A Study On The Differentiation From Neonatal Rat Neural Stem Cells To Cholinergic Neuron

PART ONE ISOLATION AND CULTIVATION OF NEONATAL RAT NEURAL STEM CELLSBackground and Objective Neural stem cell, the self-renewing multipotential cell, can generate neural tissue, including neurons, astrocytes and oligodendrocytes. It exist not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including human. Isolation and culture of NSC is the basement of the fundamental and applied research. Objective of this part is to obtain neonatal rat NSCs for further study on its proliferation and differentiation.Methods Isolation the primary NSCs from endbrain and midbrain of neonatal rats(in 24h after birth), the cells are suspended and cultured in serum free medium (containing bFGF and EGF ) for long-term survival in vitro. Technique of single-cell clone culture was used to confirm the character of self-renewing. Ability of multi-directional differentiation and specific antigen, nestin, were detected by immunocytochemistry. Cytometry was used to evaluate the proliferation capability of NSCs and the growth curve is plotted.Results NSCs can survive in vitro for long time. They show multipotential of differentiate into neurons and glial cells and the ability of self-renewing and proliferating. The NSCs are positive for nestin expression.Conclusions The NSCs are isolated from endbrain and midbrain of neonatal rats. These cells can be cultured for long time in vitro. They express nestin and have the ability of self-renewing and multi-directional differentiation. Cells can expand in serum free medium plus mitogen factors.PART TWO ATRA AND NGF INDUCED NEONATAL RAT NEURAL STEM CELLS TO CHOLINERGIC NEURONBackground and Objective NSC can be served as a source of cell transplantation for the treatment of nervous system disease, but the treatment need a great deal of neural cells with definite phenotype and function. The high point of today is to control NSC differentiate to a special needed phenotype. Alzheimer's disease is a neurodegenerative disease of cholinergic neurons. Objective of this part is to induce NSC to cholinergic neuron in vitro and provide experiment foundation for NSC-derived cholinergic neuron.Methods ATRA and/or NGF were added into the P3 NSCs induce system. We used immunocytochemical techniques to detect CHAT-positive neurons induced by ATRA, NGF, ATRA and NGF.Results CHAT-positive neurons with statistical significance(p<0.05) were detected induced by ATRA, NGF, ATRA and NGF. The effect of ATRA and NGF is not found stronger than single NGF.Conclusions ATRA, NGF, ATRA and NGF can induce NSC differentiate into cholinergic neurons from NSC in vitro. The effect of ATRA and NGF is not found stronger than single NGF.