Study On The Neural Protective Effect Of Ciliary Neurotrophic Factor (CNTF) After Optic Nerve Injury

Part 1.To research the neural protective effect of rhCNTF after cat opticnerve incomplete injuryObjective To research the neural protective effect of rhCNTF after cat optic nerveincomplete injury.Methods Completes the cat optic nerve incomplete injury model;The experimentalgroups give rhCNTF by the vein,the anterior chambe,the intravitreal to inject separately,the blank group gives the physiological saline;15 days after of operation,two-sidedanterior colliculi,lateral geniculate body microinjection DiI retrogradation nerve tracing;The different time spot completes the examination of visual evoked potential(P-VEP);30days after of operation,complete the situ heart dabbling;Retina stretched preparation,retina ganglion cell counting;Takes the optic nerve of operation side,GAP-43 weredetected with immunohistochemistry,the light microscope and the electron microscopeobservation morphologic change of optic nerve.Results The blank group P-VEP compares the experimental groups oscillationamplitude of P-100 to decrease obviously,incubation period of P-100 obvious extension,difference remarkable(P＜0.01);Compares with the blank group,the survival number ofretina ganglion cell of experimental groups increases obviously(P＜0.01),the nervestructure is more complete. Conclusion After optic nerve incomplete injury,the rhCNTF different way injectionhas the optic nerve protective effect,but effect existence difference.Part 2.The neural protective effects of rhCNTF in combination with sciaticnerve transplantation after cat optic nerve incomplete injuryObjective To research the neural protective effects of rhCNTF in combination withsciatic nerve transplantation after cat optic nerve incomplete injury.Methods Completes the cat optic nerve incomplete injury model and sciatic nervetransplantation;The CNTF group gives rhCNTF by the intravitreal to inject separately,theblank group gives the physiological saline;15 days after of operation,two-sided anteriorcolliculi,lateral geniculate body microinjection DiI retrogradation nerve tracing and sciaticnerve microinjection Fluoro-Gold retrogradation nerve tracing;The different time spotcompletes the examination of visual evoked potential(P-VEP);30 days after of operation,complete the situ heart dabbling;Retina stretched preparation,retina ganglion cell counting;Takes the optic nerve of operation side,GAP-43 were detected with immunohistochemistry,the light microscope and the electron microscope observation morphologic change of opticnerve.Results The blank group P-VEP compares the CNTF groups oscillation amplitude ofP-100 to decrease obviously,incubation period of P-100 obvious extension,differenceremarkable(P＜0.01);Compares with the blank group,the survival number of retinaganglion cell of CNTF groups increases obviously(P＜0.01),the nerve structure is morecomplete.Conclusion After optic nerve incomplete injury,the rhCNTF in combination withsciatic nerve transplantation has the coordination optic neural protective effect. Part 3.Establishment of superparamagnetic iron oxide(SPIO)and enhancedgreen fluorescence protein(EGFP) double labeled CNTF gene modifiedneural stem cellsObjectives 1.To culture and identify El4 rattus brain neural stem cells.2.Toconstruct the eukaryotic expression vector pEGFPN1-CNTF and investigate its expressionin eukaryotic cells.3.To establish SPIO and EGFP double labeled CNTF gene modifiedneural stem cells.Methods 1.The brain tissue was dissected from embryonic day 14 rat under asepticconditions.Using a fire-polished glass pipet,the brain tissue was triturated into a finesingle-cell suspension.The cells were cultured with the serum-free medium containingDMEM/F12(1:1),EGF,bFGF and N2 supplement.After primary neurospheres formed,cells were subcultured.The third-generation neurospheres were collected andimmunocytochemically identified for Nestin.At the same time,the cells were inducted todifferentiation,and the phenol types of differentiated cells were immunocytochemistricallyidentified for GFAP,CNPase andβ-Ⅲ-tubulin.2.The coding sequence(CDS) of CNTFwas emplified by RT-PCR from human astrocytoma cell line U251.By gene