波里氏囊在刺参排脏后体腔细胞恢复中的作用研究

Coelomocytes of Apostichopus japonicus are the cell type shown to be involved in cellular immune functions such as phagocytosis of pathogens, encapsulation and cytotoxicity. After evisceration, coelomocytes would also be expulsed together with visceral organs and restored to the pre-evisceration level accompanied tissue regeneration in A. japonicus. Rete mirabile, respiratory tree and polian vesicle in normal A. japonicus were the hematopoietic tissues, and polian vesicle will not be expulsed after evisceration. This project focuses on the recovery process of the ceolomocytes after evisceration in A. japonicus, the change of coelomocyte count, proportion and cellular immunity function after evisceration will be studied. The variety of tissue and cell structure, cell proliferation and coelomocytes associated antigen expression in polian vesicle of A. japonicus after evisceration will be studied. The difference of protein expression in polian vesicle of A. japonicus after evisceration by comparative proteomics technique will be analyzed, and the proteins related to the the genesis of ceolomocytes after evisceration will be identified by the technique of western-blot and RNAi referring to the genomic data of A. japonicus. Through the above studies, we hope to clarify the role of polian vesicle in the recovery of coelomocytes, and the cellular mechanism of coelomocytes genesis after evisceration in A. japonicus. It provides a basis for further understanding the mechanism of immune response and the formation of coelomocytes after evisceration in A. japonicus, and provides more references for a deeper understanding of the special mechanism of excretion and regeneration in A. japonicus.

体腔细胞是刺参体内主要的免疫反应效应器。刺参排脏时体腔细胞连同消化道等内脏被一起排出，内脏再生的同时体腔细胞也会逐步恢复。刺参的异网、呼吸树和波里氏囊具有产生体腔细胞的能力，刺参排脏时仅有波里氏囊不被排出。本项目聚焦刺参排脏后体腔细胞的恢复过程，研究体腔细胞数量、比例和免疫功能的变化；分析刺参排脏后波里氏囊的组织细胞形态结构特征变化、波里氏囊中细胞增殖变化规律及体腔细胞相关抗原表达变化规律；利用比较蛋白质组学技术进行刺参排脏前后波里氏囊中蛋白表达差异分析，参考已公布的刺参基因组数据，并结合蛋白质免疫印迹、RNA干扰等技术挖掘刺参排脏后体腔细胞发生相关蛋白。通过以上研究，以期明确波里氏囊在刺参排脏后体腔细胞恢复中的作用，初步解析刺参排脏后体腔细胞发生的细胞机制，为进一步探知刺参排脏后的免疫响应机制和体腔细胞的发生机制奠定基础，为更深入了解刺参排脏与再生这一特殊防御生存机制提供更多参考。

刺参具有独特的吐脏行为与强大的再生机能，吐脏后体腔细胞可快速再生，其对机体生理与免疫机能的恢复重构具有重要意义，但相关机制尚不清楚。本项目首先确定了正常刺参体腔细胞的组织来源，其次系统分析了刺参排脏后体腔液和体腔细胞的恢复过程，最后聚焦刺参排脏后仅留的“造血组织” ——波里氏囊，从组织、细胞、基因和蛋白多个层面，研究了波里氏囊在刺参排脏后的动态变化。研究结果发现：（1）正常刺参的异网、呼吸树与波里氏囊具有生成体腔细胞的能力，刺参水管系统（波里氏囊）内体腔细胞可向体腔内迁移。（2）刺参排脏后体腔细胞数量变化分为两个时期：快速增长期（6h内）和缓慢增长期（6h-35d），不同亚群体腔细胞数量变化趋势与总体腔细胞变化基本一致；刺参排脏后波里氏囊和体腔内体腔液上清中免疫酶活和体腔细胞中免疫基因表达水平均发生明显变化，但趋势并不一致。（3）刺参水管系统（波里氏囊）内体腔细胞向体腔内迁移速率在排脏后6h明显增强；刺参排脏后波里氏囊的形态结构发生明显变化，囊壁结缔组织在排脏后先变薄后又逐步恢复；波里氏囊壁组织和囊腔内体腔细胞中细胞增殖信号在排脏后5d较排脏前有明显增强。（4）刺参波里氏囊壁中基因表达水平在排脏后有明显变化，差异基因在细胞增殖和分化相关的3条信号通路中显示富集，包括TGF-β信号通路、Wnt信号通路和内吞作用信号通路；排脏前后的波里氏囊壁组织中共注释到222个差异蛋白，其分别和细胞增殖与分化、免疫应答与创伤修复、物质转运与能量代谢等多种生物学功能相关，其中IGFBP、beta ig-h3-like、Grb2L等生长因子和Stk10、ASPP1等造血相关因子在排脏后显著上调。研究结果为进一步解析刺参水管系统（波里氏囊）的生物学功能奠定了基础，为探知刺参排脏后的体腔细胞再生机制积累了资料。项目执行期间，共发表SCI收录论文5篇，中文核心期刊2篇，培养了4名硕士研究生，硕士生任媛的毕业论文入选辽宁省优秀硕士学位论文。