| ObjectiveA large number of studies show that traditional Chinese medicine has a unique advantage in the treatment of Chronic Hepatitis B (CHB). To detect and investigate the peripheral blood HBcAg18-27 table specific cytotoxic T lymphocyte (CTL) number and function in patients with CHB by the treatment of Nourishing Kidney and Eliminating Toxicity, parts clarify Nourishing Kidney and Eliminating Toxicity anti HBV possible target point and immune activation mechanism, and provide more comprehensive scientific basis for Nourishing Kidney and Eliminating Toxicity.Methods1. Collect the outpatient follow-up of CHB patients in Guangzhou University of Chinese Medicine First Affiliated Hospital and apply HLA-A2 as screening index. By using the method of flow cytometric fluorescence immunoassay to screen HLA-A2 (+) in chronic hepatitis B patients with a total of 32 cases, randomly divided into western medicine group (15 cases) and the Chinese-western medicine group (17 cases). Another 10 cases of HLA-A2 (+) healthy volunteers was normal control group. Western medicine in the treatment group received nucleoside analogues treatment, the Chinese-western medicine group received nucleotide analogues and Nourishing Kidney and Eliminating Toxicity.2.Collect peripheral venous blood 20ml of the normal group, western medicine group and Chinese-western medicine group when they first into the group and after 6 months of treatment, use density gradient cen-trifugation separation of peripheral blood mononuclear cells (PBMC), and frozen in liquid nitrogen.3. Cell recovery after culture, using MHC Ⅰ-pentamer and flow cytometry detection techniques to directly detect the number of HBcAg18-27 epitope specific CTL in peripheral blood.4. Cell recovery after culture, using MHC Ⅰ-pentamer and intracellular cytokine staining (ICS) flow cytometry detection techniques to detect the expression of Cytotoxic effector molecule perforin、Granzyme B and cytokine IFN-gamma on HBcAg18-27 epitope specific CTL in peripheral blood.5. Cell recovery after culture, using Enzyme Linked Immunologic Spot Assay(ELISPOT) technique to detect the response frequency and average reaction intensity of IFN- gamma which secretion by HBV core 18-27 peptide induced specific CTL.Results1. Effect of Nourishing Kidney and Eliminating Toxicity on the number of HBcAg18-27 epitope specific CTL in peripheral blood.Before treatment, compared with normal group, the frequency of spe- cific CTL (Pentamer+CD3+CD8+) was high in Chinese-Western medicine group and Western medicine group, the difference was statistically significant (P< 0.05); Compared with before treatment, the frequency of specific CTL was significantly increased both Chinese-Western medicine group and Wes- tern medicine group, the difference was statistically significant (P< 0.05); And the western medicine group compared to the Chinese-Western medicine group,, the frequency of specific CTL significantly increased, the difference was statistically significant (P< 0.05).2.Effect of Nourishing Kidney and Eliminating Toxicity on function of HBcAg18-27 epitope specific CTL in peripheral blood.(1) Combine Pentamer with ICS flow cytometry detection techniques toDetect the expression of Cytotoxic effector molecule perforin, Granzyme B and cytokine IFN-gamma.Perforin:Before treatment, the expression frequency of perforin inspecific CTL (Pentamer+Perforin+cells number accounted for the proportion of the total number of CD3+CD8+T cells) in the normal group, Chinese-Western medicine group and Western medicine group was low, almost no expression of perforin; Compared with before treatment, perforin expres-si on frequency in specific CTL of Chinese-Western medicine group and Western medicine group were increased after treatment, the difference was statistically significant (P < 0.05); And the western medicine group com pared to the Chinese-Western medicine group, the perforin expression frequency in specific CTL of Chinese-Western medicine group was nearly, the difference was not statistically significant(P> 0.05)Granzyme B:Before treatment, The granzyme B expression frequency in specific CTL of the normal group, the western medicine group and Chinese Western medicine group (Pentamer+Granzyme B+cells number accounted for the proportion of the total number of CD3+CD8+T cells) was low, almost no expression; Compared with before treatment, Granzyme B expression frequency in specific CTL of Chinese-Western medicine group and Western medicine group were increased after treatment, the difference was statistically significant (P< 0.05); And the western medicine group com pared to the Chinese-Western medicine group,, the Granzyme B expression frequency in specific CTL of Chinese-Western medicine group was nearly, the difference was not statistically significant(P> 0.05)IFN-gamma:before treatment, the expression frequency of IFN-gamma in specific CTL (Pentamer+IFN-gamma+cell number accounted for the pro-portion of the total number of CD3+CD8+T cells) of Chinese-Western medicine group and Western medicine group was higher than normal group (P< 0.05); compared with before treatment, IFN-gamma expression fre-quency in specific CTL of Chinese-Western medicine group and Western medicine group were both increased, the difference was statistically significant (P< 0.05); And the western medicine group com pared to the Chinese-Western medicine group,, the IFN-gamma expression frequency in specific CTL of Chinese-Western medicine group was higher, the difference was statistically significant (P< 0.05).(2) Use ELISPOT technique to detect the response frequency and average reaction intensity of IFN-gamma which secretion by HBV core 18-27 peptide induced specific CTL.Before the treatment, western medicine group and Chinese-Western medicine group specific CTL responses to HBcAgl8-27I epitopes frequency and average intensity of reaction is low, the difference between the two groups was not statistically significant (P>0.05);Compared with before treatment, western medicine treatment group and Chinese-Western medicine group specific CTL the HBcAg18-27I epitope peptide response frequency and average response intensity have increased, but the difference was not statistically significant (P>0.05); And the western medicine group com pared to the Chinese-Western medicine group, Chinese-Western medicine group specific CTL epitope of HBcAgl8-27I peptide response frequency and average reaction intensity were higher, but the difference was not statistically significant (P>0.05).Conclusions1. Before treatment, Chinese-Western medicine group and Western medicine group CHB patients specific CTL number and expression of cytokines level is low, we believe that, This is related to the presence of HBV specific CTL dysfunction. Normal healthy population CTL level is low, which may be because there is no sustained stimulation of HBV specific antigen in healthy people blood circulation, resulting in low level of HBV specific CTL.2. Nourishing Kidney and Eliminating Toxicity in treating CHB patients peripheral blood specific CTL cells significantly increased and perforin, Granzyme B, IFN-gamma expression in two groups of patients with CHB were showed an upward trend, especially cytokines IFN-gamma, it shows that Nourishing Kidney and Eliminating Toxicity can increase the specific CTL cell number and improve the activation of CTL secretion of cytokines to enhance the CHB patient specific T cell immune function, which may be one of the mechanisms of Nourishing Kidney and Eliminating Toxicity for treating CHB.3. Perform Granzyme B and IFN-gamma in the pentamer" CD3+CD8+cell expression levels were also high, we believe that Nourishing Kidney and Eliminating Toxicity may also have a certain degree of influence on the non-specific CTL or non HBcAgl8-27 table specific CTL. Treating by Nourishing Kidney and Eliminating Toxicity, overall CTL expression levels of perforin、 Granzyme B and IFN-gamma were elevated. Nourishing Kidney and Eliminating Toxicity through multiple targets and links affect the immune system in a variety of immune cells and cytokines play antiviral and immune regulation. |
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