Study On Treatment Of Spinal Cord Injury Using Of BMSCs Modified With NT-3 In Rat

Spinal cord injury (SCI) is very frequent in our society, the number of cases increases every year, and this pathologic condition often causes permanent and irreversible functional deficits in the patients. The devastating consequences of this affection have motivated numerous scientists throughout the world to search in experimental animal models for a repair strategy that could, in the future, be applied to humans.However, due to the limited neuronal survival, the lack of spontaneous axonal regeneration and the inhibitory glial environment, self-repair after damage of the mammalian central nervous system (CNS) is rather poor. Henceforth, the repair of brain and spinal cord injury thus far is still a major therapeutic challenge. To be gratified, in recent years,substantial evidence has suggested that the neurons and the axons proximal to the site of injury were still alive after the impairment. Therefore, external interventions such as cell transplantation and neurotrophic factors delivery overcoming the inhibitory environment at the injury site could stimulate some of the remaining intrinsic plasticity of neurons and untend the healing of the otherwise nonregenerative CNS.At present, litle of these strategies alone have been sufficient in repairment of adult CNS, two or more strategies used in combination might bring about adequate structural and functional recovery. During the past two decades, the dramatic progress in therapeutic alliance and in vivo and ex vivo gene therapy has heightened optimism about future cures for injury of CNS.Bone marrow mesenchymal stem cells(BMSCs)represent a subpopulation of non-hematopoietic pluripotent cells within the bone marrow microenvironment due to their ability to differentiate into mesenchymal lineages and neuronal lineages. This neurogenic potential of BMSCs was revealed under special culture conditions in vitro or following intracranial implantation. Intracranially implanted BMSCs can migrate away from the initial injection site towards injury site. Taken together, these findings imply that NSCs could be replaceable with BMSCs as a therapeutic vehicle for gene therapy against CNS injury . Moreover, BMSCs also have the advantage of ease of propagation in vitro and implantation of autologous BMSCs into patients with malignant glioma is ethically unproblematic. The properties of BMSCs make this cell population a very attractive candidate for the cell-based delivery of therapeutics to tumor sites. activities in vitro and in vivo.Neurotrophin-3 (NT 3), a member of neurotrophin super-family, are well known for their beneficial efects on survival of several neuronal cells, neuroprotection and neurite outgrowth.Local delivery of NT-3 can counteract pathological events and induce a regenerative response after SCI. Supplement of exogenetic NT-3 might be a new approach to SCI repair.In present experiment, we examined the effect of rat NT-3 gene modified BMSCs transplantation on SCI.Part I Isolation and Identification of BMSCsObjective: To isolate MSCs from rat bone marrow,then culture and identify the obtained cells by detecting it's biological features and ultrastructure, found a base for using of MSCs in tissue engineering.Method:BMSCs were separated from bone marrow by density gradient centrifugation and selected by plastic adhesion according to references. At near-confluence, cells were subcultured after trypsin digestion. To characterize these cells, immunocytochemistry was performed to identify specific antigens,such as CD44, CD29. We also induced BMSCs differentiation by incubation with different culture media and strain with Gomori and oil red O.Results :The appearance of these cells are similar to fibroblast.The positive BMSCs were CD29 (52%), CD44 (78%). ALP positive cells of osteogenic differentiation of BMSCs were detected by Gomori staining, adipogenic differentiation of BMSCs were detected by oil red O staining.Conclusion:These BMSCs have the capacity to differentiate into osteoblast ,adipocyte. They were stem cells but not other mature cells.Part II:A Study on theTransfection NT-3 in the BMSCs and Induced to Differentiate into Neural Cells in VitroObject: To construct and identify the BMSCs modified with NT-3 and induced to differentiate into neurons and neuroglial cells in vitro. Method: To preparate the E coli competent cells and transform pLEGFP- NT-3 and pLEGFP-C1 plasmid into E coli .Then ,the transformed E coli were inoculate in LB medium with Ampicillin for clonal expansion of the plasmid small-scally with kit.After identified with enzyme-cutting and PCR,the recombinant plasmid was abstracted large-scally.Then we transfected the pLEGFP-NT-3 and pLEGFP-C1 into BMSCs and to detect the expression of the NT-3 with immunocytochemical stain and ELISA.After preinduced for 24h with IMDM+10%FCS+1mM BME, the BMSCs/pLEGFP-NT-3 and BMSCs were to continue to induced for 7d with DMEM/F12(bFGF 20ng/ml,EGF 20ng/ml,B27 20ng/ml)To observe the morphologic change of the induced cells and identify with fluorescent immunocytochemical stain.Result:. The results showed that the fragments of NT-3 had the same size with the expected. The BMSCs were transduced with an appropriate amount NT-3 using lipofectamine method. The expression of the Rv NT-3 in the cultured MSCs was detected by anti-NT-3 immunocytochemistry and ELISA. The results of anti-NT-3 immunocytochemistry showed that a widespread, diffuse distribution of the NT-3 throughout the cell body and the processes was seen with fluorescent microscopy. A 4.3ng/106cell molecular weight ,the expected size of NT-3 could be observed by ELISA probed with antibody against NT-3. After induced, more than 20% of BMSCs exhibited neuron-like characteristics morphologically and were immunopositive for MAP,Nestin and GFAP.Conclusion:The recombinant vector,Rv-NT-3, could express NT-3 in the cultured BMSCs.The BMSCs/pLEGFP-NT-3 can be differentiated into neuron-like cells.Part III: Transplantation of BMSCs/pLEGFP-NT-3 results in improved function recovery in the spinal cords of adult rats after SCIObjective: To observe the efects of marrow stromal cells(BMSCs) modified by neurotrophic-3 (NT-3) gene on function recovery in rats with spinal cord injury.Methods: The spinal cord of adult Wistar rats was injured with the modified Allen' s method(60gcm) at T10 vertebra level, fifty four Wistar rats were randomly divided into four groups:pseudopration group(B),NaCl group(C) BMSCs/pLEGFP-C1group(D)and BMSCs/pLEGFP-NT-3 group(E). One week after SCI, the injured spinal cords in C,D and E group were injected with normal saline (NS group),BMSCs/pLEGFP-C1-suspension (BMSCs/ pLEGFP-C1 group) and BMSCs/pLEGFP-NT-3-suspension(BMSCs-NT-3 group). The function recovery of spinal cord was determined using behaviorally tested Basso, Beatie, Bresnahan(BBB) locomotor rating scale test motor function scores, spinal cord sense evoked potential and motor evoked potential and HRP tracing method.BBB scores were obtained in 3,6,9,12,15,18,21,24,27 days after injection.At 4w after cellular transplant ,the injury spinal cords were deal with immunofluorescence cytochemical staining,SEP,MEP and HRP trace for the recovery of neurons.Results: At 1w,2w and 3w after the transplantation, rats of C,D and E groups had no sign of recovery. Assessed by motor function scores, at 4w,function recovery was found in every group, there were significant diference among all groups(E superior to C and D). Amplitude of SEP and MEP was reduced and latency time was prolonged after SCI, but recovery was found in group C,D and E at 4 weeks after injection. There were significant diference among all groups,Group E was superior to group C and D (P<0.01). Amplitude of SEP and MEP was reduced and latency time was prolonged after SCI, but recovery was found in group C,D and E at 4 weeks after cells transplantation injection. There was significant diference among all groups,Group E was superior to group C and D (P<0.01).The same accident happened in HRP.Conclusion: BMSCs modified byNT-3gene and BMSCs transplantion could improve function recovery of injured spinal cord in rats, and BMSCs/pLEGFP-NT-3 is more effective than the efect of BMSCs.