| Graft-versus-host disease (GVHD) is a significant complication with a high risk of mortality and morbidity resulting from allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although we have strictly chosen donors with the human leukocyte antigen (HLA) fully coincident or basic compatriots, and the administration of glucocorticoinds with or without calcineurin inhibitors preventively, it is still difficult to hold the occurrence of GVHD back. And GVHD can take a severely impact of the survival quality and the survival rate of patients who have just suffered from chemotherapies and allogeneic HSCT.Chronic graft-versus-host disease (cGVHD) has emerged as the most troublesome complication of allogeneic HSCT. It is characterized as a high incidence, low survival rate disease, and seriously influencing on the quality of live of the patients. The pathogenesis of cGVHD is complex and not entirely understand. So an establishment of the cGVHD animal models in order to study the disease seems to be in a great necessary.Currently, the mouse model is the most commonly used animal models for cGVHD. According to the differences of the performances and the construction methods, the cGVHD mice model has been divided into four categories: scleroderma-cGVHD (Scl-cGCHD) mice model, lupus-like cGVHD (SLE-cGVHD) mice model, lupus-like-scleroderma cGVHD mice model and thymic function defected cGVHD mice model. These models are not completely apart from each other, but they still have their own individualites. In this article, insights into the establishment of the Scl-cGVHD mice model which is quite widely used. Fibrotic changes of the skin and other orgens, similar to clinical Scl-cGVHD, and fibrosis of the liver and bile duct, lung, parotid salivary gland. Moreover, the incidence of Scl-cGVHD among all long-term patients of allogeneic HSCT is nearly 10-15%. The molecular pathologensis of this model, major histocompatibility complex (MHC) matched while minor histocompatibility antigens (miHA) mismatched. The foundation of this model usually need to accept a lethal dose of total body irradiation (TBI). One of the foreign combination of this model is B10.D2. (H-2d) into BALB/c (H-2d) hosts.T cells are sufficient and necessary to generate cGVHD in the whole process. If only infusion bone marrow cells (purified T-cell populations) into the hosts, the cGVHD is unable to find in the hosts. Therefore, in order to establish the mice model with cGVHD, we always utilizes the donor’s bone marrow cells mixed with spleen cells, which have a high level of T cells. Changing of the quantity of spleen cells may affect the ratio of T cells in the graft, and impress the development of cGVHD. Further more, the discovery of mesenchymal stem cells and myeloid-derived suppressor cells in the bone marrow indicated that injection of bone marrow cells may improve cGVHD. Although people have successfully established many cGVHD mice model, but no one described wether the ratio between bone marrow cells and spleen cells will affect the successful rate of transplantation and stability of the cGVHD mice model.CD4+T cells have two independent functional subsets:Thl cells and Th2 cells. Some studies suggested that Thl cells dominate the acute graft-versus-host disease (aGVHD). Thl inflammatory response was marked by the production of IFN-gamma, IL-2 and IL-12 cytokines. Thus, they might induced a’cytokine storm’ and then made the Th2 cells secrete cytokines such as IL-4, IL-5, IL-10, which may cause the cGVHD. Studies have shown that Thl cells related cytokines usually have a low expression in cGVHD, while the Th2 cells play a crucial role in progress of cGVHD. Th2 cells might suppressed the Thl and Th17 cells’ proliferation and secretion of cytokines as the antagonism effect of related cytokines between Thl and Th2 cells. At the same time, Th2 cells produced IL-4, IL-5, IL-13 to stimulate collagen produced from fibroblasts, collectively leading to a Scl-cGVHD. Although the GVHD dominated by the Thl cells could involve an equally serious inflammatory cascade, they could not make the fibroblasts proliferate and collagen deposite. This illustrated that the performance of the fibrosis was just under the influence of Th2 cells and their related cytokines, and had nothing to do with the Thl cells and their related cytokines.Objective:Establish a cGVHD mice model similar to the characteristic of clinical cGVHD and identify its features of pathology. Study whether the quantity of the spleen cells and bone marrow cells infusion will impact on the success rate of modeling, and the performance and stability of cGVHD, in order to choose an appropriate injection scheme.After the establishment of the model, we will try to comprehend the pathology and molecular cell features of the cGVHD mice model, and observe the roles of various cytokines in this model.Method:1.8-10 weeks old female BALB/c mice (H-2d) were randomly divided into the following groups as recipient mice. The number of injected spleen cells and bone marrow cells of each group were different from any other goups:a) group A (bone marrow cells:spleen cells=8:9< 1):injected 8×106 bone marrow cells,9×106 spleen cells by recipient mice’s caudal vein after the TBI (n=20);b) group B (bone marrow cells:spleen cells= 6:9<1):injected 6×106 bone marrow cells,9×106 spleen cells by recipient mice’s caudal vein after the TBI (n=5);c) group C (bone marrow cells:spleen cells=8:8= 1):injected 8×106 bone marrow cells,8×106 spleen cells by recipient mice’s caudal vein after the TBI (n=5);d) group D (bone marrow cells:spleen cells= 6:8< 1):injected 6×106 bone marrow cells,8×106 spleen cells by recipient mice’s caudal vein after the TBI (n=5);e) group E:injected 8×106 bone marrow cells by recipient mice’s caudal vein after the TBI (n= 5);f) group F:no operation after the TBI (n=5);g) group G:no operation (n=5).On the basis of intestinal preparation (erythromycin 250 mg/L, gentamicin 320000 U/L, started from 7 days before the transplantation and maintained to 14 days after the transplantation) for recipient mice, took the 8-10 