跨突触重建绿色荧光蛋白法检测外源性神经干细胞与缺血性卒中大鼠大脑内神经元新生突触联系

Stem cell therapy is promising in neuro-rehabilitation after acute ischemic stroke. The preclinical researches have demonstrated that administered neural stem cells decreased the infarct volume and promoted the neurologic function via differentiating into mature neurons. However, there was on direct evidence to show new and functioned synapses could be built between the graft neural stem cells or their derived neurons and the host neurons. The current study is based on the fact that the maturation of synapse is mediated by the protein-protein interactions of pre- and post-synapse proteins, such as neurexin and neuroligin. Specifically, when one fragment of the split green fluorescent protein (GFP) contains the first 214 residues of GFP proteins is tethered to presynapse protein neurexin, while another split-GFP fragment consists of just 16 residues, 215-230 is tethered to postsynapse protein neuroligin, trans-binding of neurexin and neuroligin triggers GFP reconstitution and hence fluorescence onset at the place of synapses. In vitro, we intend to construct recombinant plasmids which overexpress Neurexin-GFP fragment (1-214) or Neuroligin-complementary GFP fragment (215-230), and respectively transfect these plasmids into neural stem cells and primary neurons. And we detect the GFP fluorescence which appears uniformly at the synapse where neural stem cell/ neural stem cell derived neurons contacts with the host neurons. In vivo, we intend to operate middle cerebral artery occlusion on transgenic mice which overexpress neurexin- GFP fragment, and inject neural stem cell transfected with neuroligin- complimentary GFP fragment into the infarcted area. And then the brain will be removed, and fluorescence will be detected on brain slices. Through this proposal, we intend to prove that neurorehabilitative effect of neural stem cell in cerebral ischemia brain is associated with its differentiation into neurons and further building of new and active synapse connection with the host neurons.

神经干细胞是缺血性卒中极具前景的治疗方法。临床前实验结果表明外源性给予神经干细胞可以分化为神经元，缩小梗死体积，改善神经功能评分。然而目前的实验结果均无法直接证实外源性神经干细胞可通过分化为神经元与脑内原有的神经元产生新的突触联系而发挥神经修复作用。本项目利用成熟的突触形成需要突触前、后蛋白的结合，分别将突触前蛋白Neurexin连接GFP前214个氨基酸残基，而突触后蛋白Neuroligin连接GFP后16个氨基酸残基，两者结合后发出荧光的原理，探讨神经干细胞分化神经元能否与原有神经元形成新的突触联系。在体外实验中质粒转染神经干细胞，与原代神经元共培养，探究其突触形成。在体内实验中，将表达GFP后16个氨基酸残基的转基因小鼠制作大脑中动脉梗塞模型后，在梗死区域注射转染质粒的神经干细胞，探讨外源性神经干细胞是否与宿主神经元形成新的突触。

缺血性卒中是危害人类健康的首要杀手。干细胞治疗是缺血性卒中的具有潜在价值的治疗方式之一。然而，外源性给予干细胞是否和内源性干细胞形成稳定的突触连接仍未得到很好的解决。.在本课题的资助下，我们开展了一系列工作，试图证实这个问题。我们首先给予卒中患者外源性干细胞，观察干细胞的疗效。结果显示干细胞显著改善卒中患者的神经功能的恢复，长期随访显示干细胞治疗是安全的。更进一步，我们利用跨突触绿色荧光蛋白法检测外源性干细胞分化为的细胞与内源性神经元形成突触联系。在体外试验中，分别转染绿色荧光蛋白片段的干细胞和神经元共培养后发出绿色荧光，显示两者之间形成解剖学的联系。然后，干细胞和神经元共培养，干细胞减轻神经元的缺血损害。最后，我们将外源性干细胞给予大脑中动脉模型大鼠，发现外源性给予干细胞显著减少梗死体积，减少神经元凋亡和改善了神经功能恢复。.我们的工作表明，外源性给予干细胞可与内源性神经元形成突触连接，并且改善了卒中的预后。.