To Compare The Effects Of Mesenchymal Stem Cells Derived From Different Sources Combined With Haploid Hematopoietic Stem Cell Transplantation On Hematopoietic Reconstitution

Hematopoietic microenvironment plays an important role in the growth and development of hematopoietic stem/progenitor cells.Bone marrow mesenchymal cells are an important component of bone marrow hematopoietic microenvironment.The morphology and function of bone marrow mesenchymal stem cells in patients with severe myeloid acute radiation sickness are damaged to varying degrees,and hematopoietic failure is more durable or difficult to recover.Haploid hematopoietic stem cell transplantation is an effective method for the treatment of severe bone marrow type acute radiation sickness.However,due to the severe heterogeneity of HLA matching,the transplant rejection rate is high,more complications of transplantation,the degree of heavy,graft failure rate is higher.Mesenchymal stem cells(MSCs),which have the ability of self replication and multi differentiation potential,are derived from bone marrow,umbilical cord,umbilical cord blood,dermis,adipose tissue and so on,which can promote hematopoietic reconstitution and regulate GVHD.It is not clear that whether haploid hematopoietic stem cells combined with mesenchymal stem cells which derived from different sources implanted in the body of patients with severe radiation injury can promote hematopoietic recovery,at the same time,and reduce the occurrence of GVHD and thus achieve effective treatment of bone marrow radiation sickness,and whether It is unclear whether mesenchymal stem cells derived from different sources have significant differences in promoting hematopoietic recovery.Therefore,it is important to compare the effects of mesenchymal stem cells derived from different sources combined with haploid hematopoietic stem cell transplantation on hematopoietic reconstitution in the treatment of severe bone marrow type acute radiation sickness.1.Research content and methods:1.1 Isolation,culture and identification of mesenchymal stem cells derived from different sourcesThe bone marrow mesenchymal stem cells were isolated and cultured by density gradient centrifugation.The dermal mesenchymal stem cells were isolated and cultured by enzyme digestion.The umbilical cord mesenchymal stem cells were isolated and cultured by tissue culture.The phenotypic identification of mesenchymal stem cells derived from three sources was performed by flow cytometry.1.2 Irradiation dose of animal model of severe bone marrow radiation injuryThe first generation(CB6F1(H-2b × d))mice of C57 BL / 6(H-2b)with BABL / C(H-2d)Hybridization were subjected to 60Co? 5.0 Gy,6.0 Gy,7.0Gy,8.0 Gy radiation dose.Each dose group was respectively given PBS and hematopoietic stem cell transplantation to observe the survival of F1 mice after radiation injury.To explore the appropriate radiation dose for the animal model of severe bone marrow radiation injury in this study.1.3 The effects of mesenchymal stem cells derived from different sources combined with haploid hematopoietic stem cell transplantation on hematopoietic reconstitutionPreparation of hematopoietic stem cells from donor C57 BL / 6(H-2b).The first generation(CB6F1(H-2b × d))mice of C57 BL / 6(H-2b)and BABL / C(H-2d)were harvested with 60Co?-ray(8.0Gy).The mice were divided into blank control group,HSC single transplantation group,HUCMSCs + HSC cohort group,BMSCs + HSC cohort group and DMSCs + HSC cohort group,40 mice in each group.The control group mice were injected with 200 ?l PBS solution through the tail vein.1 × 106 hematopoietic stem cells were injected into HSC single transplantation group through the tail vein.The cohort group mice were injected with 1 × 106 HSC and 1 × 106 mesenchymal stem cells.Twenty mice were used to observe the survival status and survival rate.Twenty mice were used to detect changes in peripheral blood,bone marrow pathological changes and bone marrow cell colony formation.2.Results:2.1 Isolation,culture and identification of mesenchymal stem cells derived from different sources Bone marrow mesenchymal stem cells,dermal mesenchymal stem cells and umbilical cord mesenchymal stem cells showed long spindle and polygonal cell morphology.The cells grow into a spiral,reticular or parallel arrangement.Subcultured cells quickly adhere to the state,the state is relatively uniform,the growth is rapid.With the increase of the number of passages,the cells gradually increased,and the growth rate decreased gradually.The results showed that the mesenchymal stem cells of three different origins did not express CD14,CD20,CD34,CD45 and other hematopoietic cell surface antigens,and high expression of CD73,CD90,CD105 and other mesenchymal stem cell surface antigens.They were detected by mesenchymal stem cell phenotype assay kit and flow cytometry.,2.2 Irradiation dose of animal model of severe bone marrow radiation injuryPBS transplanted mice of the 8.0Gy radiation dose group were all killed and hematopoietic stem cell transplanted mice survived more than 30 days.PBS transplanted mice survived of 5.0Gy,6.0Gy,7.0Gy radiation dose more than 30 days.The hematopoietic function in mice was recovery.2.3 The effects of mesenchymal stem cells derived from different sources combined with haploid hematopoietic stem cell transplantation on hematopoietic reconstitutionThe results showed that the recovery of peripheral hemogram,bone marrow biopsy,CFU-E,BFU-E,CFU-GM and so on of the cohort group mice were significantly different from those of single stem cell transplantation group mice(P < 0.05)The rate and extent of hematopoietic recovery in umbilical cord mesenchymal stem cells and hematopoietic stem cell transplantation group were significantly higher than those of the other two cohort groups(p <0.05).3.Conclusion:3.1 The mesenchymal stem cells isolated from bone marrow,dermis and umbilical cord have the morphological characteristics of mesenchymal stem cells and express the surface antigen of mesenchymal stem cells.3.2 8.0Gy is the appropriate radiation dose for the animal model of severe bone marrow radiation injury.3.3 The effect of umbilical cord mesenchymal stem cells combined with haploid hematopoietic stem cells on hematopoietic reconstitution in mice with severe bone marrow injury was significantly better than that of bone marrow mesenchymal stem cells and dermal mesenchymal stem cells.