| Objectives:Muramyl dipeptide(MDP)is a kind of oligo-peptide with adjuvant effects derived from bacteria.In recent years,MDP and its derivatives have been widely researched as adjuvants to enhance the efficacy of vaccines.Exploring the adjuvant effects of MDP on HIV DNA vaccine is meaningful for promoting the development of HIV DNA vaccine.Methods:18 mice were randomly divided into three groups:blank group,HIV DNA vaccine group and "HIV DNA vaccine+MDP" group,(n=6).HIV Gag DNA vaccine and HIV Env DNA vaccine were cultured in E.coli.Using MDP as an adjuvant,mice were immunized with above-mentioned HIV DNA vaccine for three times.The interval between each injection is two weeks.Two weeks after the final immunization,immune cells in mice were collected,and flow cytometry was used to detect the changes of IL-2,IL-4,TNF-?,IFN-? secreted from CD4+T cells and CD8+T cells,and detect the expression of CD80 and CD86 on the surface of dendritic cells.Serum samples were collected,and diluted by 100-,1,000-,and 10,000-fold.ELISA was used to detect the production of Gag-and Env-specific IgG.Results:Flow cytometry results showed that:?For the expression of IL-2,IL-4,TNF-? and IFN-?,there was no significant difference between the Gag-specific CD4+T cells and CD8+T cells.?The secretion of IL-2 from Env specific CD4+T cells was statistically significant in the "HIV DNA vaccine+MDP" and the blank group(P<0.05),while the secretion of other cytokines was not significantly different in the HIV Env specific CD4+T cells from the three groups(P>0.05).?For the expression of IL-2,IL-4,TNF-a and IFN-?,there was no significant difference between the Env-specific CD8+T cells.?Similarly,there was no significantly different in the expression of CD80 and CD86 of dendritic cells among the three groups(P>0.05).ELISA data showed that:?When the mouse serum was diluted by 100,1000,and 10000 folds,the Gag specific IgG response in "HIV DNA vaccine+MDP" group were significantly stronger than that in HIV DNA vaccine alone group(P<0.05);?There was no statistically significant difference in Gag specific IgG antibody response between the HIV DNA vaccine group and the blank group when the mouse serum was diluted by 100,1000,and 10000 folds(P>0.05);?When the serum was diluted by 1000 and 10000 folds,the Env specific IgG antibody response in "HIV DNA vaccine+MDP" group were significantly stronger than that in HIV DNA vaccine alone group(P<0.05).When the serum of the mice was diluted by 100 times,there was no significant statistical difference between the two groups(P>0.05);?When the serum was diluted by 100 fold,the Env specific IgG antibody response in HIV DNA vaccine were significantly stronger than that in blank group(P<0.05);There was no statistically significant difference in Env specific IgG antibody response between the HIV DNA vaccine group and the blank group when the mouse serum was diluted by 100,1000,and 10000 folds(P>0.05).Conclusions:(1)In the mice model,MDP can significantly enhance IgG response induced by HIV DNA vaccine(Gag and Env).(2)For HIV DNA vaccine-induced cell responses,there are no significant difference between "HIV DNA vaccine+MDP" group and HIV DNA vaccine alone group.Objective:Investigate the diagnostic methods and indicators of primary biliary cholangitis(PBC).Methods:Sixty-eight cases of liver perforation,including 55 cases of PBC and 13 cases of Primary Biliary Cholangitis-Autoimmune Hepatitis Overlap Syndrome(PBC-AIH OS),were collected from the pathology department of Kunming medical university from January 2015 to March 2019.The 55 patients of PBC were divided into 28 patients of AMA-M2-positive PBC group and 27 patients of AMA-M2-negative PBC group according to the titer of the patients' serum anti-mitochondrial antibody type 2(AMA-M2);And were divided into 15 cases of PBC ??14 cases of PBC ??9 cases of PBC? and 7 cases of PBC ? according to the stages of PBC.Gender,age,Alanine Aminotransferase(ALT),Aspartate Aminotransferase(AST),Alkaline phosphatase(ALP),Gamma-glutamyl transpeptidase(GGT)and other clinical data of the patients were collected to evaluate the changes of important laboratory indicators,the expressions of AMA,Kelch like protein 12(KLHL12)and Hexokinase 1(HK1)in PBC and PBC-AIH OS were detected by immunohistochemical method,and the clinical diagnostic value of AMA,KLHL12 and HK1 in PBC were analyzed,so as to provide a new autoantibody marker for the clinical diagnosis of PBC.Results:(1)There were no statistically significant differences in gender,age,ALT,AST,ALP and other clinical indicators between the AMA-M2-positive PBC,the AMA-M2-negative PBC,and the PBC-AIH OS group.(2)Immunohistochemical results showed that:AMA is mainly expressed in the cytoplasm of bile duct epithelial cells and hepatocytes;KLHL12 is mainly expressed in the cytoplasm of liver cells;HK1 is mainly expressed in the cytoplasm and membrane of Kupffer cells,bile duct epithelial cells and inflammatory cells.The difference in AMA positive expression rate and expression intensity was statistically significant between the AMA-M2-positive PBC group and the AMA-M2-negative PBC group(P value was 0.003 and 0.012,respectively),while there was no statistically significant difference between the other two groups(P>0.05).The difference in KLHL12 expression rate and expression intensity between the AMA-M2-positive PBC group and the AMA-M2-negative PBC group was statistically significant(P=0.003 and 0.012,respectively),while there was no statistically significant difference between the other two groups(P>0.05).The difference in HK1 positive expression rate between the AMA-M2-positive PBC group and the AMA-M2-negative PBC group was statistically significant(P=0.042).The difference in HK1 expression intensity was statistically significant in the AMA-M2-positive PBC group and the PBC-AIH OS group(P=0.026),while there was no statistically significant difference in the other two groups(P>0.05).In different stages of PBC,there was no significant difference in the expression intensity and positive rate of AMA,KLHL12 and HK1(P>0.05).Conclusions:(1)The expression sites of AMA,KLHL12 and HK1 in AMA-M2-positive PBC,AMA-M2-negative PBC and PBC-AIH OS groups were not different.(2)The expression of positive rate and positive intensity in AMA,KLHL 12 and HK1 had no significant correlation with PBC stages.(3)Among the three indicators tested in this study,the positive rate of KLHL12 expression was the highest in both the AMA-M2-positive PBC group and the AMA-M2-negative PBC group,which may be helpful for the clinical diagnosis of PBC.(4)KLHL 12 and HK1 have a high positive rate in the AMA-M2-negative PBC,which may be helpful for the diagnosis of patients with AMA-M2-negative PBC.(5)The results show that KLHL12 and HK1 may not be used as the differential diagnosis indicators of PBC and PBC-AIH OS. |
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