Experimental Study On Umbilical Cord Blood Hematopoietic Stem Cells Infusion To Repair Hematopoietic System Injury In Mice After Radiation

BackgroundNuclear radiation and nuclear radiation technology are increasingly used in people’s social life,but accidental exposure to nuclear radiation or exposure to radioactive substances is also a great harm to people.Radiotherapy and chemotherapy can cause severe damage to patients’hematopoietic system in medical treatment.Hematopoietic stem cells are particularly sensitive to radiation,and the radiation dose is（0.7-10Gy）which involves hematopoietic system^[1]and causes Acute radiation sickness（ARS）of bone marrow type,which is mainly manifested as anemia,hemorrhage,infection and even death.Mild to moderate bone marrow ARS can be cured by outpatient follow-up,treatment with cytokines,blood transfusion,etc.Severe and extremely severe bone marrow ARS have a very high mortality rate.Hematopoietic stem cell transplantation is an important choice for the treatment of severe and extremely severe bone marrow ARS.However,selection of donors and treatment of Graft versus host disease（GVHD）are unfavorable to the treatment of severe and extremely severe ARS patients.Timely reduction or repair of radiation damage to human hematopoietic system and improvement of patient survival rate are particularly important in the treatment process.Hematopoietic microenvironment is mainly composed of supporting cells（osteoblasts,endothelial cells,adipocytes,macrophages and mesenchymal stem cells,etc.）adjacent to hematopoietic stem cells in bone marrow^[2-3],which participate in the self-renewal,proliferation and differentiation of hematopoietic stem cells.Studies have shown that radiation not only damages Hematopoietic stem cells（HSCs）,but also affects the hematopoietic microenvironment on which HSCs depend^[4].ObjectiveUmbilical cord blood hematopoietic stem cells are mainly used in malignant and non-malignant hematological diseases.Umbilical cord blood hematopoietic stem cells can repair hematopoietic microenvironment and promote the proliferation and differentiation of hematopoietic stem cells.The purpose of this study is to explore the therapeutic effect of human umbilical cord blood hematopoietic stem cells in mice model of hematopoietic system injury after radiation.MethodsFifty-four male C57BL/6 mice were selected and irradiated by linear accelerator to establish the radiation damage model.Were randomly divided into radiation treatment group（group A）,radiation umbilical cord blood in treatment group（group B）and reinforcement radiation umbilical cord blood therapy group（group C）,group B after 12 h after radiation way by tail intravenous infusion umbilical cord blood hematopoietic stem cells（infusion dose of 5.52 to 6.07 x 106/kg）,group C respectively in the 12 h,24 h after TBI in mice by infusion of umbilical cord blood hematopoietic stem cells（each infusion dose 5.52-6 cutflower production potentials x 106/kg）.To observe the survival rate of the mice in each group after TBI and observe whether there were any symptoms of radiation injury such as hair removal,arch back,diarrhea,etc.,and to evaluate the effect of umbilical cord blood hematopoietic stem cell therapy on spleen,small intestine,skin and bone marrow injury through pathological examination.At the same time for groups of mice after radiation in1,3,7,11,14,17,21,28 d testing analysis of peripheral blood cell count,in vitro culture groups of mice bone marrow mononuclear cells and count,using flow cytometry to detect the proportion of CD34+cells in bone marrow mononuclear,and CFU-GM cloning experiments to detect short-term the proliferation of hematopoietic progenitor cells.Meanwhile,bone marrow mesenchymal stem cells were cultured in vitro,stained with alizarin red s,and the osteogenic induction ability of bm-mscs was determined by real-time quantitative PCR using OCN,ALP and RUNX2 genes.Flow cytometry was used to analyze the phenotype changes of CD68 and IL-10 labeled macrophages in bone marrow,and the concentrations of interleukin-4（IL-4）and interferon-γ（IFN-γ）in bone marrow were detected by ELISA.Meanwhile,the expression of granulocyte-single cell colony stimulating factor（G-CSF）and stem cell factor（SCF）in bone marrow microenvironment was analyzed by ELISA.Results1、The experiment found that transfusion of cord blood hematopoietic stem cells significantly increased