Transplantation Of Neural Stem Cells Into The Guinea Pig Cochlea

Part I Identification and culture of neural stem cellsfrom guinea pigObjective The research of neural stem cells has become more and more important in many medical fields all over the world. Neural stem cells is an adult stem cells.They has the powerful plasticity and ability of renew.So it can be used in the research of cell therapy and gene therapy.The aim of this experiment is to establish a culture method of neural stem cells derived from guinea pig.Materials and Methods The brain tissues from newborn (24h)hippocampus of guinea pig were isolated. After proliferation in conditional culture medium including bFGF or fetal bovine serum, observed the cells by Nestin immunohistochemistry staining ,GFAP and NSE immune fluorescence staining , MTT and Scanninn electron microscope to identification it.Results 1. The cells in primary culture became suspend neruospheres indifferent size.The border of the neruospheres were clear and the reflaction is good. The morphologic character of the cells in subculturing were as same as those in primary culture. 2. MTT detect show that NSC has the ability to proliferate successively. 3. The cell can differentiate into neuron and astrocyte. 4. All the neurospheres were express Nestin and the cells after differentiation were express NSE or GFAP. 5. A large of spherical NSCs aggregate in a big neurospheres.Conclusion The cells got from guinea pig hippocampus were neural stem cells. The method is simple, convenient and feasible.Part II Transplantation of neural stem cells into normalcochlea of guinea pigObjective Because of the strong potential in proliferation and differentiation,the neural stem cells can be used in the research of cell replacement therapy and gene therapy. The anatomy and relative immunology of inner ear inicated that cochlea is a very good target organ in cell transplantion and gene transfer. This study investigated surgical procedures for cell transplantation into the mouse inner ear at first. Then observed the feasibility of NSC transplantation into cochlea .Materials and Methods Inject NSCs into the perilymphatic space of normal guinea pig through tympanic scala in the basal turn of cochlea. HE staining, stretched preparetion of basilar membrane and ABR were used to evaluate the change in morphological and function of cochlea. Observe the proliferation and differentiation ofgrafted cells by immune fluorescence double staining.Results 1. There is no obversly change in morphologic and function. 2. AfterNSC transplantated 2 and 4 weeks,neuron and astrocyte can be seen in cochlea,and the number of those cells became more and more.Conclusion 1. These findings demonstrate that an approach through tympanic scala in the basal turn of cochlea can be utilized for cell transplantation into the perilymph without causing apparent auditory disorder. 2. Suggests that NSC can adapt to the environment of the cochlea.Part III The effect of neural stem cells transplantation into ischemia-reperfusion cochlea of guinea pigObjective All the damage of acoustic neuron and the degeneration of itbecause of hair cells'death can result in permanent sensorineural hearing loss . This study aimed to examine the possibility of restoration of spiral ganglion neurons and the effect to ischemia-reperfusion cochlea.Materials and Methods Established the models of ischemia-reperfusion.Inject NSCs into the perilymphatic space of ischemia-reperfusion guinea pig through tympanic scala in the basal turn of cochlea. After 4 weeks,counted the number of spiral ganglion neuron and observed the change of BDNF , GDNF in mRNA and protein by useing RT-PCR and Western Blotting.Results The expression in mRNA and protein all inhance after cells transplantation and the number of spiral ganglion neurons also increased.Conclusion 1. Neural stem cells can be the source of neurotrophic factor. 2. Neural stem cells transplantation can be used to restore the function of spiral ganglion neurons.