The Study Of Neural Stem Cells From Fetal Rat Infected By Recombinant Adenovirus With Green Fluorescent Protein And Transplanted Into Rat Cochlea

PartⅠCulture,identification and label of embryonic rat neural stem cellsObjective To explore the methods of culture,identification and label of embryonic rat neural stem cells.Methods The cells isolated from fetal rat hippocampus were identified with nestin immunocytochemical fluorescent staining. The cellular multiplication was observed by immunocytochemical fluorescence co-label after accession of Brdu. The neural stem cells (NSCs) were marked with fluorescent dye, bisbenzimide (Hoechest33342) and induced to differentiate.The differentiated cells were detected respectively with Neuron Specific Enolase(NSE) and Glial Fibrillary Acidic Protein( GFAP)immunocytochemical fluorescent staining.Results Nest-like clusters of neural stem cells were obtained in suspension and the cells could been differentiated into neurons and astrocytes which retaining the main characteristics of NSCs after 10 passages of culture. The label efficiency of cells with Hoechest33342 was 97% and no attenuation of fluorescent brightness was observed after 10 passages of culture. The cellular fluorescence was observed in the NSCs and the differentiated cells.Conclusion The cells from embryonic rat hippocampus possessed ability of division,multiplication and self-renew, which were believed to be NSCs of the central nervous system. The cells could be efficiently labeled with fluorescent dye, they could be used as donor cells in experimental research with transplantion of cells. PartⅡCryopreservation and resuscitation of neural stem cells from fetal rat Objective To explore the survival rate of neural stem cells(NSCs) from fetal rat and its biological properties after cryopreservation and thawing.Methods Different passages NSCs (the first passage,the third passage and the sixth passage)from fetal rat cultivated with serum-free medium in vitro were cryopreserved in the cryogen,which were neurosphere culture medium with 10%BSA and 7.5%DMSO(without neural growth factor).The cryopreserved cells were resuscitated at 1 week, 4 week, 8 week,12 week and 16 week respectively. The survival rate of cells were calculated and the cells were incubated and differentiated again.Result Different time of cryopreservation,different passages did not affect NSCs survival (P>0.05)after cryopreservation .The survival rate of NSCs was from 60% to 70% after resuscitated, which were differentiated into neurons and astrocytes in 10% embryonic bovine serum(without growth factor).Conclusion NSCs were successfully cryopreserved, resuscitated and re-cultured, which would create bases for the experimental study on the selected-date application of NSCs transplantation for nervous system disease.PartⅢConstruction and identification of recombinant adenovirus with Green fluorescence proteinObjective To construct recombinant adenovirus with Green fluorescence protein (Ad-GFP) and to create bases for the experimental study on gene transfer into the NSCs. Methods After the Ad-GFP was constructed by homologous recombination in bacteria, the adenovirus was amplified and the viral titer was determined by plaque assays, then the virus was conserved in refrigeratory(-80℃).Result The constructed recombination was confirmed correct on agarose gel after restriction digestion. After transfected by the plasmid DNA, the package 293 cells began to express green fluorescence after 48 hours. The intensity of fluorescence increased gradually along with the extension of time, and the amounts of cells expressing the fluorescence enhanced gradually. The viral titer of 5×109 plaque forming units (pfu)/ml was achieved after amplification.Conclusion The recombinant adenovirus carried green fluorescent protein gene (Ad-GFP) has been constructed successfully.PartⅣThe study of gene transfer into neural stem cells in vitroObjective To establish the genetically engineered neural stem cells (NSCs) to create bases for the experimental study on treating sensorineural deafness .Methods After isolated and cultured from fetal rat hippocampus,the NSCs were infected by the recombinant adenovirus with Green fluorescence protein(Ad-GFP)which was constructed by homologous recombination in bacteria. After re-cultured and induced to differentiate, we observed the infection rate and the expression of GFP through Flow cytometry and fluorescent microscopy.Result Above 97% of NSCs were infected by Ad-GFP and the expression of GFP could last for above 4 weeks, After infected by Ad-GFP, the NSCs were induced to differentiate. The differentiated cells could efficiently expression the GFP.Conclusion The recombinant adenovirus could more effectively infect the NSCs from fetal rat in vitro, which retained their characteristics and differentiation ability after infected. They can be used as donor cells to experimental research on treating sensorineural deafness with transplantation of cells. PartⅤThe experimental study on embryonic neural stem cells infected by Ad-GFP transplantation into natural rat cochleaObjective To explore the survival of neural stem cells (NSCs) infected by Ad-GFP in natural rat cochlea and to observed the changes of auditory electrophysiology and inner ear pathology of rat cochlea after NSCs transplantation.Methods NSCs infected by Ad-GFP were transplanted into rat cochlea in the study group through round window, in the same way, the artificial perilymph were injected in control group. Auditory function was monitored by measuring thresholds of auditory brain stem responses (ABR) in two groups before transplantation and after transplantation; At 14 days after transplantation, the survival of cells and the expression of GFP was observed by immunohistochemistry and fluorescent microscopy; the changes of inner ear pathology of rat cochlea was determined by HC nuclei stained and scanning electron microscopy (SEM).Result There was not significant difference in thresholds of ABR of the two groups between pre-transplantation and post-transplantation; At 14 days after transplantation, there was also not significant difference in thresholds of ABR between the two groups. In study group, the graft cells located mostly in the scala vestibule and the scala tympani of the basal turn of cochlea at 14 days after transplantation, and the survival cells were also observed in the scala vestibule and the scala tympani of other turns of cochlea, which all expressed strong green fluorescence; The expression of GFP was also obvious in stria vascularis and spiral ganglion cells but no obvious expression in cochlear hair cell(HC) and support cells. There were dispersive loss of out hair cell(OHC) in all turns of cochlea of every group, the loss rates of OHC increasd gradually from the first turn to the third turn and the total loss rate of every group were all under 1%. There was not significant difference in hair cells loss among these three groups(P>0.05). The inner hair cells (IHC) and the stereocilia of IHC were normal in all turns of cochlea of every group.So the loss of OHC was natural loss and was foreign to the surgery.Conclusion The embryonic NSCs infected by Ad-GFP could survive in normal rat cochlea after transplantation, which could express GFP efficiently, and NSCs infected by Ad-GFP transplantation had not obvious affection to auditory function and inner ear pathology of rat cochlea.