| Objective: The study began with fundamental research set up acute graft-versus-host disease(aGVHD) rat models of allogenic bone marrow transplantation, aimed to 1,25-(OH)2D3 and salvia miltiorrhiza as the interventional measures, to observe changes of aGVHD severity, physiology, hematopoietic recovery time, T cell subgroups and cytokines in rats after transplantation, to investigate the relationship between these changes and corresponding events and also to analyze the significance of T lymphocyte subgroups and cytokines to aGVHD diagnosis and influence of drug intervention.Methods: The study consisted of three parts: firstly, establishing aGVHD animal models of allogenic bone marrow transplantation with rats; secondly, observing influence of 1,25-(OH)2D3 and salvia miltiorrhiza on aGVHD and hematopoietic reconstitution of rats after allogenic bone marrow transplantation; and thirdly, observing influence of drugs on immune state of bodies. Random control studies and dynamic detection were adopted to observe aGVHD severity, physiological changes, hematopoietic recovery time, changes of internal cytokines (IL-2 and IFN-γ; IL-4 and IL-10) and T cell subgroups (CD4+ and CD8+) of rats in the aGVHD group and the intervention groups after allogenic bone marrow transplantation; in-situ hybridization was used to detect Y chromosomes of donor rats in recipient rats to verify the success of transplantation; a mature biological method (ELISA) was employed to detect cytokines and immunocytology technology (flow cytometer) was used to detect T lymphocyte subgroups.Results: Results of the first part indicated that spleen cells were required to establish a stable aGVHD animal mode of allogenic bone marrow transplantation. Bone marrow cells and spleen cells were mixed at 1∶1 and 1∶1.5, the attack time of aGVHD in rats was relatively centralized 2～3 weeks after transplantation; the median survival time was respectively 18.5 and 16.5 days and all subjects exhibited typical clinical and pathological manifestations of aGVHD. Results of the second part suggested that in the 1,25-(OH)2D3 group, changes of peripheral white blood cells, hemoglobin and platelets of rats were all higher than in the control group at different time points (the 7th, 14th and 21st day); on the 21st day after transplantation, results of white blood cell and hemoglobin examinations both reached normal, and the platelet count, though obviously higher than in the control group, didn't return to the normal level. The change trend in the salvia group was consistent with that in the 1,25-(OH)2D3 group with no significant difference in comparison, and the therapeutic efficacy in the combined group was not better than in the single groups. It was found that all rats in the aGVHD group and intervention groups died in the 50-day study period, but the attack time of aGVHD was delayed in the intervention groups compared with that in the aGVHD group, the clinical aGVHD records of rats in the intervention groups were also lower than those of rats in the aGVHD groups, the average survival time was also markedly lengthened compared with that of rats in the aGVHD group with difference of statistical significance (P<0.05), and the pathological manifestation of aGVHD in rats of the intervention groups was milder than in the aGVHD group. Results of the third group showed that CD4+ value of the aGVHD group was significantly increased after transplantation with statistically significant difference in comparison with that before transplantation (P<0.05). The increase peaked on the 14th day and the value of the 21st day approximated to the level before transplantation; the trend of CD8+ was similar to that of CD4+; the CD4+/CD8+ value change of the aGVHD group was also the most notable on the 14th day and confirmed to the aGVHD onset time; after interventional treatment, the increase amplitudes of CD4+ and CD8+, CD4+ in particular, were lower than in the aGVHD group, and the CD4+/CD8+ value of each intervention group increased fairly remarkably on the 21st day.In the aGVHD group, cytokines including IL-2 and IFN-γshowed increase trend while IL-10 showed decrease trend in examinations after transplantation. All of them displayed marked changes on the 14th day with statistically significant difference compared with the level before transplantation (P<0.05). All indexes approximated the levels before transplantation on the 21st day; after transplantation, the IL-4 value changes at each time point showed a decrease tendency; the change trends of IL-2, IL-10 and IFN-γwere in conformity with the fact that aGVHD occurred on the 14th day after transplantation.Cytokines including IL-2 and IFN-γof each intervention group decreased after transplantation with a slight increase on the 21st day, but the IL-10 showed an increase tendency with a slight decrease on the 21st day. All of them had statistical difference in comparison with levels before the transplantation (P<0.05); in each intervention group, the IL-4 value change displayed an increase tendency at each time points, but the change amplitude was low. After pretreatment with different interventional measures, aGVHD occurred on around the 21st day with mild severity and the IL-2, IL-10 and IFN-γchanged in a synchronous tendency. Cytokines at each time point had statistically significant difference in compared with levels in the aGVHD group.Conclusion: this study successfully setup the first aGVHD rat models of allogenic bone marrow transplantation in Xinjiang area. An ideal model can be created for studies on aGVHD after allogenic bone marrow transplantation when bone marrow cells and spleen cells are mixed at 1∶1 and 1∶1.5. 1,25-(OH)2D3 and salvia miltiorrhiza, as interventional factors, can promote hematopoietic reconstitution and prevent aGVHD in rats after allogenic bone marrow transplantation. The value of CD4+/CD8+ increases when aGVHD is severe and decreases when aGVHD is alleviated. Cytokines related to Th1, including IL-2 and IFN-γ, showed decrease tendencies generally in each intervention group, and the IL-10, a cytokine related to Th2, is in an increase tendency in general, indicating 1,25-(OH)2D3 and salvia miltiorrhiza have effects of regulating Th1/Th2 balance, inhibiting proliferation and function of Th1 subgroups, promoting proliferation of Th2 subgroups and acting for immune tolerance. No marked changes occurred to IL-4, suggesting detection of more than one cytokines and T cell subgroups is conducive to judgment of immune state. |
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