| Objective Neural stem cell (NSC) is a group of cells which exists in nervous system of adult mammals and have the ability of division and multiple differentiation. If they are isolated and cultured in growth factor or neurotrophic factor containing culture medium, they can proliferate to form neural spherical(cell cluster) and differentiate into neurons, astrocytes and oligodendrocytes. It has been reported that NSC distributes extensively in nerve system which include cerebral cortex, ependyma, subependymal zone, olfactory bulb, striatum, spinal cord etc.In the processes of differentiation from NSCs to neurons, the neuritis outgrow and extend to certain directions, then form synapses with target cells, the body, dendrites or axons of neurons. It is well known that the cellular movement and maintaining of cell shape depend on cytoskeletal system. But regarding the extending of neuritis to certain direction, the mobility of growth cone plays a key role in these processes.Growth cone is a mobile and fan - shaped extension of the end of axon or dendrite, which consists of two regions, lamellipodium and filopodium. Lamelli-podium is the fan - shaped extensive part; and filopodium is the finger - liked processes extending from lamellipodium.Cytoskeleton is a kind of thin network - like structures in cytoplasme, including microfilament, microtubules and intermediate filament. F - actin is the main component of microfilament, which plays a key role in mobility of growth cone. The activity of lamellipodium and filopodium of growth cone depend on polymerization and depolymerization of F - actin. When growth cone activemovement, more G - actin are added to the plus end of F -actin, which makes F - actin extends into the filopodium and force filopodium extends. Then the veil of lamellipodium and filopodium form adhesion junctions with extracellular matrix ( ECM) by the mediation of cellular adhesion molecules ( CAM), and pull the neurite to the direction. If veil of lamellipodium and filopodium cant adhere to the ECM, F - actin depolymerize. Then the veil of lamellipodium and filopodium withdraw, and unite with central region of lamellipodium. Both the processes are regulated by adhesion junction related proteins (AJRP).AJRP mainly include: integrin family, which mediate the formation of the cell - extracellular matrix adhesion junction - adhesion plaque, and cadherin family, which mainly mediate the cell - cell adhesion junction - adhesion belt.Both the formation of these two kinds of adhesion junctions needs the involvement of attaching proteins. Integrin or cadherin connect with attaching proteins firstly, and attaching proteins bind to F - actin, which connect the cell with other cell or extracellular matrix. The attaching proteins include a - , P - , -y-catenin, vinculin, a-actinin and plakoslobin0A - actinin is a cross - link protein which connects the F - actin into bundles or network. It has been reported that a - actinin also involves in the activities of transduction of cellular signal, formation of adhesion complexes and the polymerization of F - actin in adhesion plaque.Vinculin is another kind of action associated protein which connects F - actin to integrin or cadherin in cell - cell or cell - extracellular matrix adhesion junctions, and it also involves in the maintenance of cell shape, cell movement and cellular signal conduction from outside to inside. It has been reported that F - actin depolymerization causes the change of distribution of vinculin, and the formation of adhesion junction can initiate the polymerization of F - actin.In cultured cells, Cellular locomotion is driven by repeated cycles of protrusion of the leading edge, formation of new matrix adhesions and retraction of the trailing edge. In these processes, the cytoskeletal proteins are expressed sequentially , co - located distributed, and regulated each other.In summarize, in the processes of differentiation of committed NSCs, the adhesion junction play a important role in cellular mature, locomotion, the out-growth of neuritis and directing extension. So, in these processes, about if expression of F - actin changes, and how about the expression of A - actinin and vinculin, what is the relationship between the three cytoskeletal proteins, we didnt see the related report by now.We employed the methods of isolation, culture, committed differentiation and identification of NSCs, immunohistochemistry, confocal laser scanning microscopy , electron microscopy and x - ray microanalysis to study the expressions of F - actin, A - actinin and vinculin in committed NSCs, and ultrastructure of committed NSCs, so that we could have well understanding about the mechanism of NSC 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. Isolation, culture differentiation and identification of NSCs and observed under phase contrast microscopeNeurons were derived from cerebral cortex of newborn rat. Briefly, cerebral cortex tissue were cut into small pieces, and digested for 40 min at 37°C in the presence of 0.25% trypsin. Cells were mechanically dissociated in DMEM/F12 ( with 10%B27, EGF 20ng/ml,bFGF 20ng/ml) , distributed into culture bottles and cultured for 7 days at 37Tl in 5% C02. When the neurospheres formed, mechanically dissociated them into single cell or small clusters with same culture