Study On The Expression Of PTEN In The Process Of Neural Stem Cells Differentiation And Maturation And The Role Of PTEN To Axon Growth Inhibition

Part I Culture and identification of neural stem cells from rat hippocampus in vitroObjective: To supply neural stem cells(NSCs) for next research, hippocampal NSCs from Sprague-Dawley(SD) rats born in 1 to 3 days were extracted, passaged and identified.Methods: Hippocampus from neonatal SD rats in 1 to 3 days was isolated, cells were plated in 25cm2 culture flasks after suspension, and cultured in 37°C, 5% CO2 incubator. NSCs were passaged and gradually purified by mechanical pipetting combined with Accutase digestion. Cell morphology was observed under an inverted microscope, and immunofluorescence staining was used to identify neural stem cells and their multipotent differentiation.Results: We successfully isolated and cultured hippocampal NSCs, which grew spherically with refraction. Cells were further purified by passage, and nestin staining of the cultured cells was positive. After NSCs induced to differentiate for 7 days, glial fibrillary acidic protein(GFAP) and beta-tubulin III staining of differentiated cells were both positive.Conclusions: NSCs can be extracted from rat hippocampus. Highly purified NSCs were available through passage. NSCs can pluripotently differentiate into neurons and astrocytes.Part II The expression and significance of PTEN in the process of NSCs differentiation and maturationObjective: To explore the expression and significance of PTEN in the process of NSCs differentiation and maturation cultured in vitro.Methods: NSCs from rat hippocampus were cultured in vitro. The third generation of NSCs were collected and randomly divided into four groups: 0d, 1d, 3d and 7d group, then induced to differentiation for 0d, 1d, 3d and 7d, respectively. Under inverted microscope, the changes of cell morphology were observed. The expressions of phosphatase and tensinhomology deleted on chromosome ten(PTEN) protein and Phospho-s6 ribosomal(P-S6R) protein which reflects the activity of target of rapamycin(m TOR) were detected by western blot, and immunofluorescence staining was used to examine P-S6 R protein of 1d, 3d and 7d group.Results: Compared with 0d and 1d group, PTEN expression of 3d group was significantly increased(both p<0.01), and PTEN expression of 7d group was also significantly increased(p<0.01 and p<0.05), the P-S6 R expression of 3d and 7d group was both significantly decreased(p<0.01). Immunofluorescence staining showed that the percentage of P-S6 R positive cells in 3d group decreased sharply compared with 1d group(p <0.01); In 7d group, the proportion of of P-S6 R positive staining cells was still significantly lowered in comparison with 1d group and 3d group(p<0.01 and p<0.05); Fluorescence intensity of 3d group and 7d group decreased significantly.Conclusions: PTEN/m TOR signaling is involved in the regulation of proliferation and differentiation of NSCs and neurite outgrowth, and may decrease the capacity of proliferation, differentiation and axon growth of NSCs as to a certain stage. Thus, it’s difficult to establish effective synaptic connections.Part III The role of PTEN inhibition to axon growth in the process of NSCs differentiation and maturationObjective: To investigate the role and mechanism of PTEN inhibition to axon growth during NSCs differentiation and maturation.Methods: NSCs from rat hippocampus were cultured in vitro. The third generation of NSCs were collected and transfected, which were randomly divided into three groups: control group, negative transfection group, and PTEN transfection group. After transfection for 48 hours, the protein levels of PTEN and P-S6 R were detected by western blot. The number and length of axon and cell morphology were observed.Results: Compared with the control group and negative transfection group, PTEN expression in PTEN transfection group was significantly reduced(both p<0.01), but P-S6 R expression of PTEN transfection group was significantly increased(both p<0.01). Morphological changes were observed under light microscope. After NSCs differentiation for 7 days, the number and length of axon as well as cell body volume in PTEN transfection group increased markedly compared with control group and negative transfection group. There was no significant morphology difference between control group and negative transfection group.Conclusions: PTEN inhibition can enhance the capacity of differentiation,maturation and axonal growth of NSCs. PTEN/m TOR signaling is a critical mechanism that can hamper axon growth in the process of NSCs differentiation and maturation.