| Objectine: (1) Researching external high-purity"Wistar"cultural method of suckling rat cells. (2) Inserting BDNF schwann cell into presetting Chitosan tubes, constructing tissue engineering artificial nervous bridge connect rat 10mm SCs sciatic nerve damnify, researching the repairing effect of artificial nervous bridge and these interaction between nerve regeneration factor, providing experiment evidence for tissue engineering further studies and clinical application.Method: (1) Take 3-5 days suckling rat two sides sciatic nerve and brachial plexus, after digesting by enzyme, explants planting insolating cultivate SCs. After a week's cultivation, full of bottom, adding 0.25% trypsase general digested to each bottles to generation, using S-100 immunohistochemistry dyeing and calculating SCs purity. (2)Get such schwann cell and make them into suspension, density is 1×105/ml a 24 hole costar, divided into normal group and experimental group, Chitosan tiles on the bottom of holes, ultra-clean platform drying. SCs vaccinates into costar, cultivating is in the condition of 37 oC and 5% CO2 After 24h,adding DAPI (final concentration 50μg/ml) into incubation , After 24h,washing bottle using PBS at least 3 times, to be rid of DAPI which is not combined with cells. Adding inoculums to continue cultivate. Observing SCs and Chitosan histocompatibility. (3)Using lentivirus taking BDNF gene infected schwann cell, and infuse its into Chitosan tubes, construct Nerve tissue engineering bridge, connected Wistar rat sciatic nerve (A-group),researching the renovation effection,observe Chitosan tubes (B-group), sham operation (C-group),autogenous nerve graft(D group) as the comparison. After 6,12 weeks, taking the experiment by electrical physiological test,histology section, microscope and estimate its therapeutic efficacy.Result: (1) After enzyme digested, using explants organisms cultivate method, the creeping speed of SCs is faster than direct explants organisms cultivate method, take furthering purification and then identified SCs by S-100 immunohistochemistry, and the purity reached more than 95%. (2)dealed normal group and experimental group, and percentage of apoptosis cells each time point were calculated, the percentage of apoptosis cells at each time points were no significant(P<.0.05). (3)General observation: no rat death in the process of feeding, the condition of rats which suffer from contracture and drag-to gait would improve in four weeks. There were no Chitosan tubes remaining and no neuratrophy in the explantation nerve place, Vascular proliferation was discovered transpl surrounding. (4)Electrophysiology observation: comparisons of three group NCV, group A and B, B and C had significant differences (P<.0.05), group A and group D had no significant differences.Conclusion: (1) Digesting enzyme, the purity method which composed by backer explants organisms cultivate method and differential adherence method "produce" large number of SCs, high purity and low attached by outer atmosphere of cell physicochemical property and biological characteristics. At the same time, saving nerve organization material sources, avoiding repeated draw material processes, saving manpower and material resources sparingly and guaranteeing SCs derive from the same animal which was used in experiment.(2) The Chitosan tubes made by Freeze-drying technology had smooth surface, transparency, even texture. 0.15mm thickness, the same as Cover slip. Experiments show that both have good biocompatibility and SCs proliferation in the surface. Could serve as a nerve bridge over. (3) Using Chitosan tubes to act as supporting frame, Using lentivirus taking BDNF gene infected SCs as seeds cells, Construct tissue engineered artificial nerves, repairing nerve defection, the effect is closed to autogenous nerve graft, relative to single application Chitosan tubes, it could further raise nerve repairing effects. This study had broad application prospects and value of further investigation.
|
PDF Full Text Request