Experimental Study Of Bone Marrow Targeted Transplantation With SPIO-N-Au Magnetized Hematopoietic Cells

BACKGROUNDAt present,hematopoietic stem/progenitor cell transplantation(HSCT/HPCT)is mainly performed by intravenous infusion.Most cells are mainly deposited in extramedullary organs such as the lung,spleen,intestine,and liver,and have not been returned.Nest-to-marrow functions and reduces transplant efficiency.Intra-bone marrow bone marrow transplantation(IBM-BMT)allows hematopoietic stem cells or progenitor cells to stay in the bone marrow,thereby promoting the homing of transplanted cells and improving the efficiency of transplantation,thereby accelerating hematopoietic reconstruction.However,the improvement of the transplantation effect of IBM-BMT is still controversial.The big reason is that due to the rich vascular network in the bone marrow cavity,more cells transplanted through the bone marrow will still flow into the venous system,resulting in cell homing.After many years of exploration,our research group has established Magnetism-induced cell target transplantation(MagIC-TT)technology.Previous studies have confirmed that MagIC-TT can mediate bone marrow mesenchymal stem cells(MSCs)and hematopoietic stem/progenitor cells(HSCs/Targeted implantation of HPCs);and demonstrated that MagIC-TT mediated CD45 microbeads,a superparamagnetic nanoparticle magnetized hematopoietic stem/progenitor cell,can be promoted in mouse bone marrow in auto/allograft bone marrow transplantation models The effect of hematopoietic reconstruction.Based on previous research work,this study explored the effects of SPIO-N-Au magnetized hematopoietic cells targeted bone marrow transplantation,and studied the effects of different magnetized cell methods and the number of implanted cells on hematopoietic reconstruction.The scientific hypothesis of this study is that:MagIC-TT combined with IBM-BMT-mediated SPIO-N-Au magnetized hematopoietic cells are transplanted into the bone marrow of mice.The magnetic field can prevent the transplanted magnetized cells from staying in the bone marrow and Proliferation,thereby promoting hematopoietic reconstruction;the implantation of different numbers of hematopoietic cells into the bone marrow has an impact on the survival and hematopoietic reconstruction of mice in different experimental groups.In this study,SPIO-N-Au(superparamagnetic iron-nanoparticles-Au)was used to magnetize bone marrow hematopoietic cells of GFP transgenic mice,and then a mouse model of autologous bone marrow transplantation was used to pretreat the bone marrow Microinjection into the bone marrow cavity of mice,to investigate the effect of MagIC-TT combined with IBM-BMT-mediated targeted transplantation of magnetized whole bone marrow cells into bone marrow on hematopoietic reconstruction,and use GFP reporter gene to label donor cells and semi-solid decalcification And pathological techniques.OBJECTIVE1.In vitro experiments,explore the effect of SPIO-N-Au on the biological characteristics of magnetized bone marrow cells;2.In the autologous bone marrow transplantation model,explore the effects and mechanisms of MagIC-TT+intramedullary injection of SPIO-N-Au magnetized hematopoietic cells in different cell dose groups on hematopoietic reconstruction and survival rate.CONTENTPart 1.In Vitro Study of SPIO-N-Au on Migration and Biological Characteristics of Bone Marrow CellsMethod(1)Cell acquisition and magnetization:GFP+C57BL/6 donor mouse bone marrow cells were obtained;SPIO-N-Au magnetization was used.(2)Morphological analysis and cell viability measurement of magnetized cells:Observe cell morphology and SPIO-N-Au distribution by means of Wright Giemsa staining,Prussian blue staining,scanning electron microscope,and transmission electron microscope;monitor AnnexinV-FITC/PI for nuclear apoptosis Reveal nuclear activity;(3)Magnetic target migration and colonization characteristics of magnetized cells:Explore the ability to observe the horizontal migration and dynamic migration of magnetized and wild-type SPIO-N-Au magnetized hematopoietic cells under a fluorescent microscope under the condition of an external magnetic field;an external magnetic field is applied to the femoral environment to simulate the femoral environment,and magnetized bone marrow cells and culture medium are added to cultivate After 24h staining,observe the colonization of magnetized cells around the magnetic field.Result(1)Cell magnetic susceptibility and cell viability:The magnetic susceptibility of bone marrow cells is(33.5%);the proportion of live cells after initial and after magnetization(92.84 ± 1.28%vs 89.93±1.72%,P=0.127);(2)Morphological analysis:There is no significant difference in the morphology of the cells after magnetization.Scanning electron microscopy and energy spectrum analysis show that SPIO-N-Au can bind to the cell surface with high affinity;both magnetized and unmagnetized bone marrow cells maintain cell integrity.Found significant differences(3)Magnetic target migration and colonization characteristics of magnetized cells:Under a fluorescence microscope,SPIO-N-Au magnetized hematopoietic cells were observed to rapidly move toward the magnetic field,indicating that the ability of magnetized cells to target migration was significantly improved;A horizontal migration experiment system and the semi-solid technology of the research group,the magnetized cells scattered in the semi-solid were observed under a fluorescent microscope.As the migration distance increased,the number of cells and cell distribution density decreased accordingly.A magnetic field was applied to the culture dish to simulate the femoral environment.Magnetized cells and culture medium were added.After 24 hours of culture,the colonization of the magnetized cells around the magnetic field was observed by