CpG寡脱氧核苷酸触发中华绒螯蟹酚氧化酶原系统信号传导途径的研究

STUDIES ON THE SIGNALING PATHWAY OF CpG ODNs ACTIVATING proPO SYSTEM IN CHINESE MITTEN CRAB ERIOCHEIR SINENSIS

为探讨CpG寡脱氧核苷酸(CpG-oligodeoxynucleotides,CpG-ODN)激活中华绒螯蟹血细胞酚氧化酶原系统(prophenoloxidase system,proPO系统)的信号传导途径,使用一定剂量的CpGODN-1670、ODN-R以及几种细胞信号传导的激活剂或抑制剂体外处理中华绒螯蟹血细胞,通过检测胞内外酚氧化酶(PO)活性的变化,对CpG ODN触发proPO激活系统的信号传导途径进行评价。结果显示,ODN-1670与ODN-R均可触发蟹血细胞proPO激活系统,试验剂量的ODN-1670可促进血细胞内外已有的proPO转化为PO,而对proPO颗粒的释放具有一定的抑制作用;ODN-R则不仅可使proPO转化为PO,还可促进血细胞脱颗粒。两者的信号传导途径相似,可能都包含了G-蛋白介导的蛋白激酶C(PKC)途径,酪氨酸蛋白激酶(RTK)途径对ODN-1670的触发proPO激活系统的活化过程进行负调控。

Prophenoloxidase system（proPO system） plays a key role in the natural immune response of Crustacean animals,but the signal transduction pathway of proPO system is rarely reported.CpG-oligodeoxynucleotides（CpG-ODN） is synthetic oligodeoxynucleotide containing CpG motif that could simulate the bacterial DNA with immunostimulatory activity.Many studies has proved that it is a kind of good non-specificity immunostimulants and specific immunology adjuvant.CpG ODNs could stimulate the proPO system in haemocytes of Macrobrachium rosenbergii,but so far,it has not yet seen a similar study reported in the Chinese mitten crab（Eriocheir sinensis） at home and abroad.The experiment were processed in order to de-termine the signal transduction pathway involved in the CpG ODNs activating proPO system in Chinese mitten crab（Eriocheir sinensis）.The crab haemocytes cultured in M-199 medium was treated in vitro with different concentrations of CpG ODN-1670 and ODN-R,as following,0,5,10,15,20 and 25 μg/mL.Then we detected the effects on the proPO system of several activators or inhibitors for specific signaling components,including Sodium fluoride（NaF,G protein activator）,Phor-bol-12-myristate-13-acetate（PMA,PKC activator）,Chelerythrine（PKC inhibitor）,Genistein（tyrosine protein kinase inhibi-tor）,worked together with ODN-1670 and ODN-R in this study.Both intra-cellular and extra-cellular relative phenoloxidase（PO） activity including stimulated activity POs and total activity POT were measured to study the signaling way.The results showed that both ODN-1670 and ODN-R could activate the proPO system.The experimental dose of ODN-1670 could induce proPO transforming into PO,while inhibit the releasing of proPO to outside to certain extent.ODN-R could not only induce transforming proPO into PO,but also promote granules releasing.In addition,the signaling pathway of the proPO system activation stimulated by the two ODNs appeared to be some similar to each other,both the signaling pathways may include G-protein mediated PKC way,and ODN-1670 induced proPO activation was negatively regulated via the tyrosine kinase pathway.And some Toll-Like Receptor might exist in the cell membrane,and the first step of ODNs active proPO system was to combine with this Toll-Like Receptor protein then active G-protein mediated pathway.These results have provided basis to the immune defense mechanism study of crab.