Efficacy And Mechanisms Underlying The Effects Of Allogeneic Umbilical Cord Mesenchymal Stem Cell Transplantation On Acute Radiation Injury In Tree Shrews

Background:Mesenchymal stem cells(MSCs)have been isolated from various organ sites including bone marrow,skin,vascular and adipose tissues and form a heterogeneous population of multipotent stromal cells.They have been shown to exhibit a relative radiation resistance and retain their stem cell properties even after high doses of ionizing radiation.MSC has been widely studied in the context of ischemic or mechanical forms of tissue damage,and these stem cells may also constitute a powerful means of treating tissue lesions caused by ionizing radiation,either after accidental exposure to radioactivity.Animal studies and early clinical experiences suggest a role for MSC in the regeneration of these tissue lesions both by differentiating into functional parenchymal cells and by creating a nurturing microenvironment for other cells.Currently,studies are mainly focused on Bone marrow mesenchymal stem cell(BMSC).However,the proliferation and differentiation ability of BMSCs decreased with age.In addition,autologous transplantation of BMSC depends on the patient’s state,which limited its application.Umbilical cord mesenchymal stem cells(UCMSCs)are relatively abundant,induce no allogeneic rejection,are associated with no ethical controversy,can be mass produced.Hence,UCMSCs are ideal seeds for cell therapy.Objective:1.To establish an acute radiation injury model of tree shrews.2.To evaluate and analyze the efficacy and mechanism of allogeneic UCMSC transplantation on acute radiation injury in tree shrewsMethods:1.Isolation,culture,identification,and labeling of tree shrew UCMSC:Tree shrew umbilical cord samples were used to isolate and culture UCMSCs using tissue explant adherent method.Flow cytometry assay was used to detect cell surface markers.UCMSCs were induced to differentiate into adipocytes,osteoblasts and chondrocytes in vitro.EGFP-labeled UCMSC were traced in vivo.2.Irradiation procedure of acute radiation injury in tree shrews:unanesthetized tree shrews were placed in a homemade plastic box(25 cm×6.6 cm×4 cm)for whole-body single X-ray ionizing radiation.All of these procedures were performed at the Radiation Center of Kunming General Hospital.The size of the irradiation field was 30 cm×30 cm,the source wave distance was 100 cm,the dose rate was 480 cGy/min,and the total irradiation dose was 4.5 Gy.3.UCMSC transplantation on acute radiation injury in tree shrews and detection indicators:150 female tree shrews were randomly divided into four groups:(1)Treatment(n=40):radiation+UCMSC injection(3×10~7 cells/kg,1 mL PBS suspension cells);(2)Model(n=60):radiation+1 mL PBS injection;(3)Control(n=40):1 mL PBS injection;(4)EGFP(n=10):radiation+eGFP labeled UCMSC injection(3×10~7 cells/kg,1 mL PBS suspension cells).In all animals,the injection was via tail vein,the first injection was performed immediately after irradiation,and the injection day was defined as the first day.The tree shrews were treated once every 7 days for a total of 4 treatments.12 tree shrews(n=12)were taken from the treatment group,model group and control group,3 tree shrews were separately sacrificed on the 7th,14th,21st,and 28th day,one side of the femur was counted for bone marrow nucleated cells;One parts of spleen,lung,kidney,small intestine,thymus and femur were fixed with 4%paraformaldehyde solution,and another parts of them were frozen at-80°C for histopathological observation and mRNA detection.The remaining 3 groups of trees were used for weight and survival observation,peripheral blood cell counts,peripheral blood cytokine detection,apoptosis rate detection,transcriptomics sequencing,karyotype analysis and FISH staining.The EGFP group was used to track transplanted UCMSC.Results:1.UCMSCs with high purity,strong proliferation and multi-differentiation ability were abtained by using tissue explant adherent method.EGFP Labeled UCMSCs were tracing UCMSC in vivo.2.Acute radiation injury model of tree shrew was successfully established with a linear accelerator performing a single 4.5Gy X-ray irradiation on the tree shrew.3.UCMSC transplantation on acute radiation injury in tree shrews significantly improved survival rates,reduced lung inflammation and apoptosis,prevented pulmonary fibrotic processes,recovered hematopoiesis,and increased blood counts.A protein microarray analysis showed that serum levels of the anti-inflammatory cytokines IL-10 and IL-13 and the growth factors BMP-5,BMP-7,HGF,insulin,NT-4,VEGFR3,and SCF were significantly higher,while those of the inflammatory cytokines IL-2,TIMP-2,TNF-α,IFN-γ,IL-1ra,and IL-8 and the fibrosis-related factors PDGF-BB,PDGF-AA,TGF-β1,IGFBP-2,and IGFBP-4 were significantly lower in UCMSC-injected animals.A transcriptome analysis of Peripheral blood showed that the mRNA expression of C1q was upregulated,while that of HLA-DP was downregulated after UCMSC injection.These results confirm the immunohistochemistry results.eGFP-labeled UCMSC were traced in vivo and found in the heart,liver,spleen,lungs,kidneys,thymus,small intestine and bone marrow.Conclusions:1.A method for isolating,cultivating,identifying and labeling tree shrew UCMSC has been established.2.A method for acute radiation injury in tree shrews has been established.3.A role for UCMSC in the treatment of acute radiation injury in tree shrews both by regulating the level of cytokines and the expression of C1q and HLA-DP.UCMSC transplantation is an effective method for the treatment of acute radiation injury.