The Study of Relativity Both HBV DNA Load and Expression of Toll-like Receptor 3(TLR3) to Down-regulation Function of Dendritic Cells Derive from Peripheral Blood Monocyte of Chronic Hepatittis B Patients
|Keywords||With chronic hepatitis B Hepatitis B virus HBV DNA level Dendritic cells Dysmaturity Toll-like receptor Toll-like receptor 3 Chronic Phenotype|
PART IPhenotype and function of dendritic cells (DCs) derived from peripheral blood monocyte of chronic hepatitis B (CHB) patients with different HBV DNA loadsObjective:To investigate the association phenotype and function of dendritic cells derived from peripheral blood monocyte of chronic hepatitis B patients with different HBV DNA loads.Method:28 chronic hepatitis B patients were randomly included in the study. All patients were treated with nucleoside analogues (Lamivudine or LdT or Adeforvir) for 24 weeks. HBV DNA loads of peripheral blood and liver biopsy were assessed before and after the treatment. The patients were divided into two groups according to peripheral HBV DNA loads, before and after the treatment (28 as CHB-H: chronic hepatitis B patients with high HBV DNA loads, higher than 10~5copies/ml, and 25 as CHB-L: chronic hepatitis B patients with low HBV DNA loads, less than 10~4copies/ml). And ten healthy controls were included. DCs were enriched from peripheral blood of each subject. The phenotype of mature DC (mDC) was subjected to flow cytometric analysis. The lymphocyte allo-stimulatory capacity of DCs was evaluated through MTT assay. IL-10 and IL-12 production were qualified by EL1SA.Result:1. Liver tissues showed more severe inflammation in high-load group than that of low-load group (P<0.05) . But the degree of fibrosis differs nonsignificantly(P>0.05) .2. DCs in vitro can proliferate evidently when stimulated by cytokines; however, DCs of patients with chronic hepatitis B proliferate more slowly than healthy controls.3. Surface moleculars: The expression of DC surface molecular such as HLA class II(HLA-DR), CD80 (B7-1) ,CD86(B7-2) and CD83 has a positive rate of over 85% in normal population. But both groups showed low expressions of above surface moleculars (43.22±8.22%, 37.89±7.75%, 39.24±8.56% and 20.31 ±4.86% in CHB-H group; 45.11±7.99%, 40.37±6.52%, 37.48±7.71% and 22.87±5.68% in CHB-L group). Patients with chronic hepatitis B had low expression of HLA class II(HLA-DR), CD80 (B7-1) ,CD86(B7-2) and CD83 especially(p<0.001). But the difference was not significant between two the groups with different virus load (p> 0.05).4. The stimulatory capacity in mixed lymphocyte reaction (MLR) showed no difference between the two groups of patients (p>0.05), but both lower than that of healthy controls (p<0.01). And production of IL-12, IL-10 decreased significantly in patients (P<0.01) also.Conclusion:1. The positive correlation was found out between peripheral HBV DNA load and degree of inflammation of liver tissue in chronic hepatitis B patients.2. DCs derived from such patients had impaired function of maturation.3. Peripheral DCs of these patients had deficit in phenotype and down-regulation of stimulatory capacity.4. The changes in phenotype and down-regulation of function were not relevant to peripheral HBV DNA load.PATR IIThe association of Toll-like receptor 3 (TLR3) express on dendritic cells with down-regulative function of dendritic cells derived from peripheral blood monocyte of CHB patients withdifferent HBV DNA loadsObjective:To investigate expression of Toll like receptor 3 on peripheral blood dendritic cells in patients with chronic hepatitis B with different HBV DNA loads and to develop the dysmaturation mechanism of DCs of patients sustained infecting with hepatitis B virus.Method:Blood samples were obtained from 20 CHB patients with different HBV DNA loads. These patients were treated with nucleoside analogues for 16 weeks. The HBV DNA loads were measured in serum collected concomitantly with the liver biopsy specimens. The patients were divided into two groups according to peripheral HBV DNA loads (17 as CHB-L with a low HBV DNA load, less than 10~4copies/ml, and 20 as CHB-H with a high virus load, higher than 10~5copies/ml). And ten healthy controls were included. The monocytes isolated from peripheral blood of candidates were incubated with rhGM-CSF and rhIL-4 to induce the DCs generation and proliferation. Then the morphotype of DCs was identified by microscope. The phenotypes of immaturative and maturative DCs (HLA-DR, CD80, CD86, CD83) were characterized by flow cytometry. The lymphocyte allo-stimulatory capacity of DCs was evaluated through MTT assay. IL-10 and IL-12 production were qualified by EL1SA. Furthermore, we carried out flow cytometry and Western blot analysis to measure the expression of TLR3 on mDCs and immature DCs (imDCs) respectively.Result:1. DCs in vitro can proliferate evidently when stimulated by cytokines but grew more slowly and proliferate less than cultured in medium containing fetal calf serum (P>0.05).2. DCs of control group showed CD80, CD86, HLA-DR and CD83 expression (expressed as a positive ratio) at the 5th and 7th day (28.31±8.79, 82.35±8.67; 31.17±11.23, 79.61±10.08; 27.61±10.28, 92.79±8.48; 23.46±11.53, 83.76±5.47) differed significantly (P<0.001). CD80, CD86, HLA-DR and CD83 expression in CHB-H group was 18.57±10.22, 24.16±10.46, 17.87±10.38, 14.28±6.77 at the 5th day and 42.46±9.22, 40.72±11.24, 48.57±12.51, 22.25±11.2 at the 7th day, without significant difference in all markers(P>0.05). And the CHB-L group showed similar results (20.13±10.43, 39.48±7.63 ; 19.16±6.45, 34.58±13.44 ; 22.24±12.37, 43.73±16.87; 15.39±10.44, 21.68±6.38; P>0.05).3. TLR3 expression on mDC of control group, CHB-H group and CHB-L group were 8.89±4.21, 7.46±4.11 and 28.49+7.72 by imDC and 5.71±4.33, 6.68±3.17 and 6.15±3.88 respectively. The expression of TLR3 on imDC of control group was significantly high compared with both CHB groups (P<0.001), however it did not differ significantly between the 2 groups (P>0.05). The expression of TLR3 on imDC of control group was significantly higher than that on mDC (P<0.001) however the difference was nonsignificant in CHB patients (P>0.05).4. The products of IL-12, IL-10 decreased significantly in patients (P<0.01) also (41.54±20.75, 137.62+43.38 in control group; 26.22±11.20, 31.84±12.35 in CHB-H group and 31.59±14.16, 42.11±10.76 in CHB-L group).5. TLR3 expression on DCs of CHB patients was not correlated to peripheral HBV DNA load. And TLR3 expression on mDCs had no difference from that on imDCs (p>0.05).Conclusion1. DCs, while induced in vitro by cytokine IL-4 and GM-CSF, proliferate with its immaturative pattern, and with its maturative pattern after adding TNF- α to meadium.2. TLR3 expression on peripheral DCs of CHB patients decreased.3. TLR3 is qualified to be surface marker of imDC.4. Changes of TLR3 expression on DCs had influence on DCs phenotype and its functions.