Expression Of Tau Protein During The Committed Differentiation Into Neurons From Cortex Neural Stem Cells Of Neonatal Rat

ObjectiveThe research of neural stem cells ( NSCs) is a focus in the current neuro-science. Exploring the mechanism of NSCs' proliferation and differentiation, especially its committed differentiated mechanism, is concerned closely by scientists. These data indicate that NSCs belong to the multipotential stem cells, which can keep proliferating in the serum - free medium with EFG and/or bFGF in vivo and keep the basic biological properties of NSCs. NSCs are defined as undifferentiated cells that are able to self - renew as well as generate the three major cell types that constitute the central nervous system(CNS) : neurons, as-trocytes, and oligodendrocytes.The development of neurons are associated with the cytoskeleton including the growth and movement of the cells, the outgrow of axon and dendrites, the transport of large molecular substance and organelles and so on. Microtubule is one of important cytoskeletons in neurons, which play a important role not only in regulating cell shape but also in performing many cellular functions, for example the axonal transport and the signal transduction. tau is a microtubule - associated protein which can promote microtubule stability and assembly. Additionally , tau protein takes participate in the development of axon and plays a role in signal transduction.We employed the methods of isolation, culture, committed differentiation and identification of NSCs, immunocytochemistry, immunofluorescence, electron microscopy and analysis of images to study the expression of tau and investigate the relationship to the morphological changes of neurons during the commit-ted differentiation into neurons from cortex neural stem cells, so that we could have well understanding about the mechanism of NSCs' differentiation and its relationship to cytoskeleton, and enrich the knowledge in these areas and provide theoretical foundation for clinical treatment of some degenerated diseases of nervous system.Methods1. Primary and secondary cultured methods of NSCsNSCs were derived from cerebral cortex of newborn rat, cerebral cortex tissue were cut into small pieces, and digested for 30 min at 37°C in the presence of 0. 25% trypsin. Cells were mechanically dissociated in DMEM/F12 ( with 10%B27, 20ng/ml EGF, 20ng/ml bFGF) in the density of 1 ~2 x 105 -jVinl, incubated in 371 , 5% CO2, and changed the medium one times every other day, observed under phase contrast microscope. When the neurospheres formed after 7 days, and mechanically dissociated them into single - cell suspension, implantated them in the same density above and made secondary culture, transfer of culture one times every 7 days, total made 3 transfer of culture.2. Committed differentiation of NSCsCollecting neuroshperes obtained by primary and secondary cultured, dissociated cells mechanically, implantated cells into DMEM ( with 10% FBS, 2ng/ mlBDNF, lOng/mlNGF) and induced neural stem cells differentiation, we changed the medium one times every other day, and observed the change of cells differentiation form under phase contrast microscope.3. Identification of NSCsTo evaluate the primary and secondary cultured cortex neural stem cells, we performed the nestin immunocytochemistry with SABC method and immu-mofluorescence method. The result was observed by light microscope and fluorescence microscope.4. Identification of neuron during the committed differentiationOn the 7 th day of the committed differentiation of NSCs we took out the cov-erslips with cells , performed the NSE immunocytochemistry and observed theresults by light microscope.5. To observe the expression of tau during the committed differentiation of NSCsOn the 1st ,3 rd ,5 th ,7 th day of the committed differentiation of NSCs the cov-erslips with cells were taken. Fixed the cells with 4% paraformaldehyde, performed the tau immunocytochemistry with SABC method and immumofluores-cence method. The result was observed by light microscope and fluorescence microscope.6. To observe the expression of tau by electron microscopeThe distribution of tau protein was detected by immune electron microscopy on the 7th day of the committed differentiation of NSCs, observed the results with JEM - 1200EX transmission electron microscope.Results1. Cell culture and morphological observationUnder the phase contrast microscope, we observed that neurospheres can be seen after primary cultured for 24h, and increased in number and size gradually. By the time of cultured for 7 days, the neurospheres may consist of hundreds of cells. After secondary cultured, the small cell clusters proliferated to form new neurospheres again.In the early stage (Id) of differentiation, there are some single cells a-round the neurospheres. Cells are small and less, short processes, looks like multipolar or bipolar neurons. In the middle stage (3 or 5d) , the cells become larger, with longer processes, and a clear nucleus. By the 7th day, the cells are well - developed and near mature.2. The expression of nestin detected by immunocytochemistryThe whole neurospheres were dark - brown coloured. The positive reactions of nestin appear as dark - brown coloured precipitation, which densely distributed in cell body of NSCs. There is no positive reaction in nuclear region.3. The expression of nestin detected by immumofluorescenceNestin labeled by Texas - red appeared as red color. Red coloured fluores-cence was presented in the whole neurospheres4. The expression of NSE detected by immunocytochemistryDark - brown coloured reactions of NSE distributed in cell body and neu-rites under light microscope and the nucleus was unstained.5. The expression of tau protein detected by immunocytochemistry Under the light microscope, the positive reactions of tau appear as dark -brown coloured precipitation, which densely distributed in cell body and neuritis. There is no positive reaction in nuclear region. In the first day of differentiation , tau is slightly expressed and mainly distributed in peri - nuclear region. By the time of 3rd or 5th day of culture, expression of tau increased and extended into processes. When cultured for 7 days, positive reactive granules of tau distributed more densely in cytoplasm and neurites.6. The expression of tau protein detected by immumofluorescenceUnder fluorescent microscope, tan protein labeled by FITC appeared as green color. The fluorescence signals distributed in cell body and neurites. There is no green coloured fluorescence in nuclear region. With the time going, the distribution of tau and the morphological change of neurons consistent with the observation under the light microscope. On the 1st day of differentiation, green fluorescence was seen in peri - nucleus region, and When the neurons differentiated nearly mature, with the growth of neurites, the green coloured fluorescence extended until the end of neurites.7. The distribution of tau protein detected by immune electrical microscopeUnder the TEM, tau protein was presented black particles with highly electron density in peripherial region and lowly density in central region which were distributed in the cytoplasm and neurites. And Golgi complexes and well - developed mitochondria also can be seen .8. The result of analysis of imagesThe result of analysis of images demonstrates that the amount of expression of tau protein increased gradually on lst, 3 rd, 5 th, 7 th day of cultured and the difference is remarkable when comparing the figures between any two days.