Mechanism Of The Tumor Necrosis Factor-? Induced Protein 8 Like-2 In The Apoptosis Of CD4+T Lymphocytes In Postburn Sepsis

PART1 THE EFFECT OF TIPE2 ON THE APOPTOSIS OF CD4+ T LYMPHOCYTES IN MICE AND ITS POTENTIAL MECHANISMObjective In the present study,We isolated and cultured CD4+ T lymphocytes from spleen of normal mice,and TIPE2-small interference RNA(siRNA)/TIPE2 overexpression lentiviral vector would been transfected into CD4+ T cells to silence or overexpress expression of TIPE2 in CD4+ T lymphocytes.Through a observation of the change on apoptosis of CD4+T lymphocyte after inhibition or overexpression expression of TIPE2,the effect of TIPE2 on the apoptosis of CD4+ T lymphocytes in mice and its potential signaling pathway were furthe explored.The aim was to search for and screen the regulation of CD4+ T lymphocytes apoptosis by TIPE2,and to analyze whether TIPE2 regulates the apoptosis of T CD4+ lymphocytes in mice by the mitochondrial signaling pathway.Methods CD4+ T lymphocytes of normal Balb/c mice were isolated and cultured from spleen by using magnetic cell sorting system in vitro,and TIPE2-small interference RNA(siRNA)/TIPE2-RNA(silence/TIPE2 overexpression)lentiviral vector been transfected into CD4+ T cells.(1)Apoptosis detection of T CD4+lymphocytes:The apoptosis of CD4+ T lymphocytes was detected by fluorescence labeled Annexin-V/PI double staining method.(1)The expressions of TIPE2,Smad2/Smad3,(P)-Smad2/P-Smad3and Bcl-2/Bim protein in CD4+ Tregs were detected by Western blot.(3)The mitochondrial membrane potential in CD4+T lymphocytes was analyzed by flow cytometry.(4)Alteration of cytochrome C in CD4+T lymphocytes was detected by confocal microscopy.(5)Acticities of caspase-3,caspase-8,caspase-9 in CD4+ T lymphocytes were analyzed by chemical colorimetric assay.And analyzed data statistically in each group,then concluded.Results Separation of mouse spleen CD4+ T lymphocytes by MACS,high purity was obtained.pushing Kate inside.meanwhile the transfection efficiency of TIPE2 interference and over expression is high.Western blot assay displayed when TIPE2 gene silencing expression of TIPE2 protein was down regulated in mouse spleen CD4+T lymphocyte,on the contrary,overexpression of TIPE2 gene increased the expression of TIPE2 protein.The apoptosis rate in TIPE2 group was the highest,and the apoptosis rate of CD4+T lymphocyte in siTIPE2 group was significantly lower than that of the other 3 groups except for normal group(P<0.05).The expressions of Smad2/Smad3 did not decrease in siTIPE2 group,which the expressions of P-smad2/P-Smad3was decreased significantly(P<0.05).Expression of Bcl-2were significantly higher than that in the other groups(P<0.05),while the expression of Bim was decreased obviously(P<0.05).The mitochondrial membrane potential in CD4+T in siTIPE2 group was decreased significantly(P<0.01),expression of cytochrome C was lower than that of the other groups except for normal group(P<0.05).Activities of caspase-8,caspase-9 in siTIPE2 group was lower than that of the other groups(P<0.05).Activities of caspase-3,caspase-8,caspase-9 in TIPE2 group were significantly increased(P<0.05).Conclusions TIPE2 gene silencing was expressed,and the activation of Smad2/Smad3,TGF beta 1 downstream signal molecule,decreased in spleen of mouse spleen CD4+ T lymphocytes,and decrease the expression of Bim,promote the expression of anti apoptosis protein Bcl-2,reduce the release of cytochrome C and caspase family activity,finally reducing the apoptosis of CD4+T lymphocytes.TIPE2 might have stimulative apoptosis effects by regulating CD4+ T lymphocytes mitochondrial membrane permeability and releasing apoptosis gene.PART2 THE EFFECT OF TIPE2 ON THE APOPTOSIS OF CD4+ T LYMPHOCYTES IN MICE AFTER THERMAL INJURYObjective The lentivirusof small interference RNA(siRNA)and over expressionwere usedto knock down and up-regulatethe TIPE2 expression through tail vein injection.Two weeks following tail vein injection,we maked a model of severe burn in mice.To investigate the effect of tumor necrosis factor-a induced protein 8 like-2 on apoptosis of CD4+T lymphocyte in thermal injury murine model.With different TIPE2 treatment conditions observations,the present study was performed to investigate the potential role,regulatory mechanism and interventional strategy of TIPE2 in mediated cellular immune dysfunction after major burns,which might be of significance in understanding the novel insights into pathogenesis of abnormal host immune response in the development of postburn sepsis.Methods Male mice were randomly allocated into six groups,which were sham group,burn group,burn with lentivirus-RNAi-TIPE2 transfection group,burn with negative control group,burn with lentivirus-TIPE2over expression transfection group andburn with TIPE2 negative control group.The lentivirusof small interference RNA(siRNA)and over expressionwere usedto knock down and up-regulatethe TIPE2 expression.Through tail vein injection,the last four groupmice wereindividually