血浆铜蓝蛋白作为神经管畸形胚胎早期诊断分子标志物的价值及其机制探讨

Neural tube defects (NTDs) is the most serious congenital malformation harmful to human health and its embryogenesis is still not clear. Using proteomics technology we have found 16 upregulated proteins in serum pregnant women with neural tube defects. After further verification, ceruloplasmin (CP) has good sensitivity and specificity in the diagnosis of neural tube defects and it is better than AFP. All of those indicate CP is likely to be a very valuable molecular markers in noninvasive diagnosis of NTDs.Therefore, we will take advantages of the cooperation with a number of domestic birth defects research centers for multicenter and large sample validation, and compare with other common congenital malformations. At the same time, we will study whether the CP could be a biomarker for early screening. Our study may provide scientific basis for this new molecular markers widely used in clinical. In combination with our previous studies, we for the first time suppose that CP is involved in the occurrence of NTDs, that is due to some genetic or environmental abnormal factors, CP protein misfold, which trigger endoplasmic reticulum stress reaction, and lead to the excessive apoptosis of neuroepithelial cells. At the same time, metal ion metabolic abnormalities and oxidative stress damage caused by CP dysfunction further aggravate nerve cell damage and lead ultimately to the occurrence of NTDs. In order to verify the new hypothesis, in this study we will establish the animal model of NTDs to observe the CP expression, protein folding, copper ion metabolism and oxidative stress injury in neural tube from different embryonic developmental stage. We will explore the exact mechanism of CP in embryogenesis of NTDs, and put forward new treatments targeting key genes of endoplasmic reticulum stress reaction pathways for NTDs. This study belongs to the innovative research,and there is no similar study has been reported until now. It is great significant to reduce the occurrence and improve the quality of life in children with birth defects.

课题组利用蛋白质组学技术发现16种蛋白质在神经管畸形孕妇血清中存在差异表达，经过进一步验证确定血浆铜蓝蛋白（CP）在神经管畸形诊断中有较好的敏感性和特异性，优于目前在临床上应用的AFP检测。因此，本课题将进行多中心、大样本的临床验证，并与其它先天畸形进行比较，同时探讨CP能否作为结构异常发现之前的早期筛查指标，为这一新的分子标志物能够在临床广泛应用提供依据。同时结合课题组前期研究结果，提出CP参与神经管畸形发生的新假设，即由于CP蛋白质折叠错误引发内质网应激反应，同时由于CP功能异常引起金属离子代谢异常和氧化应激损伤又进一步加重神经细胞破坏，最后导致畸形发生。本课题将建立动物模型，从不同胚胎发育阶段观察CP在神经管组织的表达规律、蛋白质折叠、铜离子代谢和氧化应激损伤等指标变化，探讨CP导致神经管畸形的可能机制，同时提出以CP和内质网应激反应通路中的关键基因为靶点的神经管畸形治疗新方法。

神经管畸形和先心病是常见危害人类健康的严重出生缺陷，是由环境因素和遗传因素共同作用的复杂多基因疾病，尚无有效的治疗方法。目前神经管畸形和先心病产前诊断主要依靠超声和MRI等影像学方法，只能在已经出现明显的结构发育异常时给予诊断，失去治疗和预防的最佳时机。因此，研究其胚胎早期诊断新方法，使其能够在严重结构异常或神经不可逆损伤之前获得诊断，对于早期干预和预防具有重要意义。本课题完成了各种先天畸形胎儿孕妇血清标本收集工作并建立了出生队列进行畸形早期标本采集。利用iTRAQ蛋白质组学技术比较了神经管畸形胎儿母亲血清与正常孕妇血清的差异表达蛋白质，发现PCSK9、铜蓝蛋白、对氧磷酶、血浆a-球蛋白抑制因子H4、血清S 蛋白、糖基化磷脂酰肌醇特异性磷脂酶D、胎球蛋白、淀粉样蛋白P、血浆a-球蛋白抑制因子H2、血小板因子4、C9、C1R、C1S和C1QA是神经管畸形产前诊断的分子标志物。利用ITRAQ定量方法在先心病胎儿母亲血清中筛选并验证，发现LMNA、FLNA、TPM4和ACTG1四个蛋白质为先心病产前诊断蛋白质标志物。 采用联大茴香胺酶比色法和络合比色法测定神经管畸形模型孕鼠血清铜和血清铜蓝蛋白氧化酶活力的变化，发现测定血清铜蓝蛋白酶活性和铜离子含量对胚胎神经管畸形早期诊断存在一定价值。同样利用神经管畸形动物模型发现脊柱裂胎鼠总抗氧化能力和总谷胱甘肽明显降低，而氧化应激防御性通路基因Nrf2及其下游基因表达也明显降低，提示神经管畸形发生可能与Nrf2通路基因表达下调而导致氧化应激损伤有关。 利用全基因组甲基化芯片和生物信息学分析对显性脊柱裂引产胎儿以和正常引产胎儿的脊髓组织进行甲基化研究，发现CD40、TRIM4和STT3A基因甲基化异常导致其表达降低与神经管畸形发生有关。.本课题的研究已发表标注课题资助的SCI收录论文7篇，另外有6篇已标注本课题资助的英文论文和2篇中文论文正在投稿过程中，近期将会发表。在本课题研究基础上，作为课题负责人于2016年参与国家重点专项1项。申请发明专利1项。本课题培养6名博士研究生和2名硕士研究生。