| BackgroundPeripheral nerve damage is also called PNI, it is a very common clinical symptoms ofinjury. In recent years, with the continuous development of modern construction industry,transportation, the pathogenesis of peripheral nerve injury population increase gradually. Theclinical medical study found that each year more than50000cases of peripheral nerve injury;Every year in our country, an increase of600000to900000cases of peripheral nerve injury[1].In addition, compared with those of other organizations neurons in the mature stage cannot bedivided, copy, once appear, damage, if not timely treatment is likely to cause damage toexpand even lose muscle function, sensory function, which can lead to permanent disabilityparalysis[2]. This to people’s physical and mental health cause great harm, serious influencepeople’s normal life. At present, treatment of peripheral nerve injury theory based on thetheory of mainly renewable peripheral nerve, nerve fiber regeneration environment, providethe corresponding element of nerve growth factor and cell, make damaged nerve growth again,but there is no human receptor, effector neural fast connection. To solve this problem, thispaper presents a "bridge repair of peripheral nerve injury of neurons" treatment, is to useconnect neurons, the nerve is connected to the human body sensors, effector quickly, alleviatethe body disease symptoms, so as to achieve good repair treatment effect.Purpose1, extracted from rabbit bone marrow and culture preservation, the rabbit bone marrowderived neural stem cells targeted cells (NTCSC for short) as matrix, using theimmunofluorescence cytochemistry assay method, whether it belongs to the neural stem cells.2, to identify NTCSC separately, on the basis of train the third generation of stem cells,and transfection the GFP slowly with the virus, finally obtain NTCSC GFP to carry the virus.3, prompt with GFP viruses NTCSC differentiation to neurons, cultivate ability oftransplanted neural stem cell-derived cells.4, setting off cellular neural support, establish the pure nerve regeneration.5, through the nerve scaffold cultivating neural stem cell-derived cells, formingconditions in transplantation of the neurons in vitro. 6, in vitro tissue culture conditions in transplantation of engineered neurons, neuralearnest observation on changes of cells and tissue engineered neurons electrophysiologicalproperties.7, study engineering neural tissue transplantation, observe the damaged peripheralnerves of GFP transfection bone marrow neurons survival situation, analysis of tissueengineered neurons to the repair effect of peripheral nerve injury.Methods1, this study of bone marrow samples taken from three months big rabbit, extractedfrom rabbit tibia platform2to3ml of bone marrow, the rabbit bone marrow derived neuralstem cells orientation (NTCSC) as maternal cells.2, nerve tissue of bone marrow stem cell solution separation, until the cell fusion degreereached80%, then subculture, won the third generation NTCSC. Then add10mu L in newstem cell solution concentration of1.5x109pfu/L GFP slow virus suspension, the GFP virustransfection into the third generation NTCSC, thereby gaining NTCSC GFP to carry the virus.3, the GFP virus NTCSC medium to join2000nmol/L RA inducers and500ng/mLSHH inducers, prompting maternal cells into neurons.4, choose a medical silicone tube10cm in length, put it in80degrees below zero in thefridge frozen, and then remove dry forming silica gel controls into collagen-chitosanscaffold. Then cut0.5cm from the rabbit sciatic nerve, make nerve tissue natural retraction,nerve defect in1.5cm, with clean silicone tube then sew accept loss on both ends of the nerve,experimental animal models.5, make20rabbit sciatic nerve defect experimental model of1.5cm,10autologousnerve transplantation model. The average30rabbits randomly divided into three groups,experiment research.Experimental group1(USES empty stent grafts): cut0.5cm from the rabbit sciaticnerve, make nerve tissue natural retraction, nerve defect in1.5cm, the cell scaffold andstitched on both ends of the sciatic nerve defect, along the longitudinal axis in a micro syringe,to1.5mm for interval, select11point injection, the160mu L cells scattered suspensioninjection to take off the nerve scaffolds.Experiment2groups (the cell scaffold): the concentration of107cells/ml NTCSCnerve cell suspension, extraction of trace injector160mu L cell suspension, method is thesame as the above. Experiment3groups (using the neural transplantation):1.5cm interception from rabbitsciatic nerve, the nerve sewn back after two exchange.In vitro tissue culture conditions in transplantation of engineered neurons, neuralearnest observation on changes of cells and tissue engineered neurons electrophysiologicalproperties.6, by studying tissue engineered neurons in vivo transplantation, in animal behaviortests4weeks after cell transplantation, muscle strength tests after eight weeks,12weeks afterthe analysis of emg. For the damage to the GFP virus bone marrow samples of neurons in theperipheral nerve cell survival situation observation analysis, explore the "bridge repair ofperipheral nerve injury of neurons" treatment feasibility, evaluation analysis engineeringorganization for the treatment of peripheral nerve repair damaged neurons.Results1, by observing the cell transplantation in cell growth, differentiation of neural supportfound that neurons NTCSC can survive on scaffolds in nerve, and piece uniform distribution,the growth momentum. Sufficient cell SAP, cell membrane integrity is smooth, can seeclearly the nucleus contour. Suggesting that NTCSC cell-free nerve scaffolds by achievingtransplantation, maternal cells normally live in the transplant environment.: measured by theimmunohistochemical Nestin positive cells use smoothly to take off the bracket of cellularneural transplantation, and keep the neural precursor cells in a period of time stable structure,realization of self-renewal. In addition, through the transplanted cells found in the bracket ofelectron microscope, the transplanted cells have certain function structure. Transplanted cellselectrophysiological testing results showed that neuron cell transplant after8weeks after invitro culture began to show electrophysiological properties, overall, to take off the cellularneural scaffolding of the transplanted cells can be normal growth, differentiation,transplantation effect is good.2cell growth situation, through the observation in vivo transplantation found:experimental rabbit can walk normally, muscle strength, good tissue engineered neurons canrapid transmission links, the experimental rabbit muscle contains a lot of protein, after HRPretrograde tracer detection and transmission electron microscopy, immunofluorescencehistochemical detection, we found that the implanted in rabbit sciatic nerve damaged NTCSCneuron survival in good condition. ConclusionExperimental results show that, through the bridge of neurons sample cells in the bonemarrow, with GFP viruses can effectively repair in the treatment of sciatic nerve injury,neurons into the peripheral nerve after dry, can rapid transmission link neural, receptor andeffector, bridge neurons can restore nerve function, make nerve tissue better control sensorsand effector, achieve good repair effect, avoid muscle atrophy degeneration, muscle function,reduce the disability. |
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