Effect Of SEA On The T Cell Proliferation, Activation And Surface Ultrastructure

Objective:To study the effect of Staphylococcus aureus enterotoxin A (SEA) on proliferation and surface ultrastr ucture of human T lymphocyte, and the morphological or molecular biological change of CD3,ZAP-70,TCRR(?) on T cell treated with SEA. To learn the characteristic of T cell active signal pathway induced by SEA and to support the SEA applications.Methods:1, Normal human peripheral blood lymphocyte and Jurkat T was cultured with different concentrations SEA for different time in vitro; 2, Use CCK-8 for cell proliferation; 3, Change of T cell surface ultrastructure was detected by atomic force microscopy (AFM) and distribution of CD3 on T cell was learned by AFM tip modified with CD3 antibody induced by SEA; 4, Use confocal microscope (LSCM) to observe CD69 expressing and ZAP-70 assembling on T cell stimulated by SEA; 5, Real-Time PCR with SYBR Green I technique was used for detecting TCR(?) chain, ZAP-70 expression level andβ2-microglobulin gene (β2M) was used as an endogenous reference. Relative changes in TCR(?) chain, ZAP-70 expression level were used by the 2-△△Ct method between each group and the control.Results:1, SEA can stimulate peripheral blood lymphocyte to express CD69, and induced by suitable concentration SEA of 10-0.01 mg/L the cell multiplication has the maximum; 2, T cell surface ultrastructure changes with SEA treating in 4 hours to 48 hours, during the time cell surface average roughness (Ra) increases at first and then decreases, and especially different surface ultrastructures on T cell appears in the time; 3, Induced by SEA, many CD3 gets together on the surface of T lymphocyte; 4, Suitable concentration SEA of 10-0.01 mg/L can make fluorescence intensity of ZAP-70 changing quickly in T cell, and the fluorescence intensity of ZAP-70 increases firstly and decreases secondly in 1 hour treated with the same concentration SEA; 5, TCR(?) chain and ZAP-70 expression level become low,2-△△Ct less than 1, induced by SEA.0.1 mg/L SEA induce the level to be lower.Conclusion:1,0.1～10mg/L SEA makes the proliferation of Jurkat T or normal human peripheral blood lymphocyte obviously in vitro 48 hours; 2, Treated with SEA in different time, the surface ultrastructure of Jurkat T changes specially based on the treating time; 3, SEA can stimulate normal human peripheral blood lymphocyte to express CD69 in 12 hours; 4, ZAP-70, significant element of TCR signal pathway, contribute SEA treating T cell; 5, Compared the classical TCR signal pathway, TCR(?) chain and ZAP-70 have their own contributions for TCR signal pathway induced by SEA; 6, There is alternative pathway to activate T cell induced by SEA compared to the canonical TCR signal pathway.