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Purpose and significance of the hematopoietic stem cell transplantation (hematopoietic stem cell transplantation, HSCT) is the only means to cure a variety of hematologic malignancies. However, all patients to find compatriots matched donor chance only 25% instead of the blood donor transplants matched donor exists to find limited opportunities, and long waiting time problem, therefore, the application haploidentical unrelated donor stem cells as transplant donor-derived hematopoietic stem cell transplantation has become an effective way in recent years, treatment of hematological malignancies. Relative compatriots all mismatched transplantation, haploidentical donor hematopoietic stem cell transplantation (haploidentical hematopoietic stem cell transplantation, haplo-HSCT) is a major obstacle to graft-versus-host disease (Graft-versus-host disease, GVHD) have a higher prevalence According to reports, haplo-HSCT after acute and chronic GVHD incidence as high as 50% to 80%, about 30% of transplant-related mortality, seriously affecting the patient's survival time and quality of life, including in particular a high incidence of acute GVHD rate is still impact transplant survival rate, postoperative quality of life and hinder allogeneic hematopoietic stem cell transplantation widely the main reason. For haplo-HSCT problem prevention of GVHD after transplantation is a recent clinical transplantation hot area of ??research. At present, foreign primary prevention of GVHD adopt measures include removal of T cells in vitro, CD34 sorting and other major domestic taken to increase the strength of pre-treatment, a strong immune suppression and increase the input of CD34 cell count and other methods, but all have a high incidence of infection, recurrence rate after transplantation, transplant-related mortality disadvantages. Therefore, in recent years, cell therapy as prevention and treatment of GVHD experimental research and clinical application of hot spots, including bone marrow-derived mesenchymal stem cells in the prevention of GVHD effect has been widely recognized, its mechanism of action that can stabilize the hematopoietic microenvironment, immune regulation, regulation hematopoietic cell growth, etc. Mainly by the bone marrow microenvironment of bone marrow stromal cells, extracellular matrix (extracellular matrix, ECM) and a variety of hematopoietic growth factors, of which, extracellular matrix (ECM) and its related cell adhesion molecule (celladhesion molecules, CAMs) are and between the formation of hematopoietic cells and stromal cells of hematopoietic cells of the base interaction. Present study confirmed that hematopoietic stem cell transplantation pretreatment and / or the disease itself factors in patients with hematopoietic microenvironment injury involved in the occurrence and development of GVHD, but recent studies show that the migration process infusion of mesenchymal stem cells can repair hematopoietic microenvironment injury and regulate immune function to reduce or control GVHD. Therefore, through the regulation of hematopoietic microenvironment is expected to regulate GVHD. Basic fibroblast growth factor (basic fibroblast growth factor, bFGF) is a molecular weight of 18,000 - 24,000 basic polypeptide is highly conserved gene, human, bovine, murine FGF-2 nucleotide sequence highly homologous to sources, both single-copy genes. People of bFGF gene is located on chromosome 4q27, is fibroblast growth factor protein (fibroblast growth factors, FGFs) family one. bFGF can stimulate and regulate endothelial cells, epithelial cells, myoblasts, osteoblasts, and glial cells and other mesodermal origin, neuroectodermal cell proliferation and differentiation, not only to promote nerve cell repair and angiogenesis but also through collaboration with other hematopoietic growth factors to enhance hematopoietic cells in vitro colony formation in vitro studies have shown that basic fibroblast growth factor in vitro culture can stimulate mesenchymal stem cell proliferation, stable hematopoietic microenvironment, and its the same family acidic fibroblast growth factor (acid fibroblast growth factor, aFGF) has been shown to regulate GVHD, but has not been at home and abroad in vivo bFGF can promote the proliferation of mesenchymal stem cells as well as the correlation with GVHD reported, this project plans from bFGF body to promote mesenchymal stem cell proliferation, stable hematopoietic microenvironment start, first established mouse MHC haplotype of bone marrow transplantation model, and then observe the effect of bFGF intervention study bFGF can regulate GVHD and explore its possible mechanism. Materials and Methods 1. Establish MHC haplotype mouse bone marrow transplantation model (1) of male C57BL / 6 (H-2b) as for the mice, female BALB / c (H-2d) mice and male C57BL / 6 (H- 2b) female offspring mice (BALB / c × C57BL / 6) F1 (abbreviated as CB6F1) as recipient mice, thereby establishing C57BL / 6 (H-2b) → CB6F1 (H-2d / b) MHC haplotype transplanted mouse model. (2) pretreatment