recombinationtechnique,CNTF CDS was inserted into eukaryotic expression vector pEGFPN1 toconstruct recombinant plasmid pEGFPN1-CNTF.The recombinant plasmid was identifiedwith restriction enzyme digestion and DNA sequencing.COS-7 cells were transfected withthe recombinant plasmid by Fugene HD transfection regent.The expression of CNTF wasanalyzed by immunocytochemistery,RT-PCR as well as western blot.3.The NSCs of thethird passage were transfected with plasmid pEGFPN-CNTF using the nucleofectiontechnique,48 hours after transfection,the transfected cells were screened with mediumcontaining G418,the positive clones were selected,allowed to proliferate and then labeledwith SPIO,which was mediated by FuGENE HD transfection reagent.The expression of CNTF was analyzed by immunocytochemistery,RT-PCR as well as westem blot.Prussianblue stain and transmission electron microscopy was used to identify the SPIO particles incells.To identify the differentiation ability of SPIO labled CNTF gene modified NSCs,immunocytochemistry for GFAP,CNPase,β-Ⅲ-tubulin and CNTF respectively wereperformed after in vitro differentiation.Results 1.72 hours after culturing,the cells derived from El4 rat embryonic formneurospheres,which can be subcultured after culturing 6～7 days.A great many ofneurospheres can obtained by successive passage.The immunocytochemistery showed thecells were Nestin positive,after differentiation the cells expressed GFAP,CNPase andβ-Ⅲ-tubulin.2.The RT-PCR product of CNTF coding sequence was 622bp specificsegment.By restriction enzyme digestion,the recombinant plasmid vector was digestedinto 618bp and 4700bp fragments.The DNA sequence of the 618bp fragment was identicalwith human CNTF CDS in GenBank.The results of immunocytochemistery,RT-PCR andwestern blot showed the CNTF was expressed successfully in COS-7 cells.3.12 hoursafter transfection,the immunocytochemistry,RT-PCR and western blot showed that CNTFwas expressed in NSCs.Prussian blue staining indicated that numerous blue-stainedparticles appeared in the cytoplasma of the labeled cells.TEM showed that SPIO particleswere found in vacuolar structures of different sizes and the cytoplasma.Theimmunocytochemistry demonstrated that the labeled cells were nestin-positive.Afterdifferentiation,the cells expressed GFAP,CNPase andβ-Ⅲ-tubulin.Conclusion The CNTF gene eukaryotic expression plasmid pEGFPN1-CNTF isconstructed and the SPIO and EGFP double labeled CNTF gene modified NSCs areestablished successfully. Part 4.The neural protective effects of transplantation of CNTF genemodified neural stem cells and sciatic nerve after cat optic nerveincomplete injuryObjective To research the neural protective effects of transplantation of CNTF genemodified neural stem cells and sciatic nerve after cat optic nerve incomplete injury.Methods Completes the cat optic nerve incomplete injury model and sciatic nervetransplantation;The cotransplantation group gives CNTF gene modified neural stem cellsby the intravitreal to inject separately,the neural transplantation group gives rhCNTF bythe intravitreal to inject separately;165 days after of operation,two-sided anterior colliculi,lateral geniculate body microinjection DiI retrogradation nerve tracing and sciatic nervemicroinjection Fluoro-Gold retrogradation nerve tracing;The different time spot completesthe examination of visual evoked potential(P-VEP);180 days after of operation,completethe situ heart dabbling;Retina stretched preparation,retina ganglion cell counting;Takesthe optic nerve of operation side,GAP-43 were detected with immunohistochemistry,thelight microscope and the electron microscope observation morphologic change of optic nerve.Results The neural transplantation group P-VEP compares the cotransplantation grouposcillation amplitude of P-100 to decrease obviously,incubation period of P-100 obviousextension,difference remarkable(P＜0.01);Compares with the neural transplantation group,the survival number of retina ganglion cell of cotransplantation group increasesobviously(P＜0.01),the nerve structure is more complete.Conclusion After optic nerve incomplete injury,the transplantation of CNTF genemodified neural stem cells and sciatic nerve has the coordination optic neural protectiveeffect.