medium, and cultured for another 7 days.Took primary and secondary neurospheres, dissociated them mechanically with DMEM/12(with 10% FBS,2ng/ml BDNF) to suspend the cells. Then planted the suspended cells into 6 - pole culture plates with poly - L - lysine coated glass coverslips or ACLAR membrane, and cultured for 7 days.2. Immunocytochemistry and immune electron microscopyFor immunocytochemical staining of a - actinin, F - actin and vinculin, cells were fixed with 4% parafonnaldehyde. After washes with PBS, cells were soaked in diluted normal serum (1:50) for 25 min at room temperature to blockthe nonspecific reaction. Then the cells were incubated with rabbit anti rat F -actin, rabbit anti rat a - Actinin and mouse anti - human vinculin for at 4To-vernight, Then follow the usual protocol of immunocytochemistry ABC and electron microscopic methods3. Double-label Immunofluorescent methods and Confocal microscopy The cells on coverslips were fixed with 4% paraformaldehyde. After washing with PBS, cells wjyre pretreated with blocking solution consisting of diluted normal sheep serum for 25 min at room temperature to block the nonspecific reaction. Then they were incubated with primary antibodies 1:1 mixture; I, mouse anti - human vinculin and rabbit anti - rat a - Actinin; II, mouse anti -human vinculin and rabbit anti - rat F - actin for 24h at 4^C. After rinsing with PBS, they were incubated with mixed secondary antibodies, FITC - labeded sheep anti - mouse IgG and Cy3 - labeded sheep anti - rabbit IgG, for 50 min. After Washing thoroughly with PBS, the coverslips were mounted on glass slides and sealed using Anti - fade. Cells were observed with a confocal laser scanning microscope, and photograghed.4. Electron microscopy4. 1 scanning EMTook out 7 th days glass coverslips with committed NSCs grew from culture plate. Cells were fixed with cold 2. 5% glutaraldehyde for 15 mins. And then rinse with DW, dehydration, Freeze - drying, magnetron sputter - coating with silver, and obsearved with JEMT300 scanning electron microscope, photographed.4.2 Transmission EMTook out 7' days glass coverslips with committed NSCs grew from culture plate. Cells were fixed with cold 2.5% glutaraldehyde for 20 mins, and post -fixed with 2% OsO4 for 30mins. And then rinse with DW, dehydration, inverting embedded and ultra - sectionning with ultromircotome, stained and observed with JEM - 1200EX transmission electron microscope, photographed.5. X -ray quantitative microanalysis:The ultrathin sections of 1st, 3 rd, 5 th, 7 th day of cultured which has been treated by immune electron microscopy were chosen randomly, and the amountof ()s( in the form of Wt% ) in expressive areas of a - actinin were measured under analytical electron microscope which connected with a energetic analysis instrument. Then the data were analyzed with SPSS 11.5 statistical software.Results1. 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 7th 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 7l day, the cells are well - developed and near mature.2. The expression of a - actinin, F - actin and vinculin detected by immu-nocytochemistry2. 1 The expression of a - actininUnder the light microscope, the positive reactions of a - actinin appear as dark - brown coloured precipitation, which densely distributed in cell body and processes. There is no positive reaction in nuclear region. In the first day of differentiation , a - actinin is slightly expressed and mainly distributed in peri - nuclear region. By the time of 3 or 5' day of culture, expression of a - actinin increased and extended into processes. When cultured for7days, positive reactive granules of a - actinin distributed more densely in cytoplasme and processes.Under the transmission EM, the positive granules of a - actinin appear as spherical structure, which are more electron - density in peripheral part and e-lectron - lucent in the central part. The granules are distributed peripherally and extending into the processes.2.2 Expression of F - actinThe positive reaction appears as dark - brown coloured granules, which are distributed in cell body and processes, especially in perinuclear region. No positive reaction is at nuclear region. In the first day, the expression of F - actin is closely distributed in perinuclear region, and there are some pinocytotic vesicles in the cytoplasme. By the time of 3or 5 days of differentiation, the expression of F - actin increases gradually and some reactive products appear as thin filaments which extend into processes. When cultured for 7days, the thin filaments are distributed all over the cell body and extend into the processes, and in the processes, which are parallel arranged.2.3 Expression of VinculinIn the first day of differentiation, the cell body of neurons is small, and vinculin mainly is expressed in cell body and especially undermembrane region. It is weakly expressed in the processes. After cultured for 3 to 5 days, the expression of vinculin increases. By the time of 7 th day of differentiation, the cell body of neuron becomes larger with a clear nucleus, and the processes increase in number and length. Some of the processes branch and connect with each other. The expression of vinculin increases dramatically and densely distributed in peripheral part of neurons.3. Immunolocalization