| Purposes:This experiment is used the density-grads centrifugal method to apart the preceding cell of dendritic cell from the normal human peripheral blood,and the preceding cell will be forward differentiation and developed to the dendritic cell with the inducement of the cell factor rhGM-CSF and rhIL-4,and with the help of the rhTNF-αit will develop to the mature dendritic cell.We use the supernatant of the tumor cell line to make conditioned medium in order to research the expression of DC surface molecule,its immune estate in the tumor and virus exsited condition and the LBP effect to DC and the expression of NF-κB cultured in the normal and conditioned medium.We discuss the recurring and developing mechanism of the hepatic cell tumor related to the HBV and LBP effect to the exto experiment hepatic cell tumor and the immune accommodate role to the hepatic cell tumor related to the HBV in order to provide the scientific theory thereunder for the Traditional Chinese Medicine and herbs role in the HCC treatment.Methods:We take the normal person's peripheral blood from the elbow vein in the germfree condition,and use the heparin to anticoagulate.After 6 hours cultivation with the complete medium that contains 10%fetal claf serum in the 25ml volume culture bottlewe can get the mononuclear cell by removing most of the suspended lymphocyte and then induce the cell to differentiation and culture the seprated cells in complete medium that contains 1000μ/ml graulocyte-macrophage colony-stimulating factor and 1000μ/ml interleukin-4.According to the tumor cell culture superntant and the complete meduim volume ratio 1:3,we make the conditioned meduim by the gathered two different hepatica carcinoma cell supernatant(HepG2,HepG2.2.15). This experiment is divided into two parts.The first department is divided into three parts:the blank group,the conditioned medium group with 25%HepG2 cell supernatant and the conditioned medium group with 25%HepG2.2.15 cell supernatant.The second department:we divide the cell into four groups in the first stage,the DC without LBP,the DC group with 50μg/ml LBP,the DC group with 100μg/ml LBP and the DC group with 200μg/ml LBP.We choose the best effective concentration by the MTT.The second stage:total divided into six groups,the normal control group,the negative control group that DC cultured in the conditioned medium, experiment groups which CM and the LBP group and the experiment group which DC cultured in normal circumstance with LBP.The group without LBP is added to TNF-α800μ/ml before collecting the cells for 24 hours.We observe the shape of the DC by the convert microscope and detect the cell surface symbol(CD83,CD86,CD1a,HLA-DR) expression at the seventh day by the flow cytometry.Taking the MTT method to detect the mixed lymphocyte reaction,we collect the different DC groups' superntant and the MLR superntant to detect the secretion of IL-12p70 and IFN-γby the enzyme linked immunosorbent assay.We collect the different groups' DC to distil the nucleus protein and then detect the activity of NF-κB by the enzyme linked immunosorbent assay.Results:1.We get amount of the DC that are typical morphologic characteristic,high ability to stimulate lymphocyte multiplication and high expression of CD83,CD86,CD1a,HLA-DR from normal fresh peripheral vein blood mononuclear cell and cultured with CK rh GM-CSF,rhIL-4,rhTNF-α.2.The shape,the ability to stimulate the lymphocyte,the secretion of IL-12p70 and IFN-γ,and the expression of CD83,CD86,CD1a,HLA-DR of DC which are cultured in two kinds of condition medium made by the different hepatic cellular cancer are obvious lower than the DC cultured in normal circumstance,the difference is obvious and it has statistical meaning.3.The shape,the ability to stimulate the lymphocyte,the secretion of IL-12p70 and IFN-γ,and the expression of CD83,CD86,CD1a,HLA-DR of DC which is cultured in the HepG2.2.15 CM are obvious lower than the DC cultured in the HepG2 CM.4.After add to the LBP in the normal circumstance cultured DC,its surface molecule expression,the ability to stimulate the lymphocyte,the secretion of IL-12p70 and IFN-γare lower than the DC without LBP in the normal circumstance,the difference is obvious and it has statistical meaning.The shape,the ability to stimulate the lymphocyte,the secretion of IL-12p70 and IFN-γ,and the expression of CD83,CD86,CD1a,HLA-DR of DC which is cultured in the two kinds of condition medium added to LBP are obvious higher than the DC cultured in the CM without LBP,the difference is obvious and it has statistical meaning.5.NF-κB expression of DC cultured with LBP group is higher than the DC without LBP,the difference is obvious and it has statistics meaning.The NF-κB expression of DC cultured in the normal circumstance is higher than the DC in the CM,the difference is obvious(p＜0.05) and it has statistical meaning.The NF-κB expression of DC cultured with the two kinds of different hepatic cellular cancer cell superntant is no obvious difference,and it has no statistical meanings.Conclusions:1.The DC preducure cell is indeed in existence in normal peripheral blood mononuclear cell and it can be induced to DC with the rh GM-CSF,rhIL-4 and rh TNF-α。2.The ability to stimulate the lymphocyte,the secretion of IL-12p70 and IFN-γ,and the expression of CD83,CD86,CD1a,HLA-DR of DC can be decreased by the condition medium made by the hepatic cellular cancer cell superntant.This can affect the DC mature lead to its dysfunction,and its nuclear protein NF-κB expression decreased implies that the mechanism perhaps related to the NF-κB signal pathway.3.The LBP can affect the DC surface molecule expression that is cultured in the CM and its immune function,the results of this research implies this perhaps related to meliorate the NF-κB signal pathway.
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