no effective strategey against stroke caused central nervous system damage. Endothelial cells of the central nervous system after stroke can quickly response to ischemic signal to form the microenvironment which could promote nerve repair. ALK1 as one of the members of the TGF-beta receptors family was newly discovered which was found to play an important role in the development of blood vessels, but its related molecular mechanism is not yet clear. VEGF can promote the growth of nerve nutrition factor is known, and it is ALK1 targets, so we speculated that ALK1 in cerebral vascular reconstruction after stroke, and plays an important role in nerve regeneration. As a result, we proposed to build ALK1 and RNAi eukaryotic expression vector , promote ALK1 overexpression or silence in the environment of neural stem cells, observe its effect in the neurovascular unit, to explore its possible mechanisms of NSCs differentiation and neural axon regeneration, provide early basis of clinical therapeutic targeting toward ALK1.

脑卒中在人类疾病中有着较高死亡率及致残率。而迄今为止仍未有有效对抗脑卒中所导致中枢神经系统损伤的策略。卒中后中枢神经系统内皮细胞能够快速应答缺血性信号从而形成能够促进神经修复的微环境。ALK1作为TGF-β受体家族成员之一被新近发现在血管发育中起到重要作用，但它的相关分子机制尚未明确。VEGF是已知的能够促进神经生长营养因子，而它是ALK1的作用靶点，因此我们推测ALK1在脑卒中后脑血管重建及神经再生中起着重要作用。因此，我们拟构建ALK1的过表达及RNAi真核表达载体，使其在神经干细胞环境中过表达或抑制，观察其对神经血管单元功能的影响，探讨其对神经干细胞分化及轴突再生修复的可能作用机制，为ALK1作为临床治疗中风的干预手段提供前期依据。

脑卒中在人类疾病中有着较高死亡率及致残率，而缺血性脑卒中后促进神经再生及修复至今仍未有十分有效的策略。卒中后中枢神经系统内皮细胞能够快速应答缺血性信号从而形成能够促进神经修复的微环境。ALK1被新近发现在血管发育中具有重要作用，它对神经再生的作用尚不明确，VEGF是已知的能够促进神经生长的营养因子，而它正好是ALK1的作用靶点。本课题拟研究ALK1在缺血性脑卒中后神经再生中的作用。通过利用SV40T抗原制备永生化神经祖细胞（iNPCs），解决神经干/祖细胞难以体外培养及扩增的难题；并进一步证明神经干细胞经过永生化后能够持续增殖，并有进一步分化成为神经元、星形胶质细胞、少突胶质细胞的潜能。通过改良BJ-AdEasy腺病毒构建系统成功构建腺病毒AdR-siALK1、Ad-BMP9，将腺病毒感染iNPCs后，发现BMP9通过ALK1调控下游smad1/5/8分子的表达，并可使VEGF的表达受到抑制，而沉默ALK1后VEGF表达上调，提示ALK1对VEGF的表达起负性调控作用。在体实验同样表明BMP9通过ALK1通路下调VEGF表达，沉默ALK1或TGFβ抑制剂均能够上调VEGF，提示ALK1在神经再生过程中起到不利影响。最后，通过抑制BMP9/ALK1通路能够促进神经轴突再生，进而促进小鼠神经运动功能评分的改善。综上，本研究为临床上ALK1作为治疗缺血性脑卒中的干预手段提供了前期依据。