The Study On Repair Of Optic Nerve By Olfactory Ensheathing Cells Genetically Modified Secret NgR Antagonist Peptides

Part I:The study of separation,cultivation and purification of olfactory ensheathing cells in neonatal ratsObjective To explore the methods of separation, cultivation and purification of olfactory ensheathing cells(OECs) in neonatal rats, to construct the foundation of gene-modified olfactory ensheathing cells repairing the optic nerve injury. Methods The olfactory bulb organization of neonatal rat within 2d were separated under microscope. The olfactory ensheathing cells were clutured by primary culture, purified by two methods inclouding selective plating technique and selective plating technique +cytarabine + cell growth stimulates factor.The quantity and the form of different training time olfactory ensheathing cells were observed with inverted microscope, and NGFRp75 lmmunofluorescent histochemical were observed with fluorescence microscope. Olfactory ensheathing cells purity of two methods were analysed statistically. Results The purity of olfactory ensheathing cells were 60.41%±4.32% in group of selective plating technique, and 84.98%±4.03% in group of selective plating technique +cytarabine + cell growth stimulates factor average. Two sets of data were analysed by t-test, showing significant difference (P<0.05). Conclusion Drawing materials from the neonatal rats, separating meningeal with microscope, purifying with selective plating technique + cytarabine + cell growth stimulates factor were good olfactory ensheathing cells culture method. PartⅡ:Construction and Identification of Recombinant Lentivirus Carrying Rat NEP1-40 GeneObjective To constructe a recombinant lentivirus which carried SD rat VEP1-40 gene and test its ability to express the NEP1-40 gene in vitro. Methods The code sequence of TAT/NEP1-40 gene was amplified using PCR and cloned into pLV. Des2d p./puro vector,293FT cells were transfected using Lipofectamine 2000 and packaged for the recombinant lentivirus particles. When the cloned sequence was identified to be right, the recombinant lentivirus particles were amplified in a large quantity. The titter of virus was determined by gene transfer units (GTU) method. The expression of NEP1-40 gene was tested at the level of transcription and protein using PCR and western blot respectively. Results The recombinant lentivirus carrying SD rat NEP1-40 gene was constructed successfully and NEP1-40 gene could be expressed in 293FT in vitro. Conclusion The recombinant lentivirus carryingd NEP1-40 gene could express its purpose gene in vitro, which played a important role in the study of the recovery of injury optic nerve.PartⅢ:The expression of the NEP1-40 gene carried recombinant lentivirus in OECs in vitroObjective To study the expression of the rat NEP1-40 gene carried recombinant lentivirus in olfactory ensheathing cells(OECs) in vitro, and preliminary observe the effection of olfactory ensheathing cells carrying NEP1-40 gene to the retina ganglion cells (RGCs). Methods The primary olfactory ensheathing cells were transfected by lentivirus vector carrying NEP1-40 gene in vitro. The expression of NEP1-40 gene was tested at the level of transcription and protein using PCR and ELISA respectively. The growth of axons in retinal ganglion cells was observed while retinal ganglion cells were cocultured with transgenic olfactory ensheathing cells. Results The expression of NEP1-40 gene was confirmed at the level of transcription and protein using PCR and ELISA repcectively after olfactory ensheathing cells were transfected by NEP1-40 gene. The good condition of retinal ganglion cells was observed when retinal ganglion cells were cocultured with transgenic olfactory ensheathing cells.Conclusions The lentivirus carrying NEP1-40 gene could transfect primary olfactory ensheathing cells in vitro, express its purpose gene,and promote the growth of retinal ganglion cells, which laid a foundation for later further study.