| Objective:To study the impact of costimulatory molecules CD83、PD-L1, Toll-like receptor 9, CD8 cell activation-related molecule and cytokine IFN-γ、IL-10 expression on human peripheral blood mononuclear cells of HBV patients with different stages by Hepatitis B e antigen,and further clarify the effects of HBe Ag on immune cell function and which’s mechanism in chronic hepatitis B virus infection processes. Methods:collected immune tolerant phase of HBV infectiors and immune clearance phase of HBV patients with 20 cases each in the First Affiliated Hospital of Nanchang University infection outpatient and inpatient. By the way, strictly selected 20 subjects from healthy as control. Collecting peripheral blood to separate PBMC, complete medium was incubated as un-stimulated control group, recombinant Hbe Ag was incubated as HBe Ag group, CPG-ODN1826 was incubated as CPGODN1826 stimulation group, recombinant HBe Ag combined with CPG- ODN1826 as combine stimulation group. Using flow cytometry to detect the changes of CD83,PD-L1 expression on CD14+ cell surface and CD107 a, CD107 b expression on CD8+ cell surface, reverse transcription PCR(RT-PCR) to detect the changes of TLR9-m RNA, enzyme linked immunosorbent assay(Elisa) to detect the changes of Th1 type cytokines IFN- gamma and Th2 type cytokine IL-10. Results:1. With different concentrations(1ug/ml、4ug/ml、10ug/ml、20ug/ml) HBe Ag and normal PBMC were cultured for 0 、 2 、 6 、 12 、 24 、 48 hours, the level of TLR9-m RNA and CD83 expression on CD14+ cell was decreased, and the level of PD-L1 was increased, the change most significant is especially the concentration of 10μg/m L and the time of 12h; while the surface markers gradually recovered with extended incubation time,and 24 h has been restored to the level before stimulation.2. RT-PCR results: PBMC of the healthy controls, the immune tolerance, the immune clearance were stimulated by 10ug/ml recombinant HBe Ag after 12 h, the expression level of TLR9-m RNA(0.362 + 0.021, 0.342 + 0.019, 0.373 + 0.031) was lower than the un-stimulated group(t =-7,.62, 8.85, 11.63, P <0.05), obvious lower than that of CPG-ODN1826 group(t =-23.54,-37.45,-30.91, P,<0.05), the difference was statistically significant. It had a downward trend than the recombinant HBe Ag combined with CPG-ODN1826 stimulation group(t=-1.22,-1.78,-1.59, P, >0.05), but the difference was no statistically significant.3. Flow cytometry detection CD83 and PD-L1 expression on CD14 + mononuclear cells:(1) PBMC of three groups after stimulation by the reorganization HBe Ag, CD83 expression level [(18.69 ± 5.43)%,(15.09 ± 2.62)%,(21.26 ± 4.34)%] lower than un-stimulated group(t = 3.84, 6.35, 4.55, P <0.05), significantly lower than the CPG-ODN1826 stimulation group(t =-6.42,-9.57,-7.18, P <0.05), the difference was statistically significant; there was a lower trend than the combined stimulation group(t=-1.22,-1.78,-1.59, P >0.05), but the difference was no statistically significant.(2) PD-L1 expression level [(79.01 ± 6.08)%,(80.57 ± 4.21)%,(70.64 ± 4.56)%] was significantly higher than un-stimulated group(t =-5.78,-2.19,-5.16, P are <0.05); overtop than CPG-ODN1826 stimulation group(t = 7.93, 11.72, 6.87, P <0.05)], the difference was statistically significant; it was higher than the combined stimulation group(t = 1.79,1.89,2.01, P> 0.05) but the difference was not statistically significant.4. Flow cytometry detection CD107 a, CD107 b expression on CD8+T cell:(1)PBMC of three groups after recombinant HBe Ag stimulation, CD107 a expression level[(5.73 ± 52.15)%,(4.15 ± 0.79)%,(7.54 ± 2.18)%] lower than un-stimulated group(t =2.90,5.51, 2.73, P <0.05)]; significantly lower than the CPG-ODN1826 group [t =-5.88,-5.06,-7.25, P <0.05], the difference was statistically significant; there was a lower trend than the combined stimulation group(t=-0.78,-1.72,-1.83, P >0.05), but the difference was not statistically significant.(2) CD107 b expression level[(1.59 ± 1.06)%,(0.84 ± 0.43)%,(2.15 ± 0.45)%] below the CPG-ODN1826 stimulation group(t =-4.38,-3.37,-4.87, P all <0.05), the difference was no statistically significant; there was a trend less than un-stimulated group(t = 1.21,2.08,1.79, P> 0.05), It was higher than the combined stimulation group(t=-1.88,-1.53, the trend of-1.99, P >0.05), the difference was no statistically significant;(3) three groups treated with different provocative and incubated, the expression level of CD107 was consistent with CD107 a, but the percentage of CD8 + T cells did not change distinctly, the difference was no statistically significant.5.Elisa results:(1) three groups of subjects PBMC with stimulation of recombinant HBe Ag, Th1 type cytokines IFN- expression level[(75.42 + 7.04),(71.59+5.55),(101.64 + 9.93)] was lower than that un-stimulation group(t=.86, 3.15, 5.88, P, <0.05),it was significantly lower than that the CPG-ODN1826 group(t=-7.70-4.94, P,-9.50, <0.05), the difference was statistically significant; there was lower trend than the combined stimulation group(t=-0.93,-0.62,-0.58, P >0.05), but the difference was no statistically significant;(2) Th2-type cytokines IL-10 expression level [(61.67 ± 7.42),(54.57 ± 7.29),(84.96 ± 11.74)] was higher than un-stimulated group(t =-6.87,-4.59,-7.63, P are <0.05), it was significantly higher than that CPG-ODN1826 group(t = 9.33, 8.23, 9.61, P <0.05), the difference was statistically significant, there was a higher trend than the combined stimulation group(t = 1.93,1.79,1.98, P > 0.05), the difference was not statistically significant. Conclusions:HBe Ag may suppress innate immune system and adaptive immune response system by increasing PD-L1 and IL-10 expression while decreasing cytokine IFN-γ、TLR9、CD83 expression on CDl4+cells and 107a、CD107b on CD8+T cells. |
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