视网膜内源性多肽CRB1P在拮抗多氯联苯暴露致子代视网膜感光细胞发育异常中的作用机制研究

CRB1P in zebrafish fertilized eggs while under exposure of PCBs, the development of retinal structure in the retina of the offspring is normal than that of PCBs; the stratification is clear and the quantity and structure of the photoreceptor cells are normal; increased expression of photoreceptor cell specific marker Rho; It suggests that CRB1P could reverse the developmental defects of the retina in the offspring of PCBs exposure. It indicates that CRB1P could prevent the developmental defects of the retina in the offspring from PCBs exposure. Bioinformatics analysis revealed that CRB1P may have the similar function to its precursor protein, CRB1. CRB1 could inhibit the formation of photoreceptor cells by inhibiting Notch signaling. We have confirmed that CRB1P inhibits the Notch signaling pathway, suggesting that CRB1P may maintain the normal development of photoreceptor cells through the regulation of Notch signaling. Our research is designed to evaluate the role of CRB1P in retina development and the effect of CRB1P on Notch signaling under PCBs exposure. To clarify the role and mechanism of CRB1P in the retina development, the interaction protein of CRB1P is investigated by the rescue( Notch1 overexpression or Notch signal agonist) and pull-down experiments. The correlation between PCBs exposure and the level of children’s amblyopia and CRB1P is carried out. This project is expected to provide new research and development clues for the prevention and treatment of the effects of environmental pollutants on the development of retinal photoreceptor cells.

CRB1P是借助多肽组学发现的功能未知且高度保守内源性多肽，母体PCBs暴露后其在流产胚胎视网膜组织、子代斑马鱼胚胎及小鼠视网膜组织均显著下调。前期发现，在斑马鱼受精卵注射CRB1P可拮抗PCBs暴露所致的子代感光细胞异常，子代视网膜分层清晰、感光细胞数量及Rho表达增加；生物信息学和实验证实，CRB1是CRB1P前体蛋白，二者均可抑制Notch信号，提示CRB1P可能通过调控Notch信号而维持感光细胞的正常发育。研究拟阐明CRB1P在子代感光细胞发育中作用；以Notch信号为切入点，采用挽救实验策略、pull-down等实验手段，论证CRB1P影响感光细胞发育的分子机制；并通过评估PCBs暴露、儿童弱视、血清CRB1P水平之间的相关性，进一步评估该分子机制。本课题有望为促进子代感光细胞发育提供新靶标，为防治PCBs的损害提供新线索。

多氯联苯是一种广泛存在于环境中的有机污染物，与视网膜发育及视力损伤关系密切，但其作用的机制尚不明确，本课题组采用多肽组学技术研究影响胚胎期暴露PCBs致视网膜感光细胞发育异常的内源性多肽，并阐明其在视网膜发育中的作用及机制。. 本研究通过多肽液相质谱检测技术筛选多氯联苯暴露后斑马鱼胚胎内差异表达的内源性多肽。通过生物信息学分析方法和文献复习确定与眼发育密切相关的多肽ntn1aP。.本课题组首先研究了多肽对前体基因及视网膜神经节细胞特异基因表达的影响，结果提示多肽干预后其前体基因及视网膜神经节细胞特异基因的表达增加。进一步，本课题组通过动物及细胞挽救实验证明了多肽ntn1aP的保护作用。.在制研究部分，首先利用pull-down实验寻找与目标多肽结合的相互作用蛋白，质谱鉴定结果提示差异靶蛋白集中在热休克蛋白和角蛋白，但目前关于这些蛋白和眼部疾病的报道较少。通过检测了焦亡经典通路中关键分子及炎症因子在细胞中的表达量验证多氯联苯对细胞焦亡的影响，结果提示多肽ntn1aP和多氯联苯可能并不通过细胞焦亡途径对视网膜神经节细胞发挥作用。利用转录组测序技术筛选多肽干预后差异表达的靶基因，初步验证发现Bcl6在多肽ntn1aP干预后表达降低，进一步验证发现蛋白水平的结果与mRNA水平不一致。进一步对多肽ntn1aP干预后基因的表达变化做GSEA分析，表明多肽作用的靶基因可能主要通过参与p53信号通路发挥作用，进一步验证p53调控的下游基因，结果提示多肽ntn1aP通过p53/cyclinD1通路发挥作用。.本课题在原课题设计的基础上进行了拓展，从前体基因角度（NTN1）做了进一步机制研究，初步验证发现NTN1在多氯联苯暴露后低表达，沉默NTN1对细胞增殖和迁移有抑制作用，过表达NTN1可以挽救多氯联苯暴露所致的细胞毒性作用。于是进一步从RNA水平和蛋白水平验证了NTN1与眼发育、神经发育相关可能的下游通路，结果提示ERK信号通路是多氯联苯致视网膜细胞损伤的重要机制之一，过表达NTN1可以拮抗多氯联苯所致的ERK磷酸化水平的降低。