| ObjectiveAplastic anemia(aplastic anemia, AA) is a kind of bone marrow hematopoietic function prostration caused by different causes and mechanisms. The defect of hematopoietic microenvironment is one of the mechanisms leading to the onset of aplastic anemia. Hematopoietic stem cell transplantation(HSCT) is a very important method in the treatment of aplastic anemia in addition to immunosuppressive agents and androgen. However, the shortage of donor cells and the severe graft versus host disease(GVHD) is an important obstacle to the treatment. Mesenchymal stem cell(Mesenchymal stem cell, MSC) has robust ability of proliferation and multipotential differentiation, which can differentiate into hematopoietic cells both in vitro and in vivo. It can support hematopoiesis and regulate immune function, which is an important component of bone marrow hematopoietic microenvironment. Hepatocyte growth factor(hepatocyte growth factor, HGF) is a multifunctional cell growth factor, which can promote angiogenesis, cell division, etc. It can also mediate homing of stem cells and directly induce stem cells and progenitor cells to migrate to the site of bone marrow or ischemia. HGF gene can be transfected into mesenchymal stem cells by virus and other vector, and the expression of HGF will increase. The majority of the researches show that the HGF gene modification of mesenchymal stem cells enhances cell repair, regulation of immune function and so on. However, the researches on the effect of HGF gene modified mesenchymal stem cells on the hematopoietic system are limited.The purpose of this study is to explore the effects of human umbilical cord derived mesenchymal stem cells on hematopoietic function in aplastic anemia model through animal experiments on the basis of previous clinical experience, to investigate if the effects on the hematopoietic function in mice with aplastic anemia is cooperative between mesenchymal stem cells and hepatocytes growth factor. MethodsPart one: Six patients who were diagnosed as SAA in the Hematology Department of First Affiliated Hospital of Xinxiang Medical College and received the treatment of hUCMSC and PBSCT from January 2009 to December 2014, were retrospectively analyzed in this study.In these patients, two were SAA- I and four were SAA- II. They were treated with cyclosporine A(CSA)?androgen and the different numbers of blood transfusion before transplantation. The six donors(4 were males and 2 were females) from siblings were consistently matched in 6 loci of HLA. The donor- recipient blood group incompatibility was 1/6. Conditioning regiment was the rabbit antithymocyte globulin(ATG)?cyclophosphamide(CTX) and fludarabine(Flu). The graft versus host disease(GVHD) was prevented with short-range methotrexate(MTX) and cyclosporine A(CSA). The cases lived in sterile transplantation ward, and were given supportive treatment. The median value of the cells expressed CD34 was(4.18(2.75-6.46)×106) on the day of the infusion of stem cells. Four hours before the infusion of stem cells, MSC(2.71(2.4-3.0)×1010/L)was infused. This study aimed to observe the toxic side effects during the period of conditioning regiment, the adverse reactions of HSC and MSC infusion,the recovery time of hematopoietic engraftment and donors HSC implantation, GVHD,etc.Part two: Umbilical cord cells stem mesenchymal human(hUCMSC) was obtained by the method of adherent culture, and the surface antigen markers of MSC were detected by flow cytometry, and the characteristics of mesenchymal stem cells were detected by inducing osteogenic differentiation and neuronal differentiation.We used adenovirus vector containing HGF gene(Ad-HGF) to transfect mesenchymal stem cells, Western blot(Western blot) and enzyme-linked immunosorbent assay(ELISA) to detect the expression of HGF in transfected mesenchymal stem cell, and surface markers and osteogenic and neuronal differentiation to detect the effect on mesenchymal stem cells of Ad-HGF.Part three: We divided 28 BABL/C mice into experimental control group, mesenchymal stem cell injection group, Ad-HGF transfected MSC injection group, and exposed each group to 6Mv X-ray equally. On 2, 4 and 6 days after irradiation, we injected intraperitoneally to every mouse with cyclophosphamide 50mg/kg and chloramphenicol 62.5mg/kg. On the eighth day after irradiation, we injected mesenchymal stem cell injection group with 5×107 human umbilical cord derived mesenchymal stem cells through caudal vein and injected Ad-HGF transfected MSC injection group with 5×107 Ad-HGF transfected mesenchymal stem cell, and we detected the content of HGF in peripheral blood by ELISA on the second day after injection. The experimental group was injected with equal saline. After modeling we observed daily general condition of mice, on the eighth day after modeling blood was taken to observe the changes of blood in the three groups, and bone marrow taken for bone marrow aspirate and bone marrow biopsy. Anatomical observations were made of dying mice and dead mice. Observe the changes of blood for 21 days and evaluate the stability of model of aplastic anemia in mice. Three groups of data were analyzed with statistical method. ResultPart one: Six cases had rapidly hematopoietic engraftment after transplantation. In 3 months, we researched the blood types of receptors which were expressed the donors in 100%. All patients obtained WBC and PLT recovery at a median of 12 days and 16 days, no adverse reactions, no acute GVHD, no other serious complications were observed.Part two: Fibroblast like morphology of the adherent growth of human umbilical cord derived mesenchymal stem cells was obtained by adherence culture method. The phenotype characteristic analysis showed that the cells were non hematopoietic cell populations. The differentiation test proved that the cells had the ability of osteogenic and neurocytic in vitro. The results showed that the cells were in accordance with the characteristics of MSC.We used adenovirus vector(Ad-HGF) with HGF gene to transfect mesenchymal stem cells(MSC), which was not affected by the detection of the surface markers and differentiation ability. The expression of HGF in the transfected group was significantly higher than that in the non-transfected group, and the difference was statistically significant.Part thee: The three groups of experimental mice were used to make the model of X-ray and chemical medicine. We found activity reduction, the feeding reduction and weight loss, and that in the control group there was 1 case died. On the eighth day after modeling, the detection of blood RT was found that mesenchymal stem cell injection group, Ad-HGF transfected MSC injection group, the degree of decrease of white blood cells, red blood cells and platelets were lighter than in the control group, and each index of transfected group is a little higher than that of the stem cell groups, but there was no statistical difference. Bone marrow aspirate and bone marrow biopsy showed that the reduction of hematopoietic tissue and the increase of fat cells. But the pathological changes in the stem cell group and the transfection group were lighter than that in the experimental group. The number of white blood cells, red blood cells and platelets in each group were lower, and the stem cells group and transfection group recovered well up to 21 days. ConclusionPart one: The data here suggested that co-infusion of hUCMSC and PBSCT in the treatment of patients with SAA was a reliable and effective way, and had low incidence of GVHD.Part two: The human umbilical cord mesenchymal stem cells obtained by adherent culture method showed that the cells were in accordance with the characteristics of MSC.The characteristics of the surface markers and transformation function were not affected in HGF gene transfected with adenovirus vector(Ad-HGF) mesenchymal stem cells(MSC).Part three: X-ray, cyclophosphamide and chloramphenicol were combined to construct aplastic anemia mouse model and it was consistent with the clinical characteristics of aplastic anemia. Blood RT and bone marrow changes met with the typical aplastic anemia. Mortality was low in experimental animals. Model were stable and lasting.Human umbilical cord derived mesenchymal stem cells and Ad-HGF transfected mesenchymal stem cells had a significant protective effect on aplastic anemia mice hematopoietic function. Transfected mesenchymal stem cells had stronger protective effects, which suggested that HGF and mesenchymal stem cells might have synergy effect on hematopoietic function of aplastic anemia mice, which needs further research. |
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