Proteomics Of The Differentiation Of PC12 Cells And Cos-7 Stable Cell Line Express TrkA Under The Stimulation Of Nerve Growth Factor And Protective Effect Of Melatonin On Dopaminergic Neurons

The proliferation, differentiation and survival of neuron are important content for neuroscience research. In order to know the mechanism of the etiology of nerve system and the regulation of signaling network during neural cell differentiation and development, to this end several classical cell model have been set up.Our research using Cos-7 stable cell line highly expressing TrkA, successfully e stablished a model for n eural c ell d ifferentiation i n vitro, and was confirmed from morphologically to protein level.Using 2D gel, our research found proteins probably participate in neuronal differentiation in Cos-7 stable cell line highly expressing TrkA and PC 12 cell line upon the stimulation of NGF.Our research also demonstrates that melatonin has a protection effects on the dopaminergic neurons in midbrain.Part I Proteomics of Cos7 stable cell line express TrkA under thestimulation of NGF Objective Establish a neuronal differentiation model through NGFstimulation of Cos-7 stable cell line express TrkA and find proteins responsible for neuron differentiation. Methods Observation of the morphological change of Cos-7 cells stably transfected with TrkA 0, 1, 3 and 5 days after stimulation with 50ng/ml NGF. IHC examination of the Cos-7 cells using anti-MAP-2 antibodies. WB detection of tyrosine phosphorylated proteins with 4G10 antibodies. Using 2-D gel analysis to compare the proteomic changes of Cos-7 total cell lysate before and after differentiation. Results Cos-7 stable cell line express TrkA differentiate to neuronal cells under NGF stimulation. IHC staining of the cytosome and the prostration using anti-MAP-2 antibodies both have positive results. Tyrosine phosphorylated proteins belonging to NGF-TrkA signal pathway are detected by Western blot. 2-D gel analysis detected proteins involved with neural cell differentiation. Conclusions Successfully established a biological model of neuronal differentiation. 70 proteins are involved with neuronal differentiation. After NGF stimulation, 32 proteins concentration have increased more than 5 folds, another 38 proteins concentration have decrease more than 5 folds.Part II Proteomics of PCI 2 cells under the stimulation of NGF Objective to find proteins involved in neuronal differentiation in PC 12 cells. Methods compare the differences between proteins expressed in PC 12cells well differentiated after the stimulation with 50ng/ml NGF for 3 days and that of undifferentiated ones using 2DE. Results identified 116 proteins that involved i n neuronal differentiation, 8 6 p roteins increased after NGF stimulation and 30 proteins decreased at the meantime more than 5 folds. Conclusions 116 proteins may play a substantial role in the process of PC12 cells neuronal differentiation.Part III Protective effect of melatonin on dopaminergic neurons Objectives observe the influence of melatonin to survival of rat embryonic midbrain's dopaminergic cells in vitro. Methods culture the 14 days old rat embryonic midbrain's cells in vitro, then added different concentration of melatonin ranging from 10-10M to 10-5M, after 24 hours, count the number of survival cells. Results the survival cell number are obviously more than control group when the added concentration of melatonin between 10-5-10-8M , the differences are significantly. Conclusions melatonin has protective effects on dopaminergic cells.