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Objective:To explore the suitable mouse strains,and establish stable human-miceX-GVHD model.Methods:This study selected the Nude Mice and NOD/SCID, and gave them sublethal dose of γ-ray irradiation whole body , and then intraperitoneal transplanted human peripheral blood mononuclear cells ( PBMC) to establish xenogeneic acute graft-versus-host disease model.By detection of human T cells in the mice′s tail venous blood,tissues,organs of the infiltration and other indicators (By flow cy-tometry and immunohistochemistry ) ,we compared the human immune cell infiltration rates in the two mouse model and recorded the survival time.Finally,we determined the appropriate strains of mice to establish X-GVHD.Optimization means were transfer ways and the appropriate amount of human PBMC ,and the best time to observe the changes of T cell phenotype and function.Results:The NOD/SCID mouse was more suitable for inducing human-mouseX-GVHD model,and there were no significant differences between intrap-eritoneal injection and intravenous injection.Transfer human PBMC more than 5 ×107 can establish human-mouseX-GVHD model.Using the optimized experimental conditions to establish the human-mouseX-GVHD model,we found the 7-11 days was the best time to observe the changes of T cell phenotype and function ,and the average survival time was(14.16±1.77)days.Conclusion:Human-miceX-GVHD model can be successfully established by intraperitoneal injection of 5×107 human PBMC into NOD/SCID, and the best time to observe the changes of T cell phenotype and function is between 7-11 days.
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Objective To study whether the HBC-A2/scFv fusion protein mediates killing of tumor cells by viral specific cytotoxic T cells. Methods The fusion protein was attached to the CD71-expressing, HLA class Ⅰ negative tumor cells. And then, cytolysis by viral peptide-specific CTLs which were generated by co-culture of peripheral blood lymphocytes from HLA-A2 positive donors with inactivated T2 cells pulsed with the viral peptide were tested by lactate dehydrogenase (LDH) releasing. Results The fusion protein can attach the active viral peptide/HLA-A2 complex to K562, HepG2 and U937 cells through binding of CD71 scFv to CD71 (37.30% ±8.25%, 27.20% ±3.88%, 21.80% ±6.49% ) and mediate cytotoxicity of viral peptide-specific CTLs against those cells in vitro ( K562: 42.08% ± 1.14% vs 8.07%± 1.39%; HepG2: 49.72% ± 1.59% vs 12.46% ± 1.26%; U937: 39.72% ± 3.26% vs 7.13% ±1.48% ). Conclusion This viral peptide/HLA-A2 complex targeted by CD71 scFv is able to redirect viral peptide-specific T-cell mediated immune responses against tumor cells.
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HPV in remission CA,so the disease can be cured.
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Objective To construct vector expressing soluble mPDL1-hIgGFc and study its effect on the proliferation and apeptosis of cells in vitro. Methods The extrncellular domain of mPDL1 gene was amplified from pmPDL1 vector by PCR and inserted into phIgGFc vector. The recombinant pmPDL1-hIgGFc was transfected into CHO cells by LipofectAMINETM2000, and the transfected cells were named as CHOp. The expression of mPDL1-hIgGFc in the culture supernatants of CHOp was assayed by ELISA and Western blot. The effects of CHOp culture supernatants on mixed lymphocyte culture(MLC) was analysed by Flowm-etry. Results The extracellular domain of mPDL1 gene were obtained from PCR. DNA sequencing and the identification of digestion by HindⅢ and KpnⅠ indicated the recombinant plasmid pmPDL1-hIgGFc was suc-cessfully constructed. ELISA and Western blot analysis proved that the CHOp could express mPDL1-hIgG-Fc. CHOp culture supernatants could inhibit lymphocyte proliferation and induce the apoptosis of the activa-ted T cells in MLC in vitro in a dose-dependent manner. Conclusion The mPDL1-hIgGFc protein could in-hibit lymphocyte proliferation and induce the apoptosis of the activated T cells.
