Therapeutic Benefit Of Intravenous Administration Of Bone Marrow Stromal Cells After Spinal Cord Injury In Rats

PartⅠMesenchymal stem cells of rats stained by carboxyfluorescein diacetate succinimidy ester in vitroObjective:To investigate the feasibility of labeling mesenchymal stem cells(MSCs) of rats with 5,6,2-carboxyfluorescein diacetate succinimidy ester(CFSE)and the most optimal condition.Methods:MSCs obtained from 20 SD rats, bone marrow were isolated, purified and cultured in vitro by Percoll density gradient centrifugation combined with adherent method. The third generation MSCs filling in the floor of 25cm2 cultured bottles were incubated with 2.5,5,10,20,and 40μmol/L of CFSE for 1,5,10,15,and 20 min respectively.The most optimal concentration of CFSE and incubation time were selected by detecting the fluorescence intensity and adhesive rate of labeled MSCs.The proliferation ability of MSCs labeled by CFSE under the fitest condition was tested by MTT method.Results:Incubation with 10μmol/L of CFSE under 37℃for 10 minute was the most optimal condition for MSCs labeling.The labeled MSCs maintained the same proliferation ability as the non- labeled MSCs under the most optimal condition.Conclusion:With the advantages of high rate of dyeing, low cell toxicity,safety and convenience,CFSE can be used as a new method to label the rat,s MSCs, Incubation with 10μmol/L of CFSE under 37℃for 10 minute was the most optimal condition for MSCs labeling.PartⅡStudy on inflammatory cascade response after spinal cord injury and its effects on the survival and immigration of marrow mesenchymal stem cells in injuryed spinal cord in rats by intravenous administration Objective:To study on inflammatory cascade response after spinal cord injury(SCI) and its effects on the survival and immigration of marrow mesenchymal stem cells in injuryed spinal cord in rats by intravenous administration,also to evaluate the most optimal intravenous administration time for MSCs.Methods:The rat spinal cord injury model was prepared according to the modified Allen method.80 cases of SD rats were randomly divided into non-injured group,and 1 h,6 h,12 h,24 h,3 d,5 d,7 d seven injuried groups after spinal cord injury(SCI).The damaged region of spinal cord were removed in different time phases.The structure of spinal cord was observed by hematoxylin and eosin(HE) method to evaluate the histopathologic changes. The expressions of TNF-α,IL-1βand CINC-1 in the spinal cord post-injury were studyed by RT-PCR methods. The expressions of ICAM-1 in the spinal cord post-injury were studyed by imminohistochemitry method.Myoleperoxidase(MPO) activity in the spinal cord post-injury was measured by chemitry method. Other 40 cases of SD rats were randomly divided into non-injuried group,and seven transplanted groups at 0h,6h,12h,24h,3d,5d,7d after spinal cord injury(SCI).The survival counts and immigration distance of MSCs in the injuryed spinal cord of all groups of rats were measured.Results:The TNF-αand IL-1βbegan to express in the injured spinal cord at 1 h after SCI,and reached their peak value at 6 h after SCI. The ICAM-1and CINC-1 began to increase in the injured spinal cord at 6 h after SCI,and reached their peak value at 12 h after SCI.The activity of MPO began to express in the injured spinal cord at 6 h after SCI,reached its peak value at 24 h after SCI.The PMNL began to infiltrate into the injured spinal cord at 6 h after SCI ,and there was a great deal of infiltrating PMNL in the injured spinal cord at 24 h after SCI. The infiltrating PMNL in injured spinal cord began to decrease and the activity of MPO began to weaken from 3 d after SCI.There were no infiltrating PMNL in injured spinal cord and the activity of MPO resolved at 5 d and 7 d after SCI,but there was the proliferation of gial cells in the spinal cord. The survival counts of MSCs were fewer,but the migration distance of MSCs was longer at 1 h,6 h,12 h,24 h after SCI, The survival counts of MSCs were more,and the migration distance of MSCs was longer at 3 d after SCI, The survival counts of MSCs were fewer,and the migration distance of MSCs was shorter at 5 d and 7 d after SCI.Conclusions:There was existing the inflammatory cascade response in the injuryed spinal cord after SCI.Because of existing the inflammatory cascade response in the spinal cord at 1 h,6 h,12 h,24 h after SCI,the local micro-environment was not good for survival of MSCs. Because of existing the