The Effect Of Gemcitabine On Cellular Immunity Of Mice With Autoimmune Hepatitis

Objective:1.To investigate the effects of gemcitabine(GEM)on Th1,Th2,Th17,regulatory T cells(Treg)and myelosuppression cells(MDSC)and the effects on cell cycle and apoptosis in AIH mice.2.To investigate the changes of the cell of T?B and macrophage function after the treatment of GEM.Methods:8 mice were randomly selected as normal control from 40 male C57BL/6 mice aged 4-6 weeks,the other 32 mice were intraperitoneally injected with 1 ml of S-100 and Freund's complete adjuvant suspension twice on day 1 and 8,to establish the animal model of autoimmune hepatitis(AIH).The model mice were randomly divided into normal saline(NS),gemcitabine(GEM),hormone treatment groups and GEM combined with hormone group.Intervention was initiated on 15 days.Dosage of GEM and hormone group was 100 mg/kg and 1 mg/kg respectively.Hormone injection once a day,NS and GEM injection once a week,0.2 ml by intraperitoneally.After 3 weeks of continuous administration,the proportion of Th1/CD4+T,Th2/CD4+T,Th17/CD4+T,Treg/CD4+T,MDSC in splenic lymphocytes and the function of cell cycle and apoptosis were detected by flow cytometry.The m RNA expression of T-bet,Gata3,Foxp3,ROR?t of splenocytes were detected by q RT–PCR.Proliferation of T cell and B cell in spleen were detected by MTT.The ability of macrophage migration was detected through transwell assay.Results:1.The result of spleen size in each group: compared with the control group,the spleen of each group was obviously swollen;compared with NS group,the swelling degree of GEM,hormone and combination group was decreased;compared with GEM group,the degree of spleen swelling in hormone and combination group was lower.There was no difference between hormone and combination group.2.The result of Th1(IFN-?+CD4+)/CD4+T : compared with the control group,all increased except combination group;compared with NS group,GEM had no difference,while hormone and combination decreased;compared with GEM group,hormone had no difference,combination group decreased;compared with hormone group,combination group decreased.The result of Th2(IL-4+CD4+)/CD4+T: compared with control group,each group increased;compared with NS group,GEM,hormone and combination group decreased;compared with GEM group,hormone and combination group were decreased;there was no difference between hormone and combination group.The result of Th17(IL-17A+CD4+)/CD4+T was same to Th2.3.The result of Treg(CD4+CD25+CD127low/-)/CD4+T: compared with the control group,NS group decreased,there was no difference in the other groups;compared with NS group,GEM,hormone and combination group were increased.There was no difference among GEM,hormone and combination groups.4.The percentage of MDSC in splenic lymphocytes: compared with control group,all groups increased;compared with NS group,GEM,hormone and combination group were increased;compared with GEM group,no difference in hormone group,while combination group increased.There was no difference between hormone and combination group.5.Apoptosis: compared with control group,NS group increased,there was no difference in the other groups;compared with NS group,GEM,hormone and combination group decreased.There was no difference among GEM,hormone and combination groups.6.The cell cycle: compared with control group,G0/G1 phase decreased,S phase increased in all group,G2/M phase had no difference in all groups.There was no difference among NS,GEM,hormone and combination groups.7.The proliferation of T and B cells: compared with control group,all groups decreased;compared with NS group,GEM,hormone and combination group increased;compared with GEM group,hormone group and combination group increased;there was no difference between hormone group and combination group.8.The migration ability of macrophages: compared with control group,all groups were decreased;compared with NS group,GEM,hormone and combination group were increased;compared with GEM group,hormone and combination group were increased.There was no difference between hormone and combination groups.9.The expression of T-bet: compared with control group,NS group increased,there was no difference in the other groups;compared with NS group,GEM,hormone and combination group decreased.There was no difference between GEM,hormone and combination groups.The result of Gata 3 was same to T-bet.The expression of Ror?t: compared with control group,NS group and GEM group increased,hormone group and combination group had no difference;compared with NS group,GEM,hormone and combination group decreased;compared with GEM group,hormone group has no difference,and combination group decreased.There was no difference between hormone and combination groups.The expression of Foxp 3: compared with control group,NS group and GEM group decreased,hormone group and combination group had no difference;compared with NS group,GEM,hormone and combination group were increased;compared with GEM group,hormone group has no difference,and combination group increased;compared with hormone group,combination group increased.10.The ratio of Th1/Th2: compared with control group,NS group increased,there was no difference in the other groups;compared with NS group,GEM,hormone and combination group decreased.There was no difference among GEM,hormone and combination groups.The ratio of Th17/Treg: compared with control group,all groups increased;compared with NS group,GEM,hormone and combination group decreased;compared with GEM group,hormone group and combination group decreased;There was no difference between hormone group and combination group.12.The ratio of T-bet/Gata3 showed no difference among groups.The ratio of Foxp 3/ROR?t: compared with control group,NS group and GEM group increased,hormone group and combination group had no difference;compared with NS group,GEM,hormone and combination group decreased.compared with GEM group,hormone group and combination group decreased;there was no difference between hormone group and combination group.Conclusion:GEM can reduce the ratio of Th1,Th2,Th17 in AIH mice and increase Treg and MDSC.The balance of Th1/Th2 moves toward Th2,and the balance of Th17/Treg ratio moves toward Treg.GEM has no effect on cell cycle,but can reduce apoptosis.GEM can enhance the functions of T cells,B cells and macrophages.But GEM effect is not as good as hormone and combination treatment.GEM has hormone-like regulatory effect on immune cells of AIH mice.