| Penaeus chinesis is a very important commercial animal on Chinese mari-culture,however,the shrimp industry has always been affected by infectious diseases,mainly of bacterial and viral etiology. The research on the immunity mechanisms of shrimp become an urgent and basic need. We studied the ROS production in phagocytosis of P. chinensis hemocytes in vitro,in order to illuminate the cellular immune mechanisms of shrimp.In this study,We firstly study a culture condition for improving short-term in vitro maintenance of hemocytes from P. chinensis,intending to lay the foundation of research on the ROS production in phagocytosis of P. chinensis hemocytes subsequently. According to cell viability assayed with a trypan blue exclusion test,we established the optimal conditions of culture time,culture medium and culture temperature respectively. The result indicate:a significantly higher percentage of hemocytes viability was obtained following cultured in 30min,CM and SCM solution ,4C and 27 C conditions compared to other conditions,which show these conditions fit for the in vitro manipulation of P. chinensis hemocytes.On basis of the above experiments,we study the ROS production in phagocytosis of P. chinensis hemocytes in vitro using a CL method,intending to comprehend the CL phenomenon in phagocytosis. The result indicate:Zymosan A and LPS can both stimulate P. chinensis hemocytes to generate CL response,which show the hemocytes of P. chinensis undergo a respirtory burst in phagocytosis. But the cpm of CL is faint.Finally we studied the O-2 production and the affect of some environmental contamination on O-2 production in phagocytosis of P. chinensis hemocytes in vitrousing NBT reduction. The result indicate:the hemocytes of P. chinensis will undergo an obvious burst of respiratory and generate O 2,when they were stimulated in vitro to begin their phagocytic activity. With the same stimulator (Zymosan A and LPS) under different concentrations,the hemocytes showed different amount production of O";Different hemocytes concentrations that were activated with the optimal concention of Zymosan A show different amount production of O-2;SOD,NEM and lodoacetamide could produce a inhibition of O-2 production;5 kinds heavy metals and 2 kinds pesticides could also inhibite O-2 production;Individual animals showed a relatively high degree of variability in the production of 0-2,The amort shrimps showed a relatively low capability of O-2 production. It comes to the conclusion that the hemocytes of P. chinensis can generate CT2 etc. ROS to kill their pathogens in the phagocytic defensive reaction. Compared with CL,the NBT reduction is more propitious to be used as the index to determine the prawn immune state. |
