The Study On Dendritic Cell Biology In Different HBV-infected States

AIM Dendritic cells(DCs),the most potent professional antigen processing cells(APC)in vivo,play a crucial role in human immune system. DCs activate host immune response not only by their capturing and processing antigens to MHC-â… andâ…¡molecules on the cellular surface,but also by their enhancing or regulating naive T cells and priming a primary immune response. Now,more and more scholars pay close attention to the correlative study of DCs in HBV-infected patients.In order to get more insights into the immune function of DCs in HBV-infected conditions,we studied the function of DCs derived from peripheral blood in different HBV-infected stages,compared the different function of DCs in different HBV loads and invested if CpG ODN could promote DCs mature and enhance the function of DCs.METHODS(1)We incubated and induced DCs subset populations differentiated from peripheral blood mononuclear cells(PBMCs)of 10 HBV carriers,10 chronic hepatitis B patients and 10 healthy controls in vitro and then detected the expression of HLA-DR,CD80,CD86 by FACS,measured the level of IL-12 in supernatants by ELISA.(2)We detected the HBV-DNA in DCs of 12 chronic hepatitis B patients who's serum HBV-DNA was positive and 10 chronic hepatitis B patients who's serum HBV-DNA was negative by fluorescent quantitation PCR in order to study the relationships between the function of DCs and the loads of HBV in DCs in patients with chronic hepatitis B.(3)We incubated and induced DCs subset populations by adding CpG ODN to the medium to investigate the effect of synthetic immuno- stimulatory CpG ODN(unmethylated CpG motif containing oligodeo- xynucleotides)on the function of DCs in patients with chronic hepatitis B.RESULTS(1)The DCs were enriched from PBMCs and successfull. induced by incubation with interleukin-4(IL-4)and granulocytemacrophag, colony- stimulating factor(GM-CSF)in RPMI-1640 medium containing 10% FCS.On the first day incubation,the PBMCs adhered and aggregated,on the third day incubation,the adherent cells from PBMCs were found to proliferate and had a rapid increase of cell volume over time.On the forth day,part of the cells suspensed in medium.On the fifth day,the number of the cells increase(?) in number,the adherent cells achieved maximum proliferation and ha(?) apophysison on the seventh day incubation.On the seventh day incubation the cells were observated by transmission electron microscope,volume of the cell was large with affluent apophysison in surface,affluent rough endoplasmic reticulum in endochylema,distinct mitochondria structure,full lysosome,big and round nucleus and clear nuclear membrane.The expressions of CD80,CD86,and HLA-DR on DCs surface were identified by using FACS.The results showed that the expression levels of CD80,CD86, and HLA-DR were much lower in patients and HBV carriers than those in healthy volunteers,but there was no statistical significance between patients and HBV carriers.The levels of IL-12 in the supernatants were determined by ELISA on the seventh day incubation,The results showed that the levels of IL-12 were much lower in patients and HBV carriers than those in healthy volunteers,but there was no statistical significance between patients and HBV carriers.(2)HBV-DNA in DCs was detected in 7 patients among 12 chronic hepatitis B patients who's HBV-DNA could be detected in serum,the detection rate of HBV-DNA in DCs was 58.3%.The expressions of CD80,CD86,and HLA-DR on DCs surface were identified by using FACS.The results showed that the expression levels of CD80,CD86,and HLA-DR were more than 58%in healthy volunteers,however,the expression levels were much lower in patients who's serum HBV-DNA could be detected or could n't than those in healthy volunteers,and the levels were lower in patients who's serum HBV-DNA could be detected than could n't.The expression levels of CD80,CD86,and HLA-DR were much lower in patients who's DCs HBV-DNA could be detected or could n't than those in healthy volunteers,and the levels were lower in patients who's DCs HBV-DNA could be detected than could n't.The levels of IL-12 in the supernatants were determined by ELISA.The results showed that the levels of IL-12 in the supernatants were much lower in patients who's serum HBV-DNA could be detected than could n't and those in healthy volunteers, and the levels were lower in patients who's serum HBV-DNA could n't be detected than those in healthy volunteers.The expression levels of IL-12 in the supernatants were lower in patients who's DCs HBV-DNA could be detected than could n't and those in healthy volunteers,and the levels were lower in patients who's DCs HBV-DNA could n't be detected than those in healthy volunteers.The correlation analysis showed that a significantly negative correlation was noted between the expression levels of CD80,CD86, HLA-DR on DCs and the loads of HBV,and also could be found between the levels of IL-12 and the HBV load.(3)On the sixth day incubation,CpG ODN was added to the medium, then the DCs were continued culturing for 24h.The cells were observated by transmission electron microscope.We could see the cells had more affluent apophysison in surface and affluent rough endoplasmic reticulum in endochylema and less vacuolus than cells control.The expressions of CD80,CD86,and HLA-DR on DCs surface were identified by using FACS.The results showed that the expression levels of CD80,CD86,and HLA-DR were much higher when the DCs were co-stimulated with CpG ODN than complete medium controls in chronic hepatitis B patients,HBV carriers and healthy controls.When cultured with complete medium,the expression levels of HLA-DR,CD86,CD80 were much lower in chronic hepatitis B patients and HBV carriers than healthy controls,but there was no significant difference between chronic hepatitis B patients and HBV carriers.When cultured co-stimulated with CpG ODN,the expression levels of HLA-DR and CD86 were much lower in chronic hepatitis B patients than HBV carriers and healthy controls,but there was no significant difference between HBV carriers and healthy controls.The expression levels of CD80 were much lower in chronic hepatitis B patients and HBV carriers than healthy controls,but there was no significant difference between chronic hepatitis B patients and HBV carriers.The levels of IL-12 in the supernatants were determined by ELISA,The results indicated that the levels of IL-12 were much higher when the DCs were co-stimulate with CpG ODN than complete medium controls. When cultured with complete medium or co-stimulate with CpG ODN,the The levels of IL-12 in the supernatants were much lower in chronic hepatitis B patients and HBV carriers than healthy controls,but there was no significant difference between chronic hepatitis B patients and HBV carriers.CONCLUSION(1)chronic hepatitis B patients and HBV carriers had not only an immature phenotype,but also an impaired function.(2)The DCs derived from peripheral blood could be infected by HBV.There was a significantly negative correlation not only between levels of the molecules on DCs surface and the loads of HBV,but also between the level of IL-12 and the HBV load.(3)CpG ODN could promote DCs mature.The study provided experimental found for CpG ODN being a new method to treat chronic hepatitis B.