| Objective1. To understand if there was any difference on the function ofmonoeyte-derived dendritic cells between healthy donors and patients withchronic hepatitis B virus infection, and explore the mechanism of HBVpersistence infection.2. To evaluate the changes on the antigen presentation of dendritic cellsin the patients received the drug therapy of Minor Bupleurum Tablets fromthe perspectives of cellular immunology methods, and to find out thepossible methods to break the status of HBV persistence infection.3. To investigate the effect on the antigen presentation and specific CTLactivity by HBcAg-pulsed dendritic cell, thus it may be a potential methodto recover or up regulate the function of dendritic cells in patients withchronic hepatitis B infection.Methods1. Generation of monocyte-derived DCs in peripheral blood from patientswith chronic hepatitis B infection: Monocytes were isolated from bloodsamples containing heparin obtained from patients with chronic hepatitisB infection using Ficoll density gradient centrifugation. After 3 h ofculture in 6-well plates (2×10~6 cell/well) the non-adherent cells wereremoved by washing with phosphate-buffered saline (PBS) and cultured inRPMI supplemented with 10% fetal bovine serum. Recombine humangranulocyte macrophage-colony stimulating factor (100ng/ml,) and IL-4(500IU/ml) were added to DCs cultures. Also in the day 7, Recombinant human TNF-α(60ng/ml) was added in the culture. The culture was lasted to day11, and then collect the mature DCs, and the presence of DCs surfacemarkers was evaluated by flow cytometry using a panel of phycoerythrin-orfluorescein isothiocyanate-conjugated monoclonal antibodies specificfor CD1a, CD80, CD83, CD86 and HLA-DR. The allogeneic mixed leukocytereaction (AMLR) was used to test the capacity of DCs to initiate a primaryimmune response.2. The reproductive activity stimulated by dendritic cell and IFN-γsecreted by CTL were influenced by HBcAg pulse: in this part, we used HBcAgwith strong immunogenicity to pulse the DCs from patient after therapy.Then we analyzed the expression of DC's surface markers and the abilityto stimulate lymphocytes proliferation through FCM and AMLR before andafter HBcAg pulsed. ELISPOT was an effective method to evaluate the CTLactivity, which mainly reflected by the level of IFN-γscreted fromactived CTL. Therefore, we can consider if there was something to do withthe improved clinical index and the celluar immunity.3. The effect of CTL response induced by HBcAg-pulsed dendritic cell: themagnitude of CTL response could be determined by cytotoxicity assay.HBcAg-pulsed dendritic cells was as stimulus, autologous peripheral bloodlymphocytes as effector and HepG2/HepG2.2.1.5 cell lines as targets, thecytolytic activity was tested by lactate dehydrogenase releasedexperiment.Results1. We successfully induced the monocyte-derived dendritic cells from 5healthy donors and 5 patients with chronic hepatitis B infection. Thedendritic cell we generated displayed typical characteristics onmorphology. There was significant difference in the expression ofdendritic cell's surface markers such as CD1a, CD80, CD83 and CD86 fromhealthy donors or patients. But it was no significant difference in theexpression of HLA-DR between two groups. We found that the stimulateindex(SI) detected at the mix ratio 1:60(DC: autologous T cells) inhealthy donors and patients was 2.20±0.14 and 1.84±0.03 respectively,which had significant difference statistically. 