Effects Of Neurotrophic Factor-3 (NT-3) Modified Schwann Cells On Sciatic Nerve Defects In Rats

The results of the treatment to these patients with peripheral nerve injury are rarely satisfactory . Scientists these days begin to take more attention to the affection of Schwann cells peripheral nerve regeneration . Rothbone MP and his colleagus found that Schwann cells are perfect asreceptor cells and effective techniques of treating peripheral nerve regeneration in gene. Schwann cells can increase neurotrophic factors whice and has protective effects on peripheral nerve injury . Schwann cells can promote neuronal and nerve regeneration after peripheral nerve injury . Neurotrophic factors can facilitate nerve regeneration and protect neuronal survival . Especialy neurotrophic factor 3 NT-3 could facilitate nerve regeneration and increase neuronal survival and the sensory recovery , facilitate nerve-muscle relation and protect denervated skeletai muscle and motor end-plate. But NT-3 is not steady and soluble easily , the clinical effection is much lower . Therefore , the relation of Schwann cells and neurotrophic factors is the base peripheral nerve regeneration . Earlier investigation manifested that Schwann cells have a powerful attraction to the regenerating fibre of peripheral nerve after sciatic nerve, and also can enhance neuronal survival and prompt axonal growth. The project is mainly to explore this mechanisms preliminary . The purpose of this experimental study is to culture adult Schwann cells and modify them genetically by human NT-3 genes to increase the formation and promote neuronal and nerve regeneration .Firstly , Schwann cells were cultured,separated and purified with post-digestion tissue blot and time-speed difference stick . The sciatic nerves of 10 four to three-days-old Wistar rats were harvested and removed . After separating Schwann cells with typsin and collagenase , purified cell with G-418 . The purified Schwann cells were were identified through the phase contrast microscope and S-100 immunocytochemical stain every two days . Flow cytometries were carried out respectively . The prolife rating function of these SCs was assessed by MTT assay .We found that The purity of SCs was 95.6±2.5% by S-100 immunohistochemistry . The flow cytomery were significantly higher than the control group . The results have statistical significance (P<0.01 ). It is said that there are a large of Schwann cells in the peripheral nerve . Schwann cells can were separated and purified in extracellular . Asreceptor cells, SCs can be obtained easily and also can be cultured and reproduced massively in vitro . they can be transduced to secrete augmented levels and express the transgene for a prolonged time .This is a good methods of culturing and purifying Schwann cells . It is a simple , availability , worth spreading way to obtain and purify the schwann cells by the upper .Sencondly , Neurotrophic factor-3 is separated , purified and measured . The NT-3 cDNA gene was transfected into cultured Schwann cells by using cationic liposome . Plasmids expressing NT-3 genes were transfected to SC with lipofectamine,. The Schwann cells with NT-3 cDNA gene was examined by RT-PCR and immunoreactivity methods. The primary culture and purification of Schwann cells wasestablished and the efficiency of transfection of NT-3 was determined by reverse transcriptase polymerase chain reaction(RT PCT) , Gimsa and immunoreactivity methods. The result of RT-PCR was examined. Schwann cells with NT-3 gene were successfully selected by G418. NT-3 could be expressed in transfected Schwann cells for a long time, the ratio of transfection could be to 40%. Compared with nontranduced Schwann cells group, the levels of NT-3 began increasing after two weeks of gene transfer,and were increased obviously after four weeks . The results have statistical significance (P<0.01). Therefore, Schwann cells can be obtained easily and also can be cultured and reproduced massively in vitro ; they can be transduced to secrete augmented levels of NT-3 and express the transgene for a prolonged time . It was shown that NT-3 genes could efficiently promote Schwann cells by transfected into directly SCs in vivo ,Thirtly , in order to investigate the effective treatment of peripheral nerve injury with NT-3 gene modified Schwann cells,to explore the feasibility of the treatment by means of gene transfection . The nerve grafts was constructed by schwann cells modified with NT-3 harvesting from ex vitro , combined with extracellular gel and biodegradable polyCDL-lactide-co-glycolide (PLGA) conduit. The sciatic nerve was bridged by the grafts. The sciatic nerve transected model were made and sutured end to end with epineurium. Seventy-two adult rats were divided into 4 groups randomly,with 24 in each. Group A:extraceellular matrix gel and PLGA conduits, Group B:Schwwann cells,extraceellular matrix gel and PLGA conduits, Group C:NT-3 gene , extraceellular matrix gel and PLGA conduits .Group D: :NT-3 modified Schwwann cells , extraceellular matrix gel and PLGA conduits . Motor nerve conduction velocity(MNCV) and were investigated on 4 week,8week and 12 week potoperatively. Compared with group A and B,C,there was higher rates in group B,C in MNCV, the number and thickness of axon,the diameter of nerve fiber,the percentage of the nerve tissue area, survival rate of motoneuron in the ventral horn in spinal cord and sciatic nerve functional index SFI. But there is no singniificant difference in group B,C. there was higher rates in group D than B,C. Therefore , NT-3 gene modified Schwann cells could be used in the treatment of peripheral nerve injury. It can accelerate the growth of axis-cylinder, defer the atrophy of muscles. Schwann cells gene transfer techniques can promote nerve and axon regeneration, make up the shortage of the lower content in simple schwann cells and neurotrophic factors .Composite our experiment and literatures before, we can conclude that first Schwann cells can were separated and purified in extracellular . Asreceptor cells, SCs can be obtained easily and also can be cultured and reproduced massively in vitro . they can be transduced to secrete augmented levels and express the transgene for a prolonged time .This is a good methods of culturing and purifying Schwann cells . It is a simple , availability , worth spreading way to obtain and purify the schwann cells by the upper . Secondly , Schwann cells can be obtained easily and also can be cultured and reproduced massively in vitro ; they can be transduced to secrete augmented levels of neurotrophic factor -3 and express the transgene for a prolonged time . Thirtly, Schwann cells by neurotrophic factor -3modified genes transfer techniques can promote nerve and axon regeneration, make up the shortage of the lower content in simple schwann cells and neurotrophic factors . So , it is a important study for neurotrophic factors stimulate peripheral nerve regeneration .Schwann cells are source of accelerating peripheral nerve regeneration . Our investigation could provides a reliable animal model to study on gene treatment of peripheral nerve injury . The results provided with elementary theory for the genes treatment of neuotrophic factors .