内质网应激及下游靶基因chop参与先天性脊柱裂发病机制及在治疗中应用的研究

Spina bifida aperta are complex congenital anomalies of the central nervous system. However, the pathogenesis of spina bifida aperta is not clear yet, and current therapeutics are unsatisfactory. We performed a comparative proteomics study of the spinal cord from rat fetuses with all-trans retinoic acid-induced spina bifida aperta. We identified 13 proteins with differential expression between normal spinal cords and those with spina bifida aperta. Among the 13 differential expressed proteins, there were three identified to be involved in the process of protein folding, therefore indicated more drastic endoplasmic reticulum stress (ER stress) in spina bifida aperta. Unfolded protein response (UPR) is activated by excess ER stress. Then we investigated the expression of three UPR sensors (Atf6,Ire1,Perk) in normal spinal cords and those with spina bifida aperta. The result revealed abnormal expression of UPR sensors in spinal cords with spina bifida aperta. Because the Chop in the cascade of UPR is responds to the apoptosis, we also checked the spatio-temporal expression of Chop in normal spinal cords and those with spina bifida aperta. The spatio-temporal expression of Chop showed abnormalities in multiple time point in spina bifida aperta. So we deduced excessive ER stress and signaling cascade triggered by ER stress (including UPR and Chop) might play important role in the pathogenesis of spina bifida aperta. .In this study, we are going to use the cultured neural stem cell and neuron induced by neural stem cell to investigate the role Chop played in embryonic development of central nerve system. According to our previous study, we performed thapsigargin administration to give the cells ER stress, and we use the ER stressed neural stem cell to mimic the embryo development of spina bifida aperta. By using this model, we can manipulate the Chop expression easily and observe the biological changes. We next plan to manipulate the Chop expression in all-trans retinoic acid-induced spina bifida aperta rat fetus to explore the role Chop in the pathogenesis of spinal bifida and the application in therapy.

先天性脊柱裂是常见小儿先天畸形，发病机制不清，治疗尚无良策。我们应用蛋白质组学研究首次发现先天性脊柱裂大鼠胚胎脊髓处于异常的内质网应激状态，应对内质网应激的UPR通路在脊柱裂胚胎发育期出现表达规律异常，UPR下游参与凋亡路径的Chop表达失调。由此我们提出了异常的内质网应激以及下游靶基因Chop表达失调在先天性脊柱裂的发生中可能起关键的作用的新观点。推测机制为：制畸因素于神经管形成的关键期诱发过激内质网应激，导致Chop过强表达，凋亡增加，影响神经管闭合；而后期继发的Chop异常表达导致神经元细胞丧失保护，出现损伤。本课题拟采用内质网应激诱导剂处理体外培养和诱导分化的神经干细胞模拟先天性脊柱裂胚胎发育，探讨Chop在畸形发生中的作用，同时在细胞水平对Chop表达进行人工干扰，观察细胞生物学改变。并以此为基础，采用孕鼠羊膜腔注射的方法，开展动物在体研究，探讨Chop对先天性脊柱裂的治疗。

先天性脊柱裂是常见小儿先天畸形，发病机制不清,治疗效果不良。我们应用蛋白质组学筛查发现了先天性脊柱裂胚胎脊髓组织在胚胎发育阶段存在异常内质网应激。在正常胚胎发育过程中应对内质网应激的UPR关键基因Atf6,Ire1,Perk于E12-13，E17天分别出现2个生理性表达高峰。而在先天性脊柱裂大鼠脊髓组织中，第一个表达高峰（E12-13）各基因表达明显上调，第二个表达高峰（E17）Atf6,Ire1激活时间提前，峰值降低，Perk提前激活，峰值上调。UPR关键基因的下游靶基因Chop于E13，E16，E18出现表达高峰，E19天表达处于低谷。而在先天性脊柱裂胚胎的脊髓中，仅E16天出现表达高峰，但表达量低于正常发育胚胎。E12，E15，E19，E20天的表达均异常增高。为了解E11天前的早期胚胎发育情况，我们采用了原代神经上皮干细胞培养并诱导其向神经元细胞分化的方法。经鉴定符合上皮干细胞和神经元的特征，建立模拟早期胚胎发育体外研究体系。 我们发现，当神经上皮干细胞诱导分化为神经元后，除Ire1表达增高外，Atf6, Perk，Grp78,Chop表达均无明显变化，提示了神经上皮干细胞在向神经元分化过程中并未出现明显的内质网应激，内质网应激的感受基因也并未全部反应。用内质网应激诱导剂处理神经上皮干细胞后，其向神经元分化能力下降，早期凋亡明显增加。本研究通过动物模型的在体研究获得了神经关闭合后UPR关键基因Atf6,Ire1,Perk以及其下游靶基因Chop在正常和脊柱裂大鼠胚胎脊髓中的表达规律，通过体外原代干细胞培养体系考察了胚胎发育的早期UPR关键基因Atf6,Ire1,Perk以及其下游靶基因Chop在神经上皮干细胞分化前后的表达情况，获得了胚胎神经管正常发育以及脊柱裂畸形成的新信息，为下一步的干预治疗提供了有力的依据。