the radiation-induced survival rate of mice after radiation:the survival rate of mice in group a was 0%at 28 days,the survival rate of mice in group b was 78%,and the survival rate of mice in group c was90%.compared with the survival rate of mice in three groups,there was statistical significance（P<0.05）.After TBI,the body weight of group A mice continued to decline,and the average body weight at the 17th day of death was（11.21 0.24 g）.The lowest values of group b and group c reached at 11d after TBI were（14.75 0.27）g and（14.93 0.26）g respectively,and there was no significant difference between the two groups（P>0.05）.On the 3rd day after TBI,all three groups of mice showed depilation,irritability,easy attack,bowing back and other symptoms.The symptoms of group A mice continued to worsen and died of diarrhea and other causes.The mice in groups B and C basically recovered to normal on the 21st day,but still had depilation and other symptoms.2、Histopathological pictures can show that a large number of degeneration and necrosis of basal layer cells,necrosis of small intestinal mucosa structure and complete disappearance of structure can be seen in skin pathological sections of group A mice,and a large number of inflammatory cell infiltration can be seen.Only cortex can be seen in spleen pathological sections,but medulla and other structures are not seen.Group B mice showed slight hyperplasia of basal layer on skin,celluloid necrosis of intestinal villi in small intestine and uneven distribution of medulla in spleen.Only a small amount of cell necrosis was found in the skin of mice in group C,and intestinal villi were relatively intact.The medulla is evenly distributed in spleen.The sternum of mice was taken for pathological section.Hematopoietic cells in bone marrow cavity of group A mice were significantly reduced and fibrotic structures were increased.Hematopoiesis in bone marrow cavity of group B mice decreased,showing recovery trend.However,immature red blood cells and granulocyte regeneration can be seen in group C mice.There is a significant contrast between the three.3、After TBI,peripheral blood tests of mice in groups A,B and C showed that the white blood cell count decreased to the lowest in 7 days,and the comparison among the three groups had statistical significance P<0.05.On the28th day,group B was（9.12 1.22）×1012/l,group C was（10.14 1.31）×1012/l,the difference was statistically significant（P<0.05）.Hemoglobin count decreased to the lowest level on the 14th day,with group A,group B and group C being（54.00 1.42）g/l,（70.67 4.50）g/l and（98.66 2.08）g/l respectively,and the three groups were compared in pairs（P<0.01,P<0.01,P<0.05）.The hemoglobin count of mice in group B and C was 21 days,and the comparison between the two groups was statistically significant（P<0.05）.The difference was statistically significant（P<0.05）when comparing the platelet counts of mice in groups A,B and C,reaching the lowest point on the 14th day.the lowest platelet counts of mice in groups A,B and C were（56.00 4.25）×1012/l,（141.33 22.36）×1012/L,（207.33 12.10）×1012/L,respectively.The difference among the three groups was statistically significant（P<0.05）.Then the two groups basically returned to normal on the 28th day（P>0.05）.4、Detection bone marrow hematopoietic stem/progenitor cells,bone marrow mononuclear cell count,A,B,C three groups of pairwise comparison difference had statistical significance（P<0.01）,and compare the 7 d,14 d,three groups of mice bone marrow mononuclear cell population has grown,but slow growth in mice of group A,the count of two time points is not statistically significant（P>0.05）;The rapid growth of group B and group C was statistically significant（P<0.05,P<0.05）.On day 7 after TBI,FCM detected the proportion of CD34+cells in the mononuclear marrow.The proportion of CD34+cells in bm-msns in mice in groups A,B and C was 0.57%,1.56%and 1.90%,respectively.There were significant differences among the three groups（P<0.05）.On day 7 and day 14,cfu-gm in the three groups was compared,and the difference was significant（P<0.05,P<0.05）.5、On the 7th and 14th day after TBI,the expression levels of OCN,ALP and RUNX2 genes in BM-MSCs of group a mice were（1.22 0.06）,（1.66 0.08）,（1.39 0.04）lower than those of group b and group c mice respectively,with significant difference（P<0.05）.The expression levels of OCN,ALP and RUNX2genes in BM-MSCs of group c mice