detected by Confocal microscopy using double - label immunofluorescence methods3.1 The Immunolocalization of a - Actinin and vinculinCoverslips were observed with a confocal laser scanning microscope using X40 Apo oil. FITC and Cy3 - images were obtained by dual excitation at 488 and 543 nm, respectively. The results: a - Actinin is demonstrated as green coloured fluorescence and vinculin appeared as red coloured one. The co - localization of a - Actinin and vinculin appears as yellow or orange coloured depending on the amount of expression of two proteins. In the early stage of differentiation, the a - Actinin and vinculin are co - localized in cell and processes, and densely distributed at one side of peri - nuclear region. In the processes, some spot -liked yellow coloured fluorescence can be seen. When the neurons differentiated nearly mature, with the growth of processes, both a - Actinin and vinculin extend until the end of processes and segmental yellow or orange coloured fluores-cent signal can be seen along the processes.3. 2 The Immunolocalization of Actin and vineulinUnder the CLSM, F - Actin appears as green - coloured fluorescence and vineulin appears as red one. In the early stage of differentiation, both F - actin and vineulin distribute in cell body and processes, and expression of F - actin is stronger than vineulin in cell body. But in processes, some spot - liked red fluorescence can be seen. By the time of cultured for 7 days, the expression of vineulin increase and some segmental yellow - coloured fluorescent signals appear along the processes and until the end of it.4. Infrastructure observed under EM 4. 1 Ultrastructure observed under SEMUnder the SEM, by the time of cultured for 7 days, the neuron is well - developed with some processes. Some of processes branch, and some is long and thin. A kind of synapse - liked structures can be seen on the surface of cell body.4. 2 Ultrastructure observed under TEMUnder the TEM, by the time of cultured for 7 days, the neuron is well - developed. The neuron is rich in all kind of organelles, including SER, lipid droplet, glycogen granules, Golgi complexes and well - developed mitochondri-a, which crista is arranged longitudinally or obliquely. There are also a lot of cytoskeletal structures distributed in the cytoplasm, such as microfilament and microtubules. The cell nucleus is round and large with clear nucleolus and nuclear membrane. There are some microvilli - liked protrusions on the surface of neuron, and some of them enclose the pinocytotic vesicles.5. X - ray quantitative microanalysis demonstrate that the amount of expression of a - actinin in committed NSC increased gradually in 1", 3 rd, 5 th, 7 th day of cultured, and the difference is remarkable when comparing the figures between any two days.Conclusion1. A — Actinin is shown positive expression in cell body and processes ofcommitted NSCs derived for cerebral cortex, and the expression is increasing with the development of cell. This result suggests that the expression of a - Ac-tinin is related to the growth and mature, and also have relationship to the outgrowth of neuritis.2. The expression F - actin is closely related to the growth of committed NSCs derived for cerebral cortex and the growth of neuritis, which confirms that F - actin plays a key role in maintenance of cellular shape and structure, as well as the growth of neurons.3. Vinculin expresses in cell body especially perinuclear region in the early stage of differentiation. With the growth of cell, the expression of vinculin in peripheral part of neuron and processes increases gradually, which suggests that formation of adhesion junction is needed by the growth and mature of neuron in the process of differentiation. In addition, there is a vinculin "pool" in the cytoplasm which can store some vinculins, which probably means that the formation of adhesion junction is a rapid and retrogradable process.4. The expressions of a - Actinin and vinculin are always lo - localized in cell body and process during the processes of differentiation and development of committed NSCs and are increasing with the increasing of culture time. The a-mount and location of expression of two proteins are identical at all time. These results suggest that adhesion junctions in committed neuron are formed in a manner that a -Actinin firstly connects to vinculin, then vinculin binds to CAM.5. F - actin and vinculin are co - localized to express in cell body and processes in well - developed committed neurons. But in the early stage of differentiation , the expression of F - actin is stronger than vinculin, which means there is more F - actin needed by the cellular activity in this stage such as the maintenance of cell shape, constraction and non - adhesion dependent growth, besides the formation of adhesion junction during the early stage of development.6. By the time of cultured for 7 days, the neuron is well - developed with some processes. Some of processes are more branched, which looks like den-drites and some of them looks like axon. And some synapse - liked structures can be seen on the surface of neuron. In cytoplasm, there are all kind of the or-ganelles and large amount of mitochondria. These results suggest that the shape... |
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