The Regulatory Role Of Human Umbilical Cord Blood-derived Stromal Cells On Hematopoietic Damage Mice Model In Hematopoietic Function

bjective:This study isolated from human umbilical cord bloodmononuclear cells by density gradient centrifugation method, and culturedhUCBDSCs, hUCBDMSCs; To observe the morphology and proliferationcharacteristics of cells; To identify surface antigen by flow cytometry; Toimplanted cells with DiI labelled in hematopoietic damage mice model, dynamicdetect of venous blood in mice red blood cells, white blood cell, platelet; Todynamic detect of cells in the repair of bone marrow hematopoieticmicroenvironment, and compare with ability of restore hematopoiesis inhUCBDSCs and hUCBDMSCs. To establish the value of hUCBDSCs in theclinical and providing an animal experimental basis for the theory system ofhematopoietic recovery, and solve the clinical side effects of using promotehematopoietic growth factors and expensive cost; meanwhile bring new ideas tohematology workers of hematopoietic recovery method. Methods:1.Cellisolation and culture: to isolate from umbilical cord blood mononuclear cells bydensity gradient centrifugation, and use DMEM/F-12, MSCM cultivation systemto culture in vitro, then get high purity of hUCBDSCs and hUCBDMSCs,observing cell morphology and growth characteristics with the invertedmicroscope, using flow cytometry to detect the expression two classes of cell surface antigen;2.To marke the DiI on cells;3. To establish the damage micemodel: X ray irradiated BALB/c mice.4.Group experiment: the mice arerandomly divided into3groups: hUCBDSCs group, the hUCBDMSCs groupand control group;5. To inject hUCBDSCs (1.0x106/mouse), hUCBDMSCs(1.0x106/mouse), saline solution (0.2ml) to each group in caudal vein, observingthe mental state, diet and other physiological status of eath group, calculatingsurvival rate of each group; Collection venous blood samples to count red bloodcells, white blood cells, platelet in the3rd,7th,14thday; bone marrow smear inthe3rd,7th,14thday.5.The statistical analysis: using the SPSS22.0statisticalanalysis software for statistical analysis of data, measurement data are presentedasˉx±s, one-way ANOVA used among the groups,t-test used in pairwisecomparisons, inspection level of α=0.05. A value of P<0.05was consideredstatistically significant. Results:1.The density gradient centrifugation can obtainmononuclear cells, through serial subcultivation high purity hUCBDMSCs andhUCBDSCs were gained in vitro. When primitive cells cultivated with MSCM,they became irregular shape, rounded, uniform distribution, the cell body smallafter24hours; After48hours, cells were adherence and proliferaed rapidly,some began to stretch toward the poles; On the7th-12thday, it was consist ofspindle cells mainly, showing colony growth,hardly finding cell granules; Onthe14th-16thday, cells can bespread across the bottom; Cells were similar andgrew more rapidly after being passaged, entering the logarithmic growth phaseafter the1st-2ndday as uniform slender spindle type, the6thday into the platform stage. When primitive cells cultivated with DMEM/F-12, they became irregularshape, rounded, uniform distribution, the cell body small after24hours; After48hours, cells were adherence and proliferaed rapidly, some began to stretchtoward the poles; On the7th-12thday, it was consist of oval cells mainly,showing colony growth,hardly finding cell granules; Cells were similar andgrew more rapidly after being passaged, entering the logarithmic growth phaseafter the1st-2ndday as oval type, the6thday into the platform stage.HUCBDSCs grew less rapidly than hUCBDMSCs in early stage, and morerapidly in later stage in vitro.2.Surface antigen: hUCBDMSCs: CD29(+),CD44(+),CD106(+),STRO-1(+),CD34(-),CD45(-);hUCBDSCs: CD44(+),CD45(+),Fn(+),Lm(+),CD29(-),STRO-1(-).3.The establishment of the model:irradiated BALB/c mice in sublethal dose (total dose of5Gy, energy of6mev,distance of100cm, the absorption rate of100cgy/min,5min holding time) cancause acute injury of hematopoietic function in mice.4. DiI marks the thirdgeneration of hUCBDMSCs and hUCBDSCs, they shown the orange under thelaser scanning confocal microscope, cell morphology were the same under thegeneral microscope. two kinds of cells labeled were100%.5. Boood routine test:the results shown that there were no obvious difference among three groups ofmice on the3rdday(P>0.05); White blood cells, platelets, red blood cells,hemoglobin concentration of hUCBDSCs and hUCBDMSCs group weresignificantly higher than that of control group on the7thday (P<0.05), and thewhite blood cells, platelets, red blood cell of hUCBDSCs group were significantly higher than hUCBDMSCs group (P<0.05), and hemoglobinconcentration between two groups had no obvious difference (P>0.05). On the14thday, the white blood cells, platelets, red blood cells, hemoglobinconcentration of hUCBDSCs and hUCBDMSCs group were significantly higherthan that of control group (P<0.05), and white blood cell, platelet hemoglobinconcentration of hUCBDSCs group were significantly higher thanhUCBDMSCs group (P<0.05), and the red blood cell between two groups hadno obvious difference (P>0.05).6. The homing efficiency of hUCBDSCs andhUCBDMSCs: the results shown that the two types of cells can be stable in bonemarrow in the later period of transplantation, and the hUCBDSCs group wasearlier.7.Pathological section:On the3rdday after transplantation, the resultsshown that the hematopoietic function of all the mice bone marrow in threegroups had been inhibited, and non-hematopoietic cells increased; On the7thday,hematopoietic function of three groups began to recover, and nucleated cellswere tufte, myelosis of hUCBDSCs and hUCBDMSCs group were better thanthe control group, and hUCBDSCs group is better than that of hUCBDMSCsgroup; On the14thday, bone marrow hematopoietic function of control grouphad no significant changes, and hUCBDSCs and hUCBDMSCs group wereobviously recoveried, meanwhile the nucleated cell proliferatde actively, andthere were many megakaryocytes under the microscope. Conclusion:1.Byusing density gradient centrifugation can obtain high purity hUCBDMSCs andhUCBDSCs in vitro.2. hUCBDSCs and hUCBDMSCs could significantly promote the recovery of white blood cell, platelet, red blood cell andhemoglobin concentration when transplanted into mice, and the recovery ofhUCBDSCs were higher and marrow suppression were lower.3hUCBDSCsand hUCBDMSCs could be stable in bone marrow after transplantation, and thehUCBDSCs group homing was earlier; The two types of cells both couldpromote hematopoietic function recovery, and hUCBDSCs was better thanhUCBDMSCs.