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Objective To study the inhibitory effects of HLA-G1 expressed in ECV304 on the proliferation of allogeneic T cells.Methods The recombinant plasmid pcDNA3-HLA-G1 which contained a full-length cDNA of HLA-G1 was constructed, and the ECV304 cell was transfected with the pcDNA3-HLA-G1 by using the lipofectin transfection. The expressed HLA-G1 on the cell surface was checked by specific monoclonal antibody (G11E5) with indirect immunofluorescence assay and FCM. The HLA-G1 expressed in ECV304 was used as stimulator in co-culture with allogeneic T cells, to perform allogeneic T cell proliferation assay and to generate ECV304-specific alloreactive CTL. The proliferation of T cells and the CTL’s cytotoxicity against ECV304 were tested by the MTT method.Results The expression of HLA-G1 on the surface of the ECV304 was verified with the immunofluorescent staining of the pcDNA3-HLA-G1 transfected cells. The proliferation intensity of allogeneic T cells was significantly decreased after the HLA-G1 expressed in ECV304, as the stimulation index of co-culture of allogeneic T cells with plasmid pcDNA3 transfected ECV304 was 1.59?0.41, meanwhile it was 1.33?0.46 to pcDNA3-HLA-G1 transfected ECV304, with the difference being significant (P
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Objective To investigate the correlation between the relapse of condyloma acuminatum(CA)and the potential capability of tumor necrotic factor (TNF) production of the host′s peripheral blood leukocytes. Methods Forty-two CA patients and 58 normal controls were enrolled in this study. CA relapse was diagnosed clinically. EB virus-transformed B lymphoblastoid cell line(LCL)were used as TNF producing cells. The TNF producing capability of LCL was measured by bioassay using L929 (a TNF sensitive tumor cell line) as target cells. The LCL were stimulated with LPS to produce TNF. Results The average level of TNF production of LCL from all CA patients (including recurrent and non-recurrent CA patients) was similar to that of normal controls (30.14% ? 12.27 vs 34.06% ? 12.06,P = 0.1136). However, the level of TNF production of LCs from recurrent CA patients was significantly less than that from non-recurrent CA patients (24.75% ? 7.51 vs 36.62% ? 10.96,P = 0.00016). Compared with that of normal controls, recurrent CA patients showed a lower capability to produce TNF (24.75% ? 7.51 vs 34.06% ? 12.06,P = 0.00054), whereas non-recurrent CA patients showed a similar capability to normal controls (36.62% ? 10.96 vs 34.06% ? 12.06,P = 0.3517). Conclusions These results indicate that the cellular immune mechanism might play an important role in the clearance of the residual HPV from the host, in which TNF is involved.
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Objective:To investigate the relationship between the level of Bcl 2 protein in EB virus infected cells and the sensitivity of this cells to NK activity and apoptosis inducing factors.Methods:Antisense oligodexynucleotides(ODNs) were used to modulate the expression of Bcl 2 gene in Epstein Barr virus transformed B lymphoblastoid cell lines(EBV LCLs);then the change of cytotoxicity of natural killer cells and the sensitivity of apoptosis inducing elements(taking out growth factor ?dexamethasone)targeting EBV LCLs,were investigated.Results:The level of Bcl 2 expression in EBV LCLs has negative relativity with the cytotoxicity of NK cells to EBV LCLs(P
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Objective To construct a recombinant plasmid pcDNA3-sHLA-G1 expressing soluble HLA-G1. Methods Total cell RNA was extracted from the cell line Jeg-3 and the cDNA was amplified by RT-PCR； The cDNA fragment was inserted into the eukaryotic expressing vector pcDNA3 and the recombinant plasmid was identified by restriction endonucleases digestion and sequencing. Results After restriction endonucleases treatment and sequencing, it was confirmed that the pcDNA3-sHLA-G1 had been constructed successfully. Conclusion In this study, the recombinant plasmid pcDNA3-sHLA-G1 had been constructed successfully.
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Objective:To develop a new model of evaluating histocompatibility between donor and recipient.Methods:①We harvested 15 couples of blood samples of donor and recipient in human BMT and detected histocompatibility between donor and recipient using the aboved model.The time when GVHR began was recorded.②Skin grafts of KunMing hybride mice were respectively placed on inbred mice,BALB/c and C57BL/6.And then histocompatibility between donor and recipient was detected using the model.Survival time of skin allografts was recorded.Results:The smaller differences of histocompatibility evaluated by means of the model were,the later GVHR in human BMT would happen and the longer survival time of skin allografts in mice would become.Conclusion:The model could be used to detect correctly histocompatibility between donor and recipient.