weeks old male B10.D2. (Hcl H-2d H2-T18c) mice as donor mice. Obtained its bone marrow cells and spleen cells. Adjusted the concentration of the cells according to the grouping plan described above. All of the recipient mice would be injected with the cells, after they had had the 100 cm source skin distance,30 cm×30 cm radiation field,700 cGy,698 jump, one time total body irradiation (TBI) for pretreatment for 4 hours (no operation to group F mice after the TBI and no operation to group G mice).2. Observed the mice every day. Monitored the number of white blood cells at the first 14 days after transplantation to judge whether the transplantation were successful or not. Evaluated and recorded the clinical cGVHD score of the recipient mice from the 14th day after the transplantation. The evaluation would be proceeded for less than 3 days for once at the fixed time of the day.3. Set up the 15th day,25th day,35th day,45th day,55th day,65th day and 75th day after the transplantation as the evaluation days of cGVHD clinical score. After 75 days of observation, executed all the mice (5 mice in group A). For the mice in group A, we set up the 15th day,25th day,35th day,95th day after the transplantation as the evaluation days. At every one of the evaluation days,3 mice were executed and their peripheral blood serum was kept in order to detect the levels of Th subsets related cytokines levels by LUMINEX kits. All the dead or executed mice needed to take the pathological examination.4. Used the pathologic specimens to make HE staining slices. Evaluated the pathological cGVHD score under a microscope according to the pathological grading standard of cGVHD. If the cGVHD score was over 2, the mouse could be regarded as a cGVHD mouse.5. Analysis of the clinical cGVHD score and the cGVHD pathological changes of the mice in different groups. Analysis cGVHD pathological changes and cytokine levels of group A mice at their observation days.Results:1. Till the 8th day after transplantation, all the survival mice have had a white blood cells count of over 1×109/L. The mice in group A, C, E and G had an 100% survival, and the mice in group B and D had an 80% survival, and all the mice in group F died. The mice died of the hematopoietic functional failure. At the 75th day, the pathological results, especially in skin and lung, showed that all the mice, except the mice which had died in the first 14 days, in the group A, B, C and D had cGVHD. The success rate of the transplantation with the mice in group A were higher than that with the mice in group B and D (P<0.05).2. The clinical cGVHD scores of Group A (bone marrow cells:spleen cells= 8:9<1), group B (bone marrow cells:spleen cells= 6:9<1), group D (bone marrow cells:spleen cells= 6:8<1) mice were high and stable. The clinical cGVHD scores of group C (bone marrow cells, spleen cells= 8:8=1) mice began to gradually reduce from the 50th day after the transplantation, which meaned unable to maintain a severe cGVHD clinical manifestations. Their clinical cGVHD scores was lower than those of the other groups in the 55th day,65th day and 75th day after the transplantation (P<0.05). At the 25th and 35th day after the transplantation their scores was lower than those of group A (P<0.05). Group C’s pathological changes were lighter than that of other groups (P<0.05), especially the changes of the skin.3. The pathological changes of the mice in Group A at the 15th day after the transplantation was given priority to infiltration with inflammatory cells in skin, and some inflammatory cells infiltrate in lung. The pathological changes of the mice at the 25th day after the transplantation was mainly fat loss and inflammatory cells’ infiltration in skin. The structure of their lungs were intact and inflammatory cells’ infiltration were increased. The pathological changes of the mice at the 35th day after the transplantation was mainly collagen fiber hyperplasia, accompanied by fat atrophy and inflammatory cells’ infiltration in skin. The basic structure of the lung was destructed, while the interstitial tissue of the lung was infiltrated with many inflammatory cells. The pathological changes of the mice at the 95th day after the transplantation was obvious collagen fiber hyperplasia, hair follicle dramatical reduction and inflammatory cells’ infiltration. The basic pulmonary structure was almost absolutely disappeared, accompanied with the fibrosis and inflammatory cells infiltration of interstitial tissue. The cGVHD pathological scores in 95th day were higher than that in the 15th day,25th day,35th day (P<0.05).4. The levels of Thl, Th17 cells related IFN-gamma (P<0.05), IL-1beta (P< 0.05), IL-2, IL-17A reduced after the 15th day. The levels of Th2 related IL-4 and IL-5 (P<0.05) increased along with the development of cGVHD. The levels of IL-5 at the 95th days were higher than that at the other observation days (P<0.05).Conclusion:B10.D2. (Hcl H-2d H2-T18c)-BALB/c (H-2d) cGVHD mice model was successfully established. Fibrosis mainly expressed in the skin and lung, and gastrointestinal tract, too. At the 8th days after the transplantation, the hemopoietic ability of the mice was recuperative. In the development of cGVHD, the inflammatory cells’ infiltration of the target organs in the early stage shifted to the collagen deposition.The severity of this cGVHD mice model depended on the ratio between bone marrow cells and spleen cells. When the ratio was less than 1, the manifestation of the model would be severe, stable and persistent. And when the ratio approached 1, the appearance of cGVHD was relatively poorer.The results from this study suggested us to inject 8×106 bone marrow cells together with 9×106 spleen cells, in order to improve the success rate of modeling as well as guarantee the severity of cGVHD.This model was mainly dominated by the cytokines related to Th2 cells. It was characterized by fibrosis in target organs. This model could well simulate the features of the process and the cytokines expression in clinical cGVHD. |
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