were（1.61 0.05）,（3.12 0.16）,（2.71 0.09）respectively,and the difference was statistically significant（P<0.05）compared with the expression levels of OCN genes（1.44 0.07）,ALP（2.72±0.10）and RUNX2（2.61±0.33）in BM-MSCs of group b mice.Alizarin red staining and inverted microscope observation showed scattered orange mineralized nodules in BM-MSCs of group a mice,while orange mineralized nodules in BM-MSC of groups b and c mice were distributed in sheet form.on the 14th day,BM-MSC of group a mice had poor osteogenic mineralization,with red staining only at some cell aggregation sites.More uniform red-stained mineralized nodules can be seen in groups B and C,with no obvious difference.The expressions of PPAR and CEBP-αgenes in group a mice were higher than those in group b and c,and the difference was statistically significant（P<0.05）.Comparing the two time points of 7d and 14d,the three groups have statistical significance.The results of oil red O staining showed that orange-red lipid droplet particles were scattered in group A,and more uniform lipid droplet particles were visible in groups B and C,but the number of lipid droplets in group C was less than that in group B.On the 14th day,the red fat oil droplets in group A were clustered and almost covered the whole field of vision.The difference of fat oil drops between group B and group C is small,but it is obviously reduced compared with the 7th day.6、Macrophages in BM were isolated and extracted,and the expressions of CD68 and IL-10 in macrophages on 7th and 14th days were detected by FCM.At the same time point,the expression of CD68 in bone marrow of mice in group a,group b and group c had statistical significance（P<0.05）.At the same time point,the expression of IL-10 in bone marrow of mice in group a,group b and group c showed statistical difference（P<0.05）.the expression of IL-10 in mice in the three groups showed statistical difference（P<0.01）.Compared with the 7th and 14th days,the three groups all increased,and the difference in the rate of increase among the three groups was statistically significant（P<0.05）.The concentration of IL-4 in bone marrow was detected by ELISA.at the same time point,there was statistical significance in comparing group a,group b and group c（P<0.05）.The concentration of IFN-γin bone marrow was detected by ELISA,and the change value of IFN-γin three groups of mice was compared（P<0.05）.7、ELISA was used to detect the concentration changes of G-CSF and SCF in bone marrow cells.The concentration of G-CSF in bone marrow of mice was compared at the same time point,and the difference was statistically significant（P<0.05）.The change of SCF concentration in bone marrow of three groups of mice was statistically significant（P<0.05）.Conclusions1、Transfusions of umbilical cord blood hematopoietic stem cells can improve the TBI mice diarrhea,hair removal and other symptoms of radiation injury,improve the survival rate of the mice after radiation.2、Pathological sections showed that umbilical cord blood hematopoietic stem cells could improve the damage degree of skin,small intestine and spleen after TBI.3、The experiment suggested that the infusion of umbilical cord blood hematopoietic stem cells significantly increased the number of bone marrow mononuclear cells in mice after TBI,and the proportion of CD34+cells in bone marrow mononuclear cells increased.The CFU-GM results indicated that umbilical cord blood hematopoietic stem cells promoted the proliferation and differentiation of hematopoietic stem progenitor cells,thus accelerating the recovery of hematopoietic system.4、Umbilical cord blood hematopoietic stem cell transfusion after TBI can increase the ability of BM-MSCs to transform into osteogenesis,reduce its ability to induce adipogenesis,accelerate the homing of hematopoietic stem cells,and promote the recovery of the hematopoietic system.5、Umbilical cord blood hematopoietic stem cells reduce the release of bone marrow macrophage proinflammatory factor IFN-γafter TBI,increase the release of anti-inflammatory factor IL-4,enhance the two-way regulatory effect of bone marrow macrophages on the hematopoietic system,and reduce the inflammatory factors on blood cells Generated impact.At the same time,the concentrations of G-CSF and SFC after TBI are increased to promote early hematopoietic recovery,thereby alleviating the damage of bone marrow hematopoietic microenvironment by TBI.