肠道共生细菌与七鳃鳗可变淋巴细胞受体VLRA、VLRC交联对话（ Cross-talk）机制研究

Mechanisms of interaction between intestinal bacteria and host immune system play very important role in studying development of host immune system. In our previous study, the lamprey intestinal bacteria metagenomic library was constructed and analyzed. The bacterial community structure in intestine of Lampetra morii was relatively simple compared with higher vertebrates. After intraperitoneal injection of LPS, the relative abundance of Aeromonas decreased by 20%. While relative abundance of Chitinophaga and Shewanella increased by 15% and 4.8%, respectively. Besides, expression of VLRA and VLRC increased after LPS injection. Therefore, Aeromonas, Chitinophaga, and Shewanella were speculated to be important factors in lamprey adaptive immune regulation. Meanwhile, cross-talk between lamprey intestinal bacteria and adaptive immune system may be existed. Since VLRA+ and VLRC+ lymphocytes preferentially populate the intestinal epithelium, it is interesting to study the immunity regulation mechanism of specific intestinal microflora by VLRA or VLRC. Therefore, the RNA interference (RNAi) of VLRA or VLRC in the embryo stage of lamprey should be needed. The effect of lamprey intestinal microflora disbalance on differentiation of lamprey VLRA+ and VLRC+ lymphocytes cells should be studied. Besides, investigation on mechanism of VLRA and VLRC based lamprey adaptive immune signaling pathways affected by intestinal microflora disbalance should also be considered. As a result, the cross-talk pattern between lamprey intestinal microflora and VLRA/C based adaptive immune signaling pathways can be revealed. This study can offer insights into exploring the development of lamprey adaptive immune system and can provide reference for studying interaction mechanism between intestinal bacteria and host immune system of higher vertebrates.

肠道共生菌与宿主免疫系统互作对于宿主免疫系统发育研究具有重要作用。本项目前期构建了七鳃鳗肠道细菌宏基因组文库并分析了肠道细菌区系组成。对七鳃鳗腹腔注射脂多糖后，气单胞菌丰度下降了20%，而噬几丁质菌和希瓦氏菌丰度分别上升了15%和4.8%；且VLRA、VLRC表达量上调。因此推测七鳃鳗通过调控肠道VLRA、VLRC的表达调节了肠道三类共生菌的丰度，且肠道共生菌与七鳃鳗肠道免疫系统可能存在Cross-talk。基于此假设，本项目拟在胚胎阶段实现七鳃鳗VLRA、VLRC的基因沉默，分析VLRA、VLRC对肠道细菌区系建立的影响；造成七鳃鳗肠道菌群失衡，确定七鳃鳗肠道共生菌群中调控肠道类淋巴细胞分化的关键成员；揭示肠道共生菌与VLRA、VLRC信号通路间的Cross-talk模式。本研究对于深入探讨七鳃鳗适应性免疫系统发育具有重要意义，并为高等脊椎动物肠道微生物与免疫系统发育互作研究提供借鉴。

动物肠道中寄生的微生物区系与宿主的免疫及防御功能密切相关，肠道共生菌与宿主免疫系统互作机制对于宿主免疫系统发育研究具有重要作用。七鳃鳗具备与高等脊椎动物平行进化的适应性免疫系统，但对其适应性免疫系统发育研究还处于初步阶段。本项目分别分析了东北七鳃鳗（Lampetra morii）和日本七鳃鳗（Lampetra japonica）肠道细菌菌群组成，二者肠道共生细菌组成存在差异，与其他水生生物相比，七鳃鳗肠道共生细菌组成相对简单，气单胞菌为常见的优势菌群。与其他水生脊椎动物如青蛙、美国短吻鳄、草鱼和斑马鱼等相比，七鳃鳗具备独特的肠道细菌菌群组成。本项目确定了肠道气单胞菌（Aeromonas spp.）和希瓦氏菌（Shewanella spp.）均为调控七鳃鳗适应性免疫系统发育的关键菌群，两种菌免疫刺激均使得VLRA/B/C表达上调，其中希瓦氏菌免疫刺激可使得VLRA/B/C表达显著上调；此外，LPS和poly (I: C)免疫刺激也使得肠道总细菌和气单胞菌丰度产生变化，该结果提示肠道共生菌对于七鳃鳗适应性免疫系统的调控存在协同作用。本项目进一步鉴定了肠道气单胞菌和希瓦氏菌调控七鳃鳗外周血类淋巴细胞总蛋白质组的变化，获得相关差异表达蛋白质的信息，并对其中的关键蛋白质进行了功能初探。在所有鉴定的差异表达蛋白质中，L-Cofilin2和STAT3蛋白为后续研究的重点。基因功能研究显示，L-Cofilin2能够促进HEK293T细胞的增殖，STAT3则抑制了MCF-7细胞的增殖。通过综合分析肠道共生菌免疫刺激诱导的七鳃鳗外周血类淋巴细胞差异蛋白质的表达变化，进一步预测了STAT3在七鳃鳗适应性免疫调控过程中所参与的信号通路，为后续研究该蛋白的作用机制奠定了理论基础。综上，本项目通过分析肠道共生细菌与七鳃鳗适应性免疫系统的互作模式，可拓宽对七鳃鳗适应性免疫研究的途径，对于深入探讨适应性免疫系统发育提供新的研究思路。