bone marrow transplantation: the use of Co60 grant recipient mice 9-10Gy total body irradiation, dose rate 0.5Gy / min, the distance between the radiation source and the animal is about 50cm. (3) Preparation of transplanted cells: Take C57BL / 6 male mice were used as donors, were sacrificed, cut the mouse femur, tibia metaphysis, bone marrow cavity with a syringe to flush out the bone marrow cells into the bone marrow cell suspension solution (3.0 × 106/ml), the spleen was cut to prepare a spleen cell suspension (2.0 × 107/ml), the bone marrow and spleen cells were mixed in equal volumes of cell suspension. (4) bone marrow transplantation: irradiated recipient mice receiving 4h, by intravenous injection of spleen cells and bone marrow cells were mixed suspension 0.3ml, in which bone marrow cells of about 4.5 × 106 / a, spleen cells of about 3 × 107 / a. 2. BFGF GVHD and mechanism of regulation (1) does not take any measures to prevent GVHD group (A): To observe the post-transplant weight, appetite, appearance, diarrhea, bleeding or mucosal inflammation and white blood cell count, survival and liver, skin intestinal pathology (2) bFGF high-dose group (B group): Transplant day 0, bone marrow and spleen cells enter bFGF 100ug/Kg given 4 hours after subcutaneous injection once daily, body weight was observed after transplantation, appetite, appearance, diarrhea, bleeding or mucosal inflammation and white blood cell count, survival and liver, skin, intestinal pathology, flow cytometry MSC, CD31, CD44 expression. (3) bFGF low-dose group (C group): Transplant day 0, bone marrow and spleen cells after 4 hours to give input bFGF 20ug/Kg subcutaneously, once daily, observed after transplantation, weight, appetite, appearance, diarrhea, bleeding or mucosal inflammation and white blood cell count, survival and pathological examination, flow cytometry MSC, CD31, CD44 expression. (4) GVHD clinical manifestations observed in each group, survival time, pathological difference. (5) transplant 14 days, the mice were sacrificed, take the mouse limb femur bone marrow mesenchymal stem cells by light microscopy and flow cytometry, cell culture results to determine whether mesenchymal stem cells and the group of mesenchymal stem cell proliferation differences and make mesenchymal stem cells osteogenic differentiation and induction of differentiation into fat. Results 1. Successfully established murine hematopoietic stem cell transplantation model haploid 2 mouse survival: A group of 11 days after transplantation began mice died, 17 days completely dead, B group at 15 days after transplantation mice began to appear Death, B group survive more than 30 days have 2, C group at 13 days after transplantation began mice died, 19 days completely dead. Drawn from the Kaplan-Meier survival curves of view, A and C groups had no significant difference in survival curves (P gt; 0.05), and the survival rate in group B were significantly different (P lt; 0.05). 3. GVHD clinical manifestations: leukopenia after transplantation group A faster, but transplanted 15d WBC still gt; 0.5 × 109 / L, while the B group and C group decreased slowly; 7 days after transplantation, B, C group weight loss was lower than in group A (P lt; 0.05), transplanted 14 days in group B than in group A degree of weight loss (P lt; 0.05), while the C group and no significant difference between group A (P gt; 0.05) ; B group had diarrhea, arched, hair removal and other clinical manifestations of GVHD case later than the A and C groups. 4 Histopathological examination: gross specimen shows focal hemorrhage, endoscopic group A HE staining of liver tissue visible structural damage, leaf cells and lymphocytes increased, and a large focal hemorrhage, while the B group and group C, the extent and degree of bleeding lymphocyte infiltration were lighter than the A group. 5 mesenchymal stem cells: 14 days after transplantation, bone marrow-derived mesenchymal stem cells, the results show, A group and C group were scarce, can not form a colony and continue to grow, while the B group showed a large colony and grow well cells, light microscopy, B group microscopic cells grew well adherent cells, shape elongated fusiform nuclei closely united. Group B, flow cytometry results suggest that expression of CD31 and CD44-negative were positive. And the group of cells can be induced to differentiate into osteoblasts and adipocytes. Conclusions 1. Vivo applications of basic fibroblast factor (bFGF) can significantly reduce the pretreatment bone marrow microenvironment injury, stable hematopoietic microenvironment to promote hematopoietic stem cell transplantation haploid mice survived; 2 basic fibroblast growth factor (bFGF) can play to reduce GVHD after transplantation mouse MHC haplotype effects, but its prevention and treatment of GVHD alone limited effect; 3. This study suggests that basic fibroblast growth factor (bFGF) to reduce mice GVHD mechanism of action, possibly by stimulating mesenchymal stem cell proliferation in vivo and play a role in immune regulation.
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