Role Of Tissue Factor And Its Signaling Transduction In Bone Marrow Transplantation

Part I The experimental investigation the HUVEC apoptosis of the recipients induced by CD8+T cells in coordination with TNFα.Objective During hematopoietic stem cell transplantation,the number of the damaged circulating endothelial cells are distinctly increased and had a positive correlation with the vaso-complication.In the animal model ,apoptosis of the endothelial cells is a earlier events before the damage of the other tissues and organs.But the mechanism and induced factor are unclear.Our research destination to observe the apoptosis effects of allogeneic CD8+T cells of the donor to the vascular endothelial cells of the recipients induced by TNFα.Methods Human periphery mononuclear cells were isolated by density gradient technique.CD8+T cells were isolated with Human T Cell CD8 Subset Column Kit via high affinity negative selection. HUVEC lines were divided into 4 groups.They were: 1)control group cultured 1640 2)cell culture treated with low dose TNF-α3) cell culture mixed with lymphocyte without TNF-α4)cell culture mixed with lymphocyte treated with TNF-α.Apoptosis of HUVEC were observed with fluorescence microscope and quantificated with Flow Cytometry after staining with Annexin-V and PI.Results Low dose 5ng/ml of TNFαcould not induced apoptosis of HUVEC compared with control, p>0.05.The early stage of the HUVEC apoptosis was high enough to 83.6±0.42% at 24 hour time point after coculture with CD8+T cells pre-treated with TNFα,the apoptosis rate was 5.8 times compared with the coculture group without TNFα.It has a very significant statistic difference between the two groups p<0.001.Conclusion In vitro,donor CD8+T cells could induced apoptosis of the recptor vascular endothelial cells.Low dose TNF-αcould distinctly amplify the apoptosis effects. Part II Expression and Significance of A20 and Caspase 3 in Apoptosis Pathway of human umbilical vein endothelial cells triggered by human Allogeneic CD8+T lymphocytesObjective During allogeneic hematopoietic stem cell transplantation, apoptosis of vascular endothelial cells is not only the important pathological hallmark in the transplantation associated vascular complications,but also likely to mediate the damage of the tissues and organs in graft versus host diseases in early stage. In order to discover the apoptosis mechanism of human vascular endothelial, the expression of A20 and Caspase 3 in apoptosis pathway were detected.Methods Human periphery mononuclear cells were isolated by density gradient technique.CD8+T cells were isolated with Human T Cell CD8 Subset Column Kit via high affinity negative selection. HUVEC lines were cocultured with allogeneic CD8+T lymphocyte after treated with low dose TNFα.Apoptosis of HUVEC were quantificated with Flow Cytometry after staining with Annexin-V and PI.The activity of apoptosis related enzyme Caspase 3 were detected with full wave fluorospectrophotometer. Expression of protein A20 in HUVEC were detected with westen blot.Results Allogeneic CD8+T cell could induced HUVEC apoptosis after coculture with HUVEC.The maximum apoptosis rate of HUVEC in early stage was at 24 hour after co-culture measured with flow cytometry,it was 83.6±0.42%. The peak time of the activity of Caspase 3 related with the advanced stage of apoptosis was at 48 hour after co-culture,its fluorescence intensity is 13.59±0.27, and then decreased significantly.It has very significantly statistic difference compared with control group p<0.001. A20 protein was negative correlation with the degree of HUVEC apoptosis induced by allogeneic CD8+T cells.Conclusion In vitro,donor CD8+T cells could induced apoptosis of the vascular endothelial cells of the recipients.Activation of caspase 3 mediated apoptosis of HUVEC. Signaling protein A20 are likely to negatively regulate apoptosis of HUVEC. Part III Construction of the small interfering RNA vector targeting cytoplasmic domain of tissue factor and the protection role for HUVEC from apoptosisObjective To construct small interfering RNAs vectors targeting cytoplasmic domain of tissue factor ,and transfer them into HUVEC line,and to observe their protecting role for the HUVEC from apoptosis in mixed lymphocyte reaction without affecting their coagulation function.Methods Specific small interfering RNA oligos targeting cytoplasmic domain of tissue factor in vitro were designed,and insert into plasmid DNA and transforming E.coli to reproduce. The extracted plasmids were transfected into HUVEC with liposomes. Megnetic bead is used to measure APTT of the supernatant liquid in the mixed lymphocytes culture.The apoptosis rate of HUVEC tranfected with the empty control,siRNAI,siRNAII plasmid were analyzed with Flow cytometry,Results The apoptosis rate of HUVEC transfected with siRNAI plasmid was significantly decreased compared with the empty control.The apoptosis rate of HUVEC transfected with siRNAII plasmid was not so significance as siRNAI plasmid,but also has significantly difference compared with empty control P<0.05.The time of APTT in cultured supernatants had no significant difference compared with empty control, P>0.05.Conclusion The succeed constructed small interfering RNA plasmids targeting cytoplasmic domain of tissue factor could protect HUVEC apoptosis ,and did not effect coagulation function of the tissue factor.Part IV The expression and signaling cascade of tissue factor in HUVEC induced by allogeneic CD4+T cellObjective Block of protein synthesis of the tissue factor in cytoplastic domain could protect apoptosis of HUVEC induced by allogegeic CD8+T cells without effecting the normal coagulation.Apoptosis of vascular endothelial cells mediated GVHD in early stage.Therefore, downregulation of tissue factor in the cytoplasmic domain could alleviate the degree of GVHD.If blood coagulation was disturbanced ,downregulation of intra-and-extra domain of tissue factor would have dual-effects both in anti-coagulation and alleviation of GVHD.But the mechanism of the regulation of tissue factor expression in the signaling pathway are not unclear.The aim of our research is to detecte the expression of signaling moleculars intra-celluars which control tissue factor expression induced by allogeneic CD4+T cells,and provide experimental evidence to find a new therapeutics target for clinic.Methods Human periphery mononuclear cells were isolated by density gradient technique.CD4+T cells were isolated with Human T Cell CD4 Subset Column Kit via high affinity negative selection. HUVEC lines were cocultured with allogeneic CD4+T lymphocyte after they were pre-treated withγinterferon .The expression of tissue factor on the surfaces of HUVEC were quantificated with Flow Cytometry . Transcription of tissue factor mRNA was measured with real-time PCR. Signaling molecules intra-HUVEC of JNK,P38-MAPK and phosphorylation of I-καwere detected with westen blot.Results Expression of tissue factor on the surface of HUVEC was markedly increased at 12h after coculture with CD4+-T cells. The peak time was at 24h and lasted to 48h and then gradually decreased. The transcriptional activity of tissue factor was obviously increased at 6h after coculture with CD4+-T lymphocyte and then lasted gradually decreased. Time course of TFmRNA was similar with TF expression on the surfaces of HUVEC. Signaling molecular JNK,P38MAPK expression and phosphorylation I-καintra HUVEC particpated in the control of tissue factor expression .It has significant statistic difference when compared with control group,P<0.05.Conclusion: JNK,P38-MAPK,I-καare all likely to be the control mecular in the signaling pathway intra HUVEC,when it was induced to express tissue factor by allogeneic CD4+T lymphocytes.