基于比较转录组学的七鳃鳗类T、B淋巴细胞免疫应答信号传导分子机制

In this study, lamprey (Lampetra japonicum), a jawless vertebrate, is targeted to investigate its signal transduction molecules of its own or homolougues to higher vertebrates involved in the activation and proliferation processes of T and B lymphocyte-like cells by means of bioinformatics, biochemistry, molecular biology and immunology research methods. These signaling molecules were mined, screened and identified from high throughput sequencing data of transcriptomes of dorsal fat body, a hematogenous tissue, before and after immune stimulation. The identified candidates are further verified by using gene chip technology, and gene regulatory signal transduction networks are predicted by bioinformatics methods. The key node elements in signal transduction networks are selected for gene cloning, recombinant protein expression, purification and preparation of monoclonal antibodies. These monoclonal antibodies of the key molecules are used to screen and verify corresponding class of lymphocyte subsets by flow cytometry and submicroscopic method. The localization and co-localization of these key molecules in various lymphocyte subsets are identified by Confocal Laser Scanning Microscope. The interaction modes of these molecular are investigated using immunoprecipitation techniques. The signal transduction regulatory networks of each lymphocyte group during activation and proliferation processes after immune stimulation are verified and established through above analysis. The basic characteristics，the molecular genetic basis and the mechanisms of the immune response of adaptive immune cell subsets of jawless vertebrates will be clarified. Our results will provide new evidences in revealing the origin and evolution of the signaltransduction system of lymphocytes of vertebrate adaptive immune system.

本研究以无颌类脊椎动物日本七鳃鳗为对象，以生物信息学、比较基因组学、分子生物学及免疫学的研究方法,在对其成体造血组织脊椎弓脂肪体免疫前后各转录组之间及与高等脊椎动物的转录组进行比较分析的基础上，甄选在类T、B淋巴细胞活化、增殖过程中参与信号传导的其所特有的及与脊椎动物同源的信号传导分子。利用基因芯片技术检验差异表达候选基因并预测信号传导调控网络。筛选关键节点分子进行基因克隆，并进行重组蛋白表达、纯化及制备单克隆抗体；用流式细胞术根据分子标识筛选类淋巴细胞各亚群进行细胞学检验，用免疫荧光显微术检验这些分子在各类淋巴细胞亚群中的单一和共定位；采用免疫共沉淀技术研究这些分子的相互作用模式；验证并确立免疫激发后各类群淋巴细胞活化增殖过程的信号传导调控网络。阐明无颌类脊椎动物适应性免疫细胞各亚群免疫应答的分子遗传基础及作用机理。为揭示脊椎动物适应性免疫系统淋巴细胞信号转导体系的起源与进化提供新证据。

本研究以无颌类脊椎动物日本七鳃鳗为对象，以生物信息学、比较基因组学、分子生物学及免疫学的研究方法,在对其成体造血组织脊椎弓脂肪体、外周血淋巴细胞及鳃囊组织免疫前后各转录组之间及与高等脊椎动物的转录组进行比较分析的基础上，甄选出在类T、B 淋巴细胞活化、增殖过程中参与信号传导的其所特有的NICIP分子以及与脊椎动物同源的信号传导分子如LCK、LYN、FYN、TEC、VAV3、BTK、BLNK、SYK、DAPP1、CD81和CD82进行了深入研究。利用Q-PCR技术检验了这些候选基因的mRNA差异表达情况并预测了信号传导调控网络。对这些关键节点分子进行了基因克隆、蛋白重组表达、蛋白纯化及多克隆抗体的制备。利用免疫印迹方法检测了它们在免疫激发后在免疫相关各组织中的相对差异表达情况。利用共聚焦荧光显微技术对信号转导分子进行细胞单分子定位或与VLRB分子之间的多分子共定位研究；利用信号转导分子的荧光标记单克隆抗体对外周血淋巴细胞进行标记后通过流式细胞仪分选阳性标记细胞，对所筛选的细胞进行了细胞分型鉴定。通过以上各种方法检验了所甄选的信号转导分子的有效性。对经验证后的部分信号转导分子，利用免疫共沉淀技术研究和鉴定了与它们相互作用的上下游蛋白分子，对信号通路和调控网络的关键节点进行了验证。结果表明，尽管细胞表面受体及其可能偶联的跨膜蛋白分子与高等脊椎动物不同，但那些参与胞内信号转导的分子与高等脊椎动物T、B 细胞的相比具有共同的分子基础。本研究为揭示无颌类脊椎动物适应性免疫系统淋巴细胞信号转导体系的起源与进化提供新证据。