星形胶质细胞-神经元转分化治疗新生儿缺氧缺血性脑病的研究

Hypoxic-ischemic encephalopathy(HIE) remains one of the most important neurological disorders with high risks of severe morbidity and death in newborns, but the underlying mechanisms are complex. It is difficult for neonatals to recovery from cell loss and inflammatory reaction which caused by brain injury. At present, there is few effective treatment about HIE. The applicant has been engaged in study of mechanisms and therapeutic strategies about neonatal brain injury, and has already completed the National Natural Science Foundation named "effects of hypothermia on differentiation and maturation of oligodendrocyte precursor cells (OPC) after hypoxic ischemic brain injury" for youth. In that study, we found astrocyte acitivation and proliferation siginificantly increased in the hypoxic-ischemic injured brain, and it formed glia scar which may release a large number of inflammatory factors and suppresed neurotrophic factors secretion. This poor environment caused by astrocyte activation may lead to delayed neuron death in gray matter and impede oligodendrocyte precursor cells (OPC) differentiation and maturation in white matter of neonatal brain. This phenomenon suggested that the excessive astrocyte activation may suppressed brain repair after hypoxic ischemic injury. We prepare to transduct Lentiviral vectors containing cDNA of PAX6/neurog2/Dlx2/Ascl1 to cultured atrocytes. After that, we will detect astrocyte trans-differentiate into neuron. In vivo, we will Three-dimensionally injected lentivial vectors containg transcriptional factors and green fluorecent protein (GFP) into AST specifically in rat brain. Then, trans-differentiaiton of the transfected astrocyte will be tracked, and the numbers of newbron neurons will also be tested in the experiment. At the same time, this lentivial injection will also be injected into the area surrounding the severe injured infarct in the striatum. we will investigate if astrocyte in injured brain can transdifferentiated into neuron. The acitvation and proliferation of astrocyte, glia scar formation and neuron loss will be tested in the experiment. At the same time, the differentiation and maturation of oligodendrocyte precursor cells will also be explored in this study. We will also examined animals behavior by open filed and rotarod test in long term after brain injury in rat brain.

新生儿窒息后缺氧缺血性脑损伤（HIBD）是导致新生儿死亡及伤残的主要原因之一。HIBD发病机制复杂，脑损伤引起的细胞丢失和炎症反应较难恢复。申请人一直从事新生儿脑损伤机制和治疗策略的研究，并提前完成了青年基金项目。前期研究发现，HI脑内神经元大量丢失伴随着星形胶质细胞（AST）活化增生形成胶质瘢痕并释放炎症因子，明显抑制了少突胶质前体细胞的分化及损伤脑的修复。我们拟通过体外培养AST并分别转染转录因子PAX6/neurog2/Dlx2/Ascl1，观察AST是否能够转分化至神经元。同时通过构建携带转录因子的腺相关病毒-9，立体注射到正常或损伤动物脑内使其特异感染AST，观察AST的原位转分化。检测不同转录因子作用下，AST的转分化效率，及转分化的神经元是否具有功能。此外，观察AST转分化对损伤脑内胶质瘢痕的形成、神经元的数目、少突胶质前体细胞的分化成熟的影响，和动物行为学是否得到提高。

中重度新生儿缺氧缺血性脑病（HIE）有近50%死亡或留有重度神经系统后遗症,目前临床尚无有效的治疗措施。中重度HIE是导致新生儿死亡及伤残的重要原因之一。本课题组前期研究结果提示，新生鼠缺氧缺血后内源神经干细胞能被诱导增殖、分化，促进神经再生以修复损伤。而对于中重度HIE脑，大量神经细胞的死亡，脑自身的修复能力不足。通过移植外源神经干细胞/前体细胞来修复中重度损伤脑是最具潜力的治疗策略之一。要想实现干细胞移植治疗脑损伤，首先要明确移植入宿主脑内的细胞是否能够发挥功能、通过何种途径与宿主细胞建立环路连接、如何避免移植细胞副作用的产生从而提高细胞的安全性，使其达到治疗的最佳效果。因此，本课题在移植新生鼠HIE模型动物之前，我们先通过crispr/cas9基因编辑的方法将人工设计的G蛋白偶联受体DREADD（激活型和抑制型）表达在人的胚胎干细胞中，通过干细胞的定向分化技术我们得到不同脑区的神经元。当给与DREADD配体CNO药物作用时，我们可以在体外检测到CNO对不同类型神经元电生理活性的调节作用。同时，当我们将这些表达DREADD的人神经前体细胞移植到损伤动物脑内，同样可以检测到CNO可调控移植细胞神经递质的释放从而引起宿主纹状体GABA能神经元兴奋性突出前电流频率的增加。说明移植入的人神经元可整合入宿主神经环路且其活性可被外源药物远程操控。进一步的动物行为学实验证实，CNO可以调控动物的行为。本研究首次实现了对移植到宿主脑内人神经元的远程操控，为我们后期开展干细胞移植治疗新生鼠HIE模型奠定了基础。且本研究首次揭示了干细胞移植治疗的新机制，为未来开展干细胞临床转化的前期研究奠定了基础。