The Research Of CGVHD Murine Model After MHC-matched Hematopoietic Stem Cell Transplantation

BackgroudsAllogeneic hematopoietic stem transplantation (allo-HSCT) is an intensive therapy for high-risk haematological malignant disorders, genetic diseases and solid tumors.Especially with the development of nonmyeloablative transplantation (NST), allo-HSCT has been expanded its clinical indications. But Chronic graft-versus-host disease (cGVHD) remains the major complication of allo-HSCT, which causing transplantation failure and patient death. Clinical manifestations of cGVHD are variable and resemble other recognized autoimmune diseases (e.g. Sjogren syndrome and scleroderma).Now there are no systematic and effective strategies to prevent and treat the disease, just because the pathophysiology of cGVHD is poorly understood. In order to further the study of the pathogenesis of cGVHD and seeking for new methods to prevent and treat of the disease, a proper animal model of cGVHD is necessary.ObjectivesIn clinical practice, the mismatch of major histocompatibility antigen(MHC) can certainly increase the likelihood of developing cGVHD. However, nearly 40% of recipients of HLA-matched sibling grafts developed the syndrome. Thus, in the search for ways to accurately model the cGVHD syndrome, we select the MHC-identical, miHA-mismatched murine model of allo-transplantation, which is analogous to most human allo-BMTs.MethodsThe donors are inbred strain male DBA/2H2d mice, and the recipients are inbred strain female BALB/c H2d mice. All mouse are at age of 8-12 weeks. All recipients mouse were given 60Co Whole-body irradiation (5.5 Gy) prior to transplantation. Then the recipients were randomly divided into three groups(n=10):2 experimental groups were injected intravenously with different numbers of spleen cells(5×107 and 10×107, respectively), andcontrol group injected intravenously with RPMI1640.①Hematopoietic reconstitution were observed.②Karyotype analysis was carried out at the end of experiment in order to detect the donors'Y chromosome in recipients'peripheral blood mononuclear cells.③After 14 days of transplantation, the general condition and survival time of mice were observed and scored every 3 days.④At the end of the experiment, the pathological changes of target organs were observed and scored, according to pathological results, to determine the rate of the mice changing into the model.⑤Serum anti-dsDNA and BAFF (B cell activating factor) levels were measured by ELISA kit at the end of the experiment.Statistical Methods:Experimental data were processed with SPSS16.0 statistical software. The ANOVA for repeated measures were used to analysis the data of the WBC in different times. The significance of differences between clinical scores and pathology scores were calculated by Wilcoxon rank sum test of the two independent samples. The significance of differences in cGVHD incidence was described by Kaplan survival curve and calculated by log-rank Mantel-Cox. A value of P<0.05 was considered significant.Results①Hematopoietic reconstitution:The number of the white blood cells (WBC) recovered 4 weeks after irradiation. And in transplantation group, the recovery of the number of WBC was slower, relatively.②Chimerism:Each mouse in experiment groups were partial chimeras.③GVHD clinical scoring:all mice in group A scored 0 points, which did not develop GVHD, the differences on clinical scoring between group B and C were statistically significant(P< 0.05).④Tissue histopathology analysis: Group A mice did not develop GVHD; There was no statistical significance in severity in tissue histopathology scoring between group B and C by Wilcoxon rank sum test (P> 0.05). The incidence of A, B and C group are 0%,60%,80%. The log-rank test revealed that there was no significant different between the two morbidity curves (χ2=3.369, P=0.066).⑤ELISA for BAFF and anti-dsDNA in serum:there was significant different of blood BAFF and anti-dsDNA between model mouse and control mouse (P<0.05) Blood BAFF was closely correlated with blood anti-dsDNA in model mouse.Conclusion1. Female BALB/cH2d mice which were given 60Co 5.5Gy TBI and injected of 5×107, 10×107 splenocyte of DBA/2H"2d mice had clinical features of cGVHD.5×107 group was stable, and survival time of mice was relatively long. This mouse model is useful for intervention study of cGVHD.2. Blood BAFF in model mouse raised with the development of cGVHD. Excess BAFF may was correlated with the incidence and the development of cGVHD, which could be used to study the pathophysiology and new treatment of cGVHD.