Protective Effects Of Bone Marrow Mesenchymal Stem Cells On Spinal Cord Injury In Rats And Its Mechanism Study

Spinal Cord Injury (spinal cord injury, SCI) is a common surgical traum a,treatment on which is one of unresolved important problems in modernmedical field. One is using medicines such as Methylprednisolone,Calciumchannel antagonists, naloxone or part low-temperature protection or highpressure irrigation of manpower stream to lease or eliminate Secondarypathological reaction during the acute injury to protect the remnants of theaxon and neurons from injury, The other is to promote nerve tissueregeneration and repair during the chronic injury, including the treatment ofSchwann cell transplantation and gene therapy and hyperbaric oxygen therapy.Although many treatment methods, but there is no obvious means of effectivetreatment.Research shows that a variety of methods can be used fortransplantation to promote regeneration and repair spinal cord injury.In recent years, Stem cell researchers have aroused widespread concern onthe prospects of the MSCs Polyembryony direction of differentiation, and inthe cell and gene therapy application.As rich source of MSCs, drawing simple,easy separation, purification and culture, it can be rapidly expanded in vit rounder certain conditions, with more differentiation potential, autologoustransplantation to overcome the ethical and immunological rejection.Thisdamage to the central nervous system and the nervous system diseases such asParkinson's disease, Alzheimer's disease, multiple sclerosis, cerebral infarction,such as transplantation in the treatment of the injury provided the possibility for the central nervous system function and nerve regeneration reconstructionprovides a new technology, has become a clin ical cell therapy and cellengineering ideal seed cells.But its role in the repair of spinal cord injurymechanism is not yet very clear.Objective:this study in the use of home -made devices against spinal cord injurymodel--- on the basis of preparation of spinal cord injury in rats,apply bonemesenchymal stem cells to treat them, Observation on the protection andspinal cord injury repair,and through the impact of its neural cell apoptosis toimpact of its mechanism.Research Methods:1．According to the adherent of MSCs in vitro Purification of MSCs.Morphological changes were observed by flow cytometry to detect cell cycle,MTT assay its growth characteristics, flow cytometry to detect cell surfacemarkers identified by the separation of MSCs；2．The experiment made use of combat equipment, application ofimproved methods of Allen animal model of spinal cord injury model in rats；3．According modified Tarlov function evaluation standards, observe theMSCs rat model of spinal cord injury treatment；4．After Laser scanning confocal microscopy in vitro DAPI labeled MSCsinjected into the tail vein, to the injury settlement and chemokine；5．Histopathology and immunohistochemical methods to observe MSCsafter treatment, histopathological changes, and the Bcl -2, Bax and theexpression of Nogo.Results:1．MSCs rat model of spinal cord injury protection role After spinalcord injury in rats showed double Ruantan hindlimb muscle tension reduction, acupuncture, lower extremity non -response, confirmed that the rat spi nal cordinjury model successfully established；Rats behavioral score results show that:the first day of spinal cord injury model group, the treatment group MSCsmotor function score lower than the control group, the difference wassignificant; MSCs for seven days, some recovery of motor function in rats wassignificantly higher than that model group, but there are still large differencesbetween the normal control group.To continue to observe the first 15 days,MSCs motor function in rats treated group hig her than the model group andlower than the control group, and motor function was further recovery thanthose in the first seven days.Through the Pathologic observation we found thatthe normal control group of rats are with spinal cord structural integrity,uniform distribution of nerve cells, and normal morphology. Spinal cord injuryin the first two days, a large tissue injury become necrotic foci andinflammatory response increased; 7d spinal cord injury and the surroundingarea, the inflammatory cell infiltration reduced, and some spinal cord tissueinjury were cyst-like changed.After 15 d, softening lesions occurring at thelarge kitchen, and bleeding lesions still visible;Inflammatory cellsdisappear.White matter edema reduced, we can see that gliosi s.The MSCs aftertreatment, edema was reduced, inflammatory cell infiltration reduced,degeneration reduced, neuronal cells were rearranged.On the 15 days, nervecell morphology and structure of restoring order in the first seven days are asignificant improvement.2．MSCs on spinal cord injury in rats protective mechanism DAPIMSCs in vitro labeling, tail vein injection on spinal cord injury in model rats,after laser scanning confocal microscope observations, during the 24 hoursafter injected DAPI marker into MSCs, 5 days after injection, vascular injuryand its surrounding tissues marked MSCs dispersion; into the 10th day afterinjury, the MSCs are fluorescent widespread dispersion of normal rats injected in the first 24 hours, 5 days and 10 days. No marked MSCs DAPI.Results showthat: 24 hours of normal control group shows no expression of Nogoexpression, a cell Sedan. This shows that a large number of model group Nogoexpression, and deep coloring.Nogo seventh day of the normal control groupthere has been no change in color, model group Nogo a large number ofpositive cells, the treatment group MSCs Nogo positive cells less than themodel group.To 15 days, model group Nogo positive cells are less than theseventh day；MSCs treatment group compared with the model group, theNogo-positive cells significantly reduced and the treatment group than theseventh day of MSCs reduction; 24 hours normal control group, a smallamount of Bcl-2 expression, and the cells in Sedan. MSCs surgery and thetreatment group were Bcl-2 expression and cell deeper coloring.Seventh day ofthe model group continued high Bcl -2 expression, the treatment group MSCsBcl-2 positive cells increased, the number of positive cells more than 24 hoursa corresponding dose group. And after 15 days, the group expression of Bcl -2and coloring still demonstrate this phenomenon. 24 hours normal control groupshows no expression of Bax, model group and expression of Bax is visible inthe treatment group MSCs. Bax seventh day of the model group conti nued highexpression of MSCs treatment group Bax reduce t he number of positive cells tothe 15th day after, still show this phenomenon.Conclusion:1．Using the adherent MSCs to successfully separate and purify from bonemarrow,After repeated passages and t o maintain its original shape andcharacteristics of differentiation, Show still has the characteristics of stem cells;Low-density vaccination can be inoculated more is high density of the cellcloning and the number of confirmed results suggested that a strongproliferation；2．Application of self-made model of spinal cord injury against devices - combat-successful model of spinal cord injury in rats；3．Laser scanning confocal microscope also confirmed in vitro DAPIlabeled MSCs injected into the tail vein , the injury to chemokines and settlers,suggesting that it may adopt, migration, settlement, transforming, and theproliferation completion of the process of the spinal cord injury repair；4．This study confirmed that MSCs injected into the tail vein after spinalcord injury in rats can promote the restoration of motor function in the ratspinal cord injury repair；5．MSCs can show that in vivo by inhibiting the Nogo expression,regulation of apoptosis gene expression, and promote the repair of spinal cordinjury. The increased expression of Bcl -2, Nogo, Bax expression downturn,further confirms that MSCs for spinal cord injury repair mechanism；6．The rich MSCs tissue repair both the theory and experimental data,MSCs also provide data for clinical and theore tical basis.Innovation:1. This study confirmed the donor MSCs in the body of movement,homing. Tip MSCs may be through injury in vivo tissue proliferation anddifferentiation to repair the injury；2. This study confirmed the MSCs of theorganization has a spinal cord injury repair, Spinal cord injury in rats and topromote the restoration of motor function；3. his study confirmed the NogoMSCs can be expressed through the downward effect to the treatment of spinalcord injury；4. This study confirmed the MSCs can inhibit the expression ofBax and Bcl-2 on the role of the upward adjustment to the treatment of spinalcord injury.