proliferation of gial cells in the spinal cord at 5 d and 7 d after SCI, the local micro-environment was not good for migration of MSCs. It maybe the most optimal intravenous administration time for MSCs at 3 d after SCI.PartⅢDifferentiation of transplantating MSCs in rat injured spinal cordObject:To investigate the possibility that transplantating MSCs differentiate into functional neurons in injured spinal cord in rats.Methods:The ultrastructure of transplantating MSCs in injured spinal cord was observed at 1,3,5 week after transplantating by using immuno-electron microscopic study with the method of low temperature embedding.The expressions of ChAT,GAD,Synapsins,MBP and PLP of transplantating MSCs in the injured spinal cord were observed at 1,3,5 week after transplantating by using immunofluorescence staining and laser confocal scanning microscopey methods.Results:The organelles in MSCs migrating into grey matter of spinal cord were not developed at 1 week after transplanlating,there were no the feature of ultrastructures of neuron. The organelles in MSCs immigrating into white matter of spinal cord were developed at 1 week after transplanlating,there were the feature of ultrastructures of oligodendrocytes. The organelles in MSCs migrating into grey matter of spinal cord were developed at 3 week after transplanlating,there were the feature of ultrastructures of neuron.The MSCs migrating into grey matter of spinal did not express ChAT,GAD and synapsins at 1 week after transplanlating,but the MSCs migrating into white matter of spinal cord expressed MBP and PLP. The MSCs migrating into grey matter of spinal expressed ChAT,GAD and synapsins at 3 week after transplanlating.The numbers of MSCs migrating into white matter of spinal cord were more than which MSCs migrating into grey matter of spinal cord.Conclusion:The transplantating MSCs in injured spinal cord can partly differentiate into both neurons and oligodendrocytes.There was difference at time between MSCs differentiating into neurons and oligodendrocytes.The time of MSCs differentiating into neurons is later than that MSCs differentiating into oligodendrocytes.The numbers of MSCs migrating into white matter of spinal cord were more than which MSCs migrating into grey matter of spinal cord.PartⅣEffect of bone marrow mesenchymal stem cells on the proliferation of oligodendrocytes and remyelination of axons after spinal cord injury by vein transplantationObject:To investigate the effect of MSCs on proliferation of oligodendrocytes and remyelination of axons after spinal cord injury by vein transplantation .Methods:60 cases of SD rats were divided into non-injuried group,control group and experimental group.The rats in the non-injuried group and experimental group were infused with MSCs through tail vein, the rats in the control group were infused with PBS.The ultrastructure of myelin sheaths and oligodendrocytes were observed by electron microsecope at 1 and 3 week after transplanlating.The expression of MBP and PLP were examined by immunofluorescence staining methods at 1 and 3 week after transplanlating. The expression of MBP and PLP of transplantating MSCs in the injured spinal cord were observed at 1,3 week after transplantating by using immunofluorescence staining and laser confocal scanning microscopey methods.Results:In control groups at 1 week after SCI, the myelin sheaths were seriously swellen,and part of them were disrupted.Some oligodendrocytes were necrotic and resolved,many proliferated oligodendrocytes were detected. At 3 week after SCI,the swelling myelin sheaths were palliative,and a few proliferated oligodendrocytes were also detected.In experimental groups at 1 week after SCI,the swelling myelin sheaths were obviously palliative,few oligodendrocytes were necrotic,and a great many of proliferated oligodendrocytes were detected. In experimental groups at 3 week after SCI,the structure of myelin sheaths and oligodendrocytes was normal.The expression of MBP and PLP in control group was lower than that in non-injuried group, the expression of MBP and PLP in experimental group was more than that in control group. The MSCs migrating into white matter of spinal cord expressed MBP and PLP at 1 week after transplanlating.Conclusion: After spinal cord injury the transplanting MSCs by vein may inhibit degeneration of nervous fibers,and promote proliferation of oligodendrocytes and remyelination of regenerated and demyelinated axons.