staining.The observation results under the microscope showed that there were a large number of bone marrow cells retained in the magnetic field,indicating that the magnetized cells can Localized by magnetic field.summaryIn vitro experiments,SPIO-N-Au can efficiently magnetize hematopoietic cells.Through transmission electron microscopy and other means,it was observed that SPIO-N-Au can bind to the cell surface with high affinity,and there is no statistical difference in cell activity detection before and after magnetization;and It was observed under the electron microscope that the magnetized hematopoietic cells maintained the cell integrity;the fluorescence microscope observed that under the action of an external magnetic field,high-speed targeted migration and horizontal migration of the magnetized hematopoietic cells can be achieved.Stay and colonize a lot.Part 2.The autologous bone marrow transplantation model explores the effects and mechanisms of MagIC-TT+intramedullary injection of SPIO-N-Au magnetized hematopoietic cells in different cell dose groups on hematopoietic reconstruction and survival rates.method(1)Experimental grouping:According to the number and type of transplanted cells,the experiment is divided into 2 series-5 × 106 SPIO-N-Au magnetized hematopoietic cell transplantation series,1× 106 SPIO-N-Au magnetized hematopoietic cell transplantation series;in each series,mice were randomly assigned to MagIC-TT,non-MagIC-TT And Control(n=19,17,6 VS n=13,14,3),MagIC-TT applies a magnetic field before IBM-BMT;(2)Rat pretreatment:I.Experimental study on the optimal lethal dose of radiotherapy pretreatment:Set multiple radiation dose groups of 7.0Gy,7.5Gy,7.7Gy,and 8.0Gy.Each group of radiation dose corresponds to 10 C57BL/6 mice.To compare the deaths of mice in multiple groups of radiation doses.In order to explore the optimal dose of radiotherapy in rats.Radiation pretreatment of ? recipients:C57BL/6 recipients received lethal radiotherapy and oral antibiotics(levofloxacin)+intraperitoneal antibiotics(meropenem)preventive medication before IBM-BMT transplantation;(3)IBM-BMT:self-made bone puncture(Chinese Patent No.CN201620090904)for mouse bone Intramedullary puncture injection(4)Evaluation of transplantation effect:with 21-day survival,+5 d,+ 8d,+10d,+12d,and+18d bone marrow and peripheral blood eGFP+cell ratio,blood routine(WBC,HGB,PLT),bone marrow and lung,spleen Histological observations of other organs were used to evaluate hematopoietic reconstruction and the distribution of transplanted cells.ResultResults of pretreatment of radiotherapy radiation dose:After treatment with radiation doses of 7.7Gy and 8.0Gy,the mice died on the 10th to 12th days,and the mortality rate was 100%;after 7.0Gy radiation treatment,observation was performed for 21 days The mice died 0%;after 7.5Gy radiotherapy treatment,the mortality of mice was observed at 16.7%for 21 days.Therefore,this experiment uses a radiation dose of 7.7 Gy.(2)Survival rate:The survival rate of the two series of MagIC-TT group is higher than that of the non-MagIC-TT group,and there is no statistical difference in the survival rate of the two groups;and 1 x 106 SPIO-N-Au magnetized hematopoietic cell transplantation series There is no significant difference between the survival rate of the MagIC-TT group and the survival rate of the 5 × 106 SPIO-N-Au magnetized hematopoietic cell transplantation series,even the 1× 106 SPIO-N-Au magnetized hematopoietic cell transplantation series.Survival rate is higher.All patients in the control group died within 10-12 days(3)Proportion of eGFP+cells in peripheral blood and bone marrow for 1h:The ratio of eGFP+cells in the bone marrow for 1h in the MagIC-TT group suggests that the cells can be locally fixed to the femur of the transfusion side of the mouse under the action of magnetic field(5 ×106 P=0.014&1 × 106 p=0.001);(4)Blood routine:5×106 SPIO-N-Au magnetized hematopoietic cell transplantation series WBC(p=0.025),HGB(p=0.003),PLT(p=0.021)MagIC-TT group had excellent growth quantity and speed In the non-MagIC-TT group,there is a statistical difference between the two;1 × 106 SPIO-N-Au magnetized hematopoietic cell transplantation series HGB(p=0.042),PLT(p=0.049)MagIC-TT group growth number and The speed is better than the non-MagIC-TT group,there is a statistical difference between the two,WBC(p=0.907)MagIC-TT group growth number and speed are better than non-MagIC-TT group,but there is no statistical difference between the two.(5)Histology of bone marrow,lung,intestine,and spleen:GFP+cells in the MagIC-TT group are mainly distributed in the bone marrow and are rare in the periphery;non-MagIC-TT group can observe GFP+cells in the bone marrow,lung,intestine and spleen.SummaryIn an autologous bone marrow transplantation model,MagIC-TT combined with IBM-BMT mediates SPIO-N-Au magnetized hematopoietic cells for targeted transplantation into mouse bone marrow.Observed for 21 days,5×106 SPIO-N-Au magnetized hematopoietic cells The survival rates of the MagIC-TT group of the transplant series and the 1×106 SPIO-N-Au magnetized hematopoietic cell transplant series were higher than those of the non-MagIC-TT group.There was no significant difference in survival rates between the two series of MagIC-TT groups,even at low doses.The survival rate was better in the group;the ratio of GFP+in bone marrow and peripheral blood on the transfusion and non-transfusion sides suggested that the magnetized hematopoietic cells implanted in the bone marrow under the magnetic field could stay and colonize the femur on the transfusion side,and promote hematopoietic reconstruction.Blood routine results suggest that the WIC,PLT,and HGB growth numbers and rates of the MagIC-TT group are higher than those of the non-MagIC-TT group,but not all of them have statistical differences.