| Objective:(1).To set up the method of high-purity Wistar suckling rat OlfactoryEnsheathing Cells (OECs) cultural.(2).To research the biocompatibility of chitosan tubesand OECs.(3).To research the repairing effect of artificial nervous which was constructedfrom tissue engineering artificial nervous repaired rat10mm brachial plexus injury andthese interaction between nerve regeneration factor, providing experiment evidence fortissue engineering further studies and clinical application. Methods:(1).Differentialadherence method and chemical inhibitors method were used to isolate and culture OECs:Bulbus cinereus which come form3-5days old Wistar suckling Rats were purified by2times differential adherence method and cytarabine treatment. NGFRp75immunohistochemistry dyeing was used to calculate OECs purity.(2)To research thebiocompatibility of chitosan tubes and suckling rats OECs. There were two groups in thisstudy: The culture plates were treated with chitosan or not and the serial subcultivationolfactory ensheathing cells were cultured onto them. DAPI method was used todeterminate survival and proliferation of OECs.(3).Nerve-repair experiment wasperformed by graft of tissue engineered artificial nerves. Animal models were randomlydivided into4groups: A: chitosan conduct+NT4-OECs group, B: Auto-nervetransplatation group, C: chitosan conduct group, D: negative control group.The survivalof NSCs and function recovery of PN was observed by means of EMG, HE stain, andIMF stain at the time of6,12weeks after operation. Results:(1).NGFRp75immunohistochemical stain identification showed that OECs were isolated and culturedsuccessfully, its purity reached95%.(2).The chitosan tubes have good biocompatibilitywith OEC. There was no and percentage of apoptosis cells each time point werecalculated, the percentage of apoptosis cells at each time points were nosignificant(P>0.05).(3).General observation: ⅰ. No rat death took place in the process offeeding; ⅱ. Different degree plantar ulcer\contracture and drag-to gait were observed inall three groups(recuperation:Group A,B>C>D)after reparation of long segment ofbrachial plexus nerve by artificial nerve. Those would improve after six weeks. ⅲ.The chitosan tube was absorbed when rats were sacrificed after12weeks.(4).Electrophysiology observation: comparisons of NCV, group A and C, A and D hadsignificant differences,but there weren’t significant differences in group A and group B.(5).Histological observation：the recovery of nerve in Histological observation of12weeks was significantly better than6weeks. The nerve fiber were tidiness andenlargement with the surface covered with thicker medullary sheath in GroupA andB,but raritas with little collagen tissue and new blood vessels in Group C, while muchwave-shaped fiber tissue can be observed in Group C. The nerve fiber in Group D weretangle with lots of collagen tissue. Fluorescence microscope illustrated that GFPfluorescence was A group expressed after12weeks,which showed that the NSCs werestill alive after transplantation. Conclusions:1.Chemical inhibitors and differentialadherence methods can produce large number of OECs, high purity and low attached byouter atmosphere of cell physicochemical property and biological characteristics.(2).TheChitosan tubes both have good biocompatibility and OECs proliferation in the surface. Itcan serve as a nerve bridge over.(3).Using Chitosan tubes as supporting frame and usingOECs which transfected and expressed NT-4gene as seeds cells, to construct tissueengineered artificial nerves has a good effect in repairing nerve defection. The effect of itis closed to autogenous nerve graft. So it could further raise nerve repairing effects. Thisstudy had broad application prospects and value of further investigation. |
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