2. It was no significant difference in the expression of dendritic cell'ssurface markers such as CD1a, CD80, CD83, CD86 and HLA-DR from patientsbefore or after therapy, and the same to DCs pulsed by HBcAg in theexpression of CD1a, CD80, CD86 and HLA-DR. But we found that it wasdifferent in the expression of CD83 after HBcAg pulsed. The SI of healthygroup, before therapy group, after therapy group and HBcAg-pulsed groupwas 2.20±0.14, 1.85±0.03, 1.95±0.03, 2.06±0.02 and 1.95±0.03,respectively, which had difference between groups statistically.HBcAg-pulsed DC could active more cytotoxic T lymphocytes, whichsecreted high level IFN-γ, compared to the DC without antigen loaded.3. The peripheral blood monocyte-derived dendritic cells of five treatedCHB patients were induced by HBcAg, the level of specific lysis of twotarget cells, HepG2 and HepG2.2.1.5, are (57.93±3.00)%, (77.73±3.63)% respectively, there was a significant difference between them.The peripheral blood monocyte-derived dendritic cells which were notinduced by HBcAg could lyse the two target cells as same as the inducedgroup, the level of specificlysisare (47.42±2.02)%, (64.45±1.76)% respectively, and there was also a significant difference betweenthem. Autologous PBLs was induced by HBcAg-non pulsed dendritic cellsand not induced by dendritic cells could lyse the two target cells, HepG2and HepG2.2.1.5. There was also a significant difference between thelysis from two groups. Autologous PBLs was induced by HBcAg-pulseddendritic cells and not induced by dendritic cells could lyse the twotarget cells, HepG2 and HepG2.2.1.5. There was also a significantdifference between the lysis from two groups. The peripheral bloodmonocyte-derived dendritic cells of five treated CHB patients wereinduced HBcAg or not. The percentage of CTL cytotoxin which activatedby the dendritic cells and HepG2 were (56.40±1.57)%VS (46.60±4.12)%,(59.17±2.64)%VS (49.20±0.46)%,(55.67±0.71)%VS (48.00±1.44)%,(58.23±1.10)%VS(46.60±0.89)%和(62.00±0.44)%VS(46.70±0.75)% respectively. There wasn't significant difference in No. 1 patient,otherwise in other patients. The monocyte-derived DCs in peripheralblood from five patients with chronic hepatitis B infection. Thepercentage of CTL cytotoxin which activated by the dendritic cells andHepG2 were (74.67±1.29)%VS (61.90±0.60)%,(81.77±0.72)%VS (63.73±0.21)%,(74.17±1.40)%VS (64.47±0.85)%,(76.33±1.34)%VS(65.73±1.21)%和(81.70±1.45)%VS(64.40±0.70)%respectively,and there was a significant difference in two groups.Conclusions1. The ability of peripheral blood dendritic cell from patients withchronic hepatitis B infection to stimulate reproduction of lymphocyteswas down regulation. Considering the comparison research of DC'ssurface marker, we believed that the down regulation was related to thelower expression level of adhesive molecules of DC for the low levelexpression of adhesive molecules may lead to down regulation of antigenpresentation ability of DC and hence affect its stimulative ability.Probably, an enhanced antigen presentation ability of DC is beneficialto the clearance of virus in HBV infecting patients.2. There is no obvious difference of expression of CHB patient's DC'ssurface marker between pre-therapy group and post-therapy group thattreated by Minor Bupleurum Tablets, but the level of expression is upregulated. This phenomenon suggested that Minor Bupleurum Tablets can upregulate the expression of adhesive molecules of DC. The up regulationof CD83 expression level of DC after stimulated by HbcAg may relate tothe degree of maturity and the antigen presentation ability. The resultsof surface marker expression level of DC, AMLR and ELISPOT suggested thatHbcAg charge could up regulate the antigen presentation ability of DC andenhance humoral and cellular immunological response induced by DC.3. The DC of CHB patients could present HbcAg effectively and induce HbcAgspecific CTL response after stimulated by HbcAg. Our experiment confirmedthat monocyte-derived DC of CHB patients could induce specific CTL againstHBV after stimulated by HbcAg. The result suggested that DC may act asan immune adjuvant in the immune therapy of CHB.4. The Minor Bupleurum Tablets was a effective remedy in the therapy ofCHB and it could regulate the expression of a series of cell factor. Ourwork suggested that Minor Bupleurum Tablets could up regulate the functionof cellular immunity of patients which probablly relates to theenhancement of antigen presentation ability of DC. The prospect of usingantigen-charge DC as an immune adjuvant in the immune therapy of CHB was promising.
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