Properties Of Biodegradable Electrospun Nanofiber-PLGA Membrane Combined With Olfactory Ensheathing Cells And GDNF

Nerve repair is a clinical difficult problem in the recent years.As the progress of science,there is a great progress in the nerve injury repairing.At present,the incidence of prostate cancer increased year by year.The gold standard of treatment is radical prostatectomy.But in the operation of radical prostatectomy,there will be some damage such as clamp、burning、pull to the cavernous nerve.Further more,cavernous nerves have a unique structure looked like a neural network.There is still no ideal way to repair the cavernous nerve injury.According to the special anatomical structure of the cavernous nerve,we adopted a membranous tructure in the cavernous nerve damaged section of full coverage on order to supporting,protecting and guiding the cavernous nerve regeneration.Electrospinning technology is a new technology for the scaffold structure.The nanofiber scaffold created by this technology has a larger specific surface area and higher porosity.This structure does not only conducive to growth factor,but also can adsorption of enzymes and other biological macromolecules.On the other hand,this tructure can help the seeding cell exchange with the microenvironment and help the cell attachment and spreading.Poly lactic acid-glycolic acid polymerization of poly(DL-lactic-co-glycolicacid,PLGA)has good biological compatibility and degradation,the combination of PLGA and nanofibre is an ideal material for the manufacturing cell scaffold.The OECs(Olfactor Ensheathing cells)is original from the olfactory epithelial basement membranes of glial cells.OECs locates in the regional of olfactory nerve and olfactory bulb.OECs can secrete various neurotrophic factors and neural cell adhesion factor and play an important role in the survival of injured neurons.OECs can promote axonal regeneration and myelination and can help the nerve regeneration.GDNF(glial cell-derived neurotrophic factor)is the most effective neurotrophic factor for motoneuron in vitro.GDNF can protect injured nerve,decrease the death of neurons,promote functional reconstruction of the nerve fiber regeneration and help the movement effect.In the first part,we use the modified Nash method to cultivate high purity OECs,and use immunofluorescence identification of its purity.In the second part,we used lentivirus transfected olfactory ensheathing cells,which have high expression of GDNF in olfactory ensheathing cells,and GFP fluorescence expression,Western blot and PCR assay confirmed it.In the third part.We part the high expression GDNF of OECs in the PLGA electrospun membrane.We use both in vivo and in vitro experiments to prove its biocompatibility.Finally we got the electrospinning nanofibre PLGA membrane combined with olfactory ensheathing cells and GDNF,which is an ideal biological engineering nerve graft.Part one:Separation,cultivation and identification of theratolfactory bulb original OECsObjective:Separate,cultivate and purify the ratolfactory bulb cord original OECs.Identify the OECs by morphological observation and immunofluorescence technology.Methods:By using the modified Nash method to separate OECs from the 120 g rat’s olfactory bulbs.Using a whole culture medium which contain bFGF,Forskolin,20%FBS and DMEM/F12 to culture of OECs.Observating the cell proliferation and morphological by the microscope.Elevate the purity of OECs by p75 NGFR immunofluorescence.Results:By using the modified Nash method,we successfully obtained high activity,good condition and strong proliferation ability of olfactory ensheathing cells in vitro.The longest incubation time can be up to 30 days,but the days of activity of cells well within 14 days.P75 NGFR immunofluorescence showed the purity of olfactory ensheathing cells is more than 95%.Conclusion:We can obtain high purity OECs by the modified Nash method.Olfactory ensheathing cells are mature somatic cells,characteristic does not have unlimited proliferation in vitro,although can proliferate,but the survival time is limited.It is necessary to carry out related experiments in 30 days,more than 30 days after the cell activity and cell number death is the best days.The best relevant experiments were carried out within 14 days.Part two:Lentiviral transfection to obtain the high expressionGDNF OECsObjective: Obtain the high expression GDNF OECs.Identify the OECs’ s ability to high expression GDNF.Combination GDNF and OECs to neural repair.Methods:GDNF gene was transfected into OECs with slow virus.The virus transfection efficiency was determined by the fluorescence expression of GFP under fluorescence.The expression of GDNF gene was verified by PCR and the GDNF protein was confirmed by Western blot.Results:Fluorescence observed GFP expression in mol of 50:1 for 50%,showed that the virus transfection rate of 50%.At the same time PCR was performed to validate the GDNF gene expression was 130 times of control group of normal cells.Western blotting analysis showed that high expression of GDNF OECs can produce GDNF protein,and Western blotting of normal OECs negative results.Conclusion:High expression GDNF OECs can be obtained form slow virus which cantained GDNF gene transfection.The cell state is good and have strong proliferation activity.The cells are good enough to repair the injuried nerve.Part three:Biocompatibility of electrospun nanofiber-PLGAmembranewith olfactory ensheathing cellsObjective:To explore the biocompatibility of olfactory ensheathing cells(OECs)in rat with electrospun nanofiber-PLGA(DL-lactic-co-glycolicacid)membrane.Methods : The olfactory ensheathing cells were seed on the electrospun nanofiber-PLGA membrane.Evaluate the growth and adhesion to the membrane of OECs by immunofluorescent and phase contrast microscopy.Electrospun nanofiber-PLGA membrane were implanted in rats to observe the biocompatibility.Results:The morphological feature and adhesion of OECs is good on the electrospun nanofiber-PLGA membrane.Cell count analysis showed that the cell proliferation was normal.The in vivo transplantion experiments confirmed that the electrospun nanofiber-PLGA membrane was fused with the surrounding tissue and partially degraded.Conclusion:The electrospun nanofiber-PLGA membrane show great biocompatibility with OECs.It is a suitable tissue engineering nerve graft combined with OECs.