| Background and Objective:Hematopoietic stem cell transplantation (HSCT) is an effective method which can cure a variety of hematologic malignancies. However, For many patients we can not find a full-matched donor or only one locus mismatched related donors. In high risk patients , haploidentical donors are suitable alternatives.. Haploidentical stem cell transplantation has become an effective method for treatment of hematologic malignancies in recent years since we can easily get haploidentical donors from parents or child .Compared with full-matched transplantation, the GVHD (Graft-versus-host disease) is the main obstacle for the haploidentical hematopoietic stem cell transplantation (haplo-HSCT). The occurrence of a-GVHD after haplo-HSCT was reported from 50% to 80%, and the transplant-related mortality rate was 30% , which seriously affected the survival time and the quality of life .,And it is the main causes why haplo-HSCT can't be widely apllied. In this study we want to find a new way to decrease the occurrence of a-GVHD after haplo-HSCT so as to decrease the complication and mortality rate of haplo-HSCT.The prevention methods for GVHD after Haplo-HSCT is mainly consist of removal of T cells in vitro, CD34 sorting, strengthing conditioning regimen and strengthing immune suppression taking. However, all the above methods have the disadvantages of high incidence of infection rate,recurrence rate,and transplant- related mortality rate. In recent years, cell therapy as a-GVHD prevention method in experimental study and clinical application got satisfactory effective, and the bone marrow-derived mesenchymal stem cells to prevent the effects of GVHD has been widely recognized.Present study confirmed that injury of hematopoietic microenvironment which caused by conditioning regimen and / or the primary diseases was associated with occurrence and development of GVHD. Recent studies showed that MSCs infusion during transplantation can repair the damaged hematopoietic microenvironment so as to decrease the occurrence rate of GVHD.Basic fibroblast growth factor (bFGF) is a member of the protein fibroblast growth factor (FGFs) family which can stimulate the proliferation of mesenchymal stem cells(MSCs) and stable hematopoietic microenvironment. Acidic fibroblast growth factor (aFGF), the same family member of FGFs, have been proven to regulate GVHD. While bFGF promoting the mesenchymal stem cells proliferation in vivo and the correlation with GVHD have not been reported. Based on the above evidence, we firstly want to establish a mouse model of MHC haploidentical hematopoietic stem cell transplantation , secondly we want to observe the effect of bFGF's intervention , then study the bFGF regulating GVHD and the associated Mechanism .Method:1. Establish a mouse model of MHC haploidentical bone marrowtransplantation(1) Male C57BL / 6 (H-2b) as the donor mice, BALB / c (H-2d)male mice and C57BL / 6 (H-2b) female mice were hybrids (BALB / c×C57BL / 6 ) ,F1 (referred to as CB6F1) as the recipient mice, thus establishing C57BL / 6 (H-2b)→CB6F1 (H-2d / b) MHC mouse model of haploidentical hematopoietic stem cell transplantation.(2) Conditioning regimen of bone marrow transplantation: give the recipient mice 9-10Gy total body irradiation, dose rate 0.5Gy / min, the distance between the radiation source and the animal is 50cm.(3) Prepared transplanted cells: Take C57BL / 6 male mice as donors, kill the mice by cervical, cut mouse femur, tibia metaphysis, the bone marrow cavity with a syringe flush out bone marrow cells into bone marrow cell suspension (3.0×106/ml ), the spleen was cut to prepare spleen cell suspension (2.0×107/ml), the bone marrow and spleen cell suspension an equal volume of cell suspension mixed.(4) Bone marrow transplantation: the above mixed suspension was injected into the recipient mice after 4 hours'total body irradiation,with bone marrow cells about 4.5×106 , spleen cells about 3×107.2. Basic fibroblast growth factor and the mechanism of regulating GVHD(1) No treatment for GVHD group (group A):after transplantation, the following items were observed:body weight, appetite, appearance, diarrhea, bleeding or inflammation of mucous membranes and peripheral blood white cell count, survival and liver, skin, intestinal pathology(2) bFGF high dose group (group B): the 0 days after transplantation, in the input given bFGF 100ug/Kg after bone marrow and spleen cells subcutaneous injection 4 hours,everyday . observe body weight, appetite, appearance, diarrhea,bleeding or mucous membrane Inflammation and WBC count, survival and liver, skin, intestinal pathology, flow cytometry MSC,CD31, CD44 expression.(3) bFGF low dose group ( group C): the 0 days after transplantation in the input given bFGF 20ug/Kg after bone marrow and spleen cells subcutaneous injection 4 hours,everyday . observe body weight, appetite, appearance, diarrhea,bleeding or mucous membrane Inflammation and WBC count, survival and liver, skin, intestinal pathology, flow cytometry MSC,CD31, CD44 expression.(4) Observe the clinical manifestations of GVHD, the survival time and pathological differences.(5) +14 Days of transplantation, the mice were sacrificed by cervical, cultured cells from femoral bone marrow of mice limbs, determine the mesenchymal stem cells by light microscopy and flow cytometry cell and the group Mesenchymal stem cells differ, and mesenchymal stem cells for osteogenic differentiation and induction of differentiation into fat.Result1. The mouse model of haploidentical hematopoietic stem cell transplantation was uccessfully established.2. Survival of mice: the mice of group A were dead at 11 days after transplantation, and completely dead at 17 days, the mice of B group were dead at 15 days after transplantation, 2 mices of B group were survive more than 30 days , the of mice C group were dead at 13 days after transplantation and completely dead at 19 days. Drawn from the Kaplan-Meier survival curve, A group and C group no significant difference in survival curves (P> 0.05), and B significant difference in survival rate (P <0.05)3. Clinical manifestations of GVHD: white blood cells of A group decreased fast after transplantation , but still portable WBC> 0.5×109 / L at 15d , while the B group and C group decreased slowly; 7 days after transplantation, B, C group level of weight loss Lower than the A group (P <0.05), group B 14 days of transplantation weight losslower than A group (P <0.05), while the C group and A group was no significant difference (P> 0.05); B group had diarrhea, Arched, hair removal and other clinical manifestations of the situation later in the A group and C group.4. Histopathological examination: gross specimen shows focal hemorrhage, endoscopic large number of points were observed in leaf cells and lymphocytes, as well as a large number of focal hemorrhage in group A , and B group and C group the bleeding extent and the lymphocyte infiltration were light A group.5. Mesenchymal stem cells culture : bone marrow mesenchymal stem cells culture at 14 days after transplantation , the results show, A group and C group were scarce, not form colonies and continue to grow, while the B group showed a large number of cell colonies and the growth of good cells. B group cells grew well adherent cell microscopy, elongated spindle morphology, the nucleus closely united. Group B were negative flow cytometry results suggest that expression of CD31 and CD44 positive expression. And that the cells can be induced to differentiate into bone cells and into the fat cells.Conclusion1. Basic fibroblast growth factor can reduce the injury of bone marrow micro environment which caused by pretreatment, stable hematopoietic microenvironment to promote haploid mouse hematopoietic stem cell survival after transplantation, compared to in vitro culture, theneed for higher concentrations (> 100ug/Kg).2. Basic fibroblast growth factor can reduce GVHD after haploidentical hematopoietic stem cell transplantation. But it can not completely prevent and treat GVHD alone.3. The mechanism of basic fibroblast growth factor reducing GVHD in mice is still by stimulating stem cell proliferation in vivo . |
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