injected with lentivirous vector via the tail vein.Two weeks following tail vein injection,they were sacrificed at 24 hours after postburn.CD4+ T were purified from spleen of the BALB/c mice by using magnetic cell sorting system.The expressions of TIPE2,Smad2/Smad3,(P)-Smad2/P-Smad3and Bcl-2/Bimprotein in CD4+ Tregs were detected by Western blot.The mitochondrial membrane potential in CD4+T was analyzed by flowcytometry.Alteration of cytochrome C in CD4+ T was detected by confocalmicroscopy.Acticities of caspase-3,caspase-8,caspase-9 in CD4+ T wereanalyzed by chemical colorimetric assay.Results Down-regulation of TIPE2 promoted apoptosis of CD4+T lymphocytein siTIPE2-burn group,which the expressions of P-smad2/P-Smad3was decreased significantly.Expression of Bcl-2were significantly higher than that in the rest groups(P<0.05),while the expression of Bim was decreased obviously(P<0.05).The mitochondrial membrane potential and expression of cytochrome C in CD4+T in siTIPE2-burn group were down regulated except for sham group,Activities of caspase-3 was lower than that of TIPE2-burn group,caspase-8,caspase-9 in CD4+T decreased compared with other groups.The apoptosis rate of TIPE2-burn group is the highest,Smad2/Smad3 in TIPE2-burn group was higher than that of sham group,the expression of P-smad2/P-Smad3 was significantly increased compared with the other groups,The mitochondrial membrane potential in CD4+T in TIPE2-burn group was decreased significantly(P<0.01),expression of cytochrome C markedly elevated(P<0.01).Activities of caspase-3,caspase-8,caspase-9 in CD4+T were obviously higher than those of others(P<0.05).Conclusions TIPE2 gene silencing was expressed,Smad2/Smad3 phosphorylation and the expression of Bim decreased,the expression of anti apoptosis protein Bcl-2 increased,which reduced the release of cytochrome C and caspase family activity,finally reducing the apoptosis of CD4+T lymphocytes after thermal injury.On the contrary,TIPE2 gene promoted the apoptosis of CD4+T lymphocytes after thermal injury.PART3 THE EFFECT OF TIPE2 ON THE APOPTOSIS OF CD4+ T LYMPHOCYTES IN PERIPHERAL BLOOD LEUKOCYTES AMONG HEALTHY INDIVIDUALSObjective CD4+ T lymphocytes of healthy people were isolated and cultured from peripheral blood by using magnetic cell sorting system,and TIPE2-small interference RNA(siRNA)/TIPE2-RNA(silence/TIPE2 overexpression)lentiviral vector been transfected into CD4+ T cells.With both The main research contents include various aspects as follows:(1)to investigate changes on apoptosis of CD4+T lymphocyte in peripheral blood of healthy people.(2)to study the apoptosis and its signaling pathway of CD4+T lymphocyte mediated by TIPE2.(2)to evaluate its clinical significance and practical value.Methods Adult healthy people were divided into 5 groups,which were normal group,RNAi-TIPE2 transfection group(siRNA-TIPE2),siTIPE2-negative group,TIPE2overexpression transfection group and TIPE2 negative control group.CD4+ T lymphocytes of healthy people were isolated and cultured from peripheral blood by using MiniMACS immunomagnetic cell sorting system.The expressions of TIPE2,Smad2/Smad3,(P)-Smad2/P-Smad3and Bcl-2/Bimprotein in CD4+ Tregs were detected by Western blot.The mitochondrial membrane potential in CD4+T was analyzed by flowcytometry.Alteration of cytochrome C in CD4+ T was detected by confocalmicroscopy.Acticities of caspase-3,caspase-8,caspase-9 in CD4+ T were analyzed by chemical colorimetric assay.Results The apoptotic rate of CD4+T lymphocytes of healthy people from high to low was TIPE2 group,normal group and siTIPE2 group(P<0.05).There was no significant difference in the expressions of Smad2/Smad3 among siTIPE2 group and other groups,which the expressions of P-smad2/P-Smad3 was decreased significantly(P<0.05).Expression of Smad2/Smad3 were significantly higher than that in the other groups,while the expression of P-smad2/P-Smad3 TIPE2 group also elevated obviously.Expression of Bcl-2 in siTIPE2 group were significantly higher than that in the other groups,while the expression of Bim was decreased obviously,and vice-versa in TIPE2 group(P<0.05).The mitochondrial membrane potential in CD4+T in siTIPE2 group was elevated significantly,expression of cytochrome C was lower than that of the other groups(P<0.05),and vice-versa in TIPE2 group.Activities of caspase-3,caspase-8 and caspase-9 in siTIPE2 group was lower than that of the other groups(P<0.05).Activities of caspase-3,caspase-8,caspase-9 in TIPE2 group were significantly increased(P<0.05).Conclusions TIPE2 gene silencing was expressed,Smad2/Smad3 phosphorylation and the expression of Bim of CD4+T lymphocyte in peripheral blood of healthy people decreased,the expression of anti apoptosis protein Bcl-2 increased and the expression of Bim reduced,which decreased the release of cytochrome C and caspase family activity,finally reducing the apoptosis of CD4+T lymphocytes.On the contrary,TIPE2 gene promoted the apoptosis of CD4+T lymphocytes in peripheral blood of healthy people.