Research On The Function Of Peripheral Blood Dendritic Cells From Tradional Chinese Medicine Type Of Syndrome That Stagnation Of The Liver-Qi And Deficiency Of The Spleen And Liver And Gallbladder Damp-Heat From Patients With HBV Chronic Infection Of Diff

Part-ⅠCulture and Identification of Dendritic Cells From Healthy Human Peripheral Blood In VitroObjective:To establish the cultivated method of dendritic cells (DCs) in vitro.Methods:The adherent PBMCs were obtained from the flesh blood from normal adult was isolated by ficoll density gradient centrifugation, and cultured with granulocyte/macrophage colony stimulating factor (GM-CSF)1000u/ml interleukin4(IL-4) 500u/ml, The morphologic characteristics of these cells were observed with invert optical microscope.Electron microscope was employed to analyse the morphological features of DCs,The expression and identifition of phenotypic molecules of DCs was detected with flow cytometry (FCM).Results:Large numbers of dendritic cells could be obtained from human peripheral blood by combination of GM-CSF and IL-4, whose typical morphology was tested by electron microscope, By using flow cytometric cellsurface marker analysis, the level of expressing FITC-CD1a 49.52±5.11%、PE-HLA-DR 91.19±4.29%. FITC-CD80 59.42±7.05%. PE-CD86 86.21±2.39%.Conclusion:The results indicate that by combination of GM-CSF and IL-4, large numbers of dendritic cells could be obtained from human peripheral blood,which may facilitate further studies of DCs and its clinical application. Part-ⅡResearch on the function of peripheral blood dendritic cells From Tradional Chinese Medicine Type of Syndrome That Stagnation of the Liver-Qi and Deficiency of the Spleen and Liver and Gallbladder Damp-Heat from patients with HBV chronic infection of different statusObjective:To study the function of DCs derived from peripheral blood in the patients with both liver stagnation and spleen deficiency and liver and gallbladder damp-heat syndrome of different HBV-infected status in traditional Chinese medicine(TCM),to learn the Immune expression trait between the two card-type in chronic HBV infection ,Provide an objective basis for TCM differentiation of symptoms and signs, and further understand the essence of chronic HBV infection.Methods:In this study, We incubated and induced DCs from peripheral blood mononuclear cells (PBMCs) of the patients with both liver stagnation and spleen deficiency and liver and gallbladder damp-heat syndrome of different status HBV-infected in TCM, Then the morphological features were observed by invert optical microscope and electron microscope,The expression of HLADR, CD80,CD86 and CDla were determined with flow cytometry, and the level of IL-12 produced by DCs were analyzed by ELISA. The lymphocyte allo-stimulatory capacity of DCs was evaluated through MTT assay,compared with 10 case of healty persons.To analyse the deference between two card-type DCs which treated by statistical method.Result:1. Morphological observation:Typical dendritic cells could be obtained from the chronic HBV infected patients and normal human peripheral blood mononuclear cells by combination of GM-CSF and IL-4,but there was no statistical significant between chronic HBV-infected patients and healthy persons on morphology.2. The expression rate of DCs surface molecules、the level of IL-12 in the supernatants and induction of MLR in patients with chronic HBV-infected was significantly lower than healthy persons (P<0.05), especially in HBV carriers was decreased obviously;3. In Period of immune tolerance (chronic HBV carriers), the expression rate of CD86 (66.83±6.53%), CD80 (15.19±3.37%), HLA-DR (78.40±6.68%)in liver stagnation and spleen deficiency syndrome was lower than (79.35±3.15), (25.69±6.13%), (85.70±7.34%)those of liver and gallbladder damp-heat syndrome group,and the difference was statistically significant (P <0.05), while the expression rate of CDla was no statistical significance (P> 0.05); The levels of IL-12 produced by DCs and the ability of DCs to stimulate T cells respond liver stagnation and spleen deficiency syndrome lower than those in liver and gallbladder damp-heat syndrome group, but they are no significant difference (P> 0.05). In Period of Immune clearance (chronic hepatitis B), the expression rate of CD86 (81.22±3.39%), CD80 (30.56±6.55%), CD1a (45.19± 6.99%), HLA-DR (86.26±4.91%) in liver stagnation and spleen deficiency syndrome was lower than CD86 (87.04±2.70%), CD80 (36.60±11.79%), CD1a (47.52±6.17%), HLA-DR (92.66±2.51%) those of liver and gallbladder damp-heat syndrome group,but only CD86、HLA-DR was statistical significance (P<0.05), while CD80 and CD1a was no significant difference(P>0.05);The levels of IL-12 produced by DCs and the ability of DCs to stimulate allogeneic T cells response in liver stagnation and spleen deficiency syndrome lower than those of liver and gallbladder damp-heat syndrome group, and there is statistically significant difference between the two groups (P<0.05).4. The expression rate of CD86 (66.83±6.53%vs81.22±3.39%), CD80 (15.19±3.3%vs30.56±6.55%), CDla (45.43±7.56%vs45.19±6.99%), HLA-DR (78.40±6.68%vs86.26±4.91%); belong to the same period of liver stagnation and spleen deficiency syndrome in the immune tolerance and immune clearance phase,and the expression of CD86, CD80, HLADR difference with statistical significance between the two groups(P<0.05);the expression rate of CD86 (79.35±3.14%vs87.04±2.70%), CD80(25.69±6.13%vs36.60±11.79%), CD1a(46.94±7.02%vs47.52±6.17%), HLADR(85.70±7.34%vs92.66±2.51%) belong to the same period in liver and gallbladder damp-heat syndrome in Immune tolerance and the immune clearance phase; and the expression of CD86, CD80, HLADR difference with statistical significance between the two groups(P <0.05). The levels of IL-12 produced by DCs and the ability of DCs to stimulate allogeneic T cells response in immune tolerance phase that syndrome of liver stagnation and spleen deficiency lower than those of the immune clearance phase,but there was no significant difference between the two groups(P> 0.05); The levels of IL-12 produced by DCs and the ability of DCs to stimulate allogeneic T cells response in immune tolerance phase that syndrome of liver and gallbladder damp-heat lower than those of the immune clearance phase,and there was significant difference between the two groups (P<0.05).Conclusions:Studies have shown both liver stagnation and spleen deficiency and liver and gallbladder damp-heat syndrome of different HBV-infected status in traditional Chinese medicine(TCM) have the defective function and immature phenotype of dendritic cells, especially in liver stagnation and spleen deficiency syndrome was decreased obviously,thus difficult to clear the HBV, and lead to chronic.In word, DCs phenotype and function of detection of disease at the same stage to provide objective indicators for the dialectic of Chinese medicine, to reflect the immune expression and immunological characteristics of the connotation of two-card-type, with a view to better clinical Diagnosis and Treatment Services; From the dialectical perspective of Chinese medicine, belong to the same syndrome of HBV infection in different status, and its immunological characteristics Whether or not exist significant differences worthy of further study,to better understand the essence of TCM differentiation of symptoms and signs.