| The discovery of neural stem cell have changed the traditionary viewpoints that neurons can't regenerate whenever it is injured, which lead a brand new cognition to the structure and function of nervous system. Cultured neural stem cells represent not only a useful model for development studies, but also a potential cell source for transplantation to cure nervous system diseases in clinic. If neural stem cells had been thoroughly studied as hematopoietic stem cells have been achieved, that is, considerable amounts of neural stem cells can be obtained for xenomal transplantation successfully, it will be a great promotion to the medical science advancement. At present, although many studies about neural stem cells have been achieved, but there still have a great deal of works need to be done for its application in clinic. For this reason, this study tried to pay more attention to the growth of neural stem cell and its morphologic characteristic, as well as to find an effective method to induce the cells directed differentiation into neurons. At the same time, the study also concern about the DNA topoisomerase Ⅱ(TopoⅡ) expression in neural stem cells in different stages, in order to finding a new target, which might be used to regulate the proliferation and differentiation of neural stem cells in future. Four parts are included in this study as follows. Part 1: In vitro growth character of cultured neural stem cells Objective: To introduce a method for effective culture of rat neural stem cells in vitro and observe their growth character. Methods: 1 Dissociation and culture of neural stem cells Collected the newborn (within 1d) rats'brain tissues around the anterior horn of the lateral ventricle, and digested it with EDTA and trypsin (1:1) mixture to obtain single cell suspension. Then, cultured the cell in serum-free medium (DMEM/F12 medium containing B27, 20ng/ml bFGF, 20ng/ml EGF) and the effection of culture density and passage time on the neural stem cells growth were investigated. 2 Identification of rat neural stem cells 2.1 Nestin immunocytochemistry:Collecting the cells 3 days after three passages, immunocytochemistrical staining was carried out to detect nestin. The primary antibody was the rabbit anti rat nestin polyclonal antibody (1:50), 4℃, 24h;the FITC labeled second antibody(1:30),37℃,2h。2.2 The growth curve of neural stem cells:Feed the 3rd passage cells with density of 5×10~5 cells/ml into 96-well microtiter plates, 200μl per well to draw the growth curve of neural stem cells with MTT assay. 2.3 Induced differentiation: Neurospheres were plated on the poly-L-lysine (PLL) coated glass coverslips. To evaluate the potential differentiation ability of neural stem cells, the cells were cultured in medium containing 1% fetal bovine serum (FBS) and 10ng/ml glial cell line derived neurotrophic factor (GDNF) for 7d (37℃, 5%CO2, fully humidified atmosphere), and then the GFAP (special marker of astrocytes ) and NSE (special marker of neurons) immunocytochemistry were carried out to determine the character of the spreading cells. 3 AO/EB staining Cultured neural stem cells were stained with acridine orange (AO) / ethidium bromide (EB) mixed fluorescent dye to estimate its viability. 4 Fluorescence labeling Cultured neural stem cells were labeled by DAPI, a kind of specific fluorescent dye to nucleus, and observed under fluorescent microscope to estimate the label ratio. The growth curve of DAPI labeled cells was also drawed according to the method described above. After passaged for 1 month, those labeled cells were observed under fluorescent microscope to estimate theResults: 1. The dissociated neural stem cells formed floating neuraspheres in suspension cultures. In 5×10~5 cells/ml culture density: Typical neurospheres (regular round and compact) could be seen after primary cultured in selective serum-free medium for 3~4d, with different size (diameter is between 5~10μm). Many cells with small round bodies, similar in size, and growth state being well could be seen on the surface of neurospheres. Black plaques were noticed in the neurospheres, after primary cultured for 9d without passage. In 1×10~5 cells/ml culture density: The number and size of neurospheres in this group was less than that in 5×10~5 cells/ml group at the parallel culture time. Most of the cultured cells were single cells. Passaged the cells 3~4d after primary culture, a great deal of neurospheres with well growing state could be continuously obtained. The doubling time of those cells was 2d. Passaged the cells 9d after primary culture,growth state of the cells became worse, and its doubling time was near 5d. 2. The cultured cells are nestin-positive cells, and have the proliferative ability. Cells became adherence growth with increasing size and stretching processes in different style, when they were induced to differentiation. Both GFAP-positive and NSE-positive cells could be identified among those adherence cells. 3. Cells among neurospheres are heterogeneous. Small round cells located on the outer edge of neurospheres; however, the cells have processes located in the core of neurospheres. AO/EB staining showed that most of the cells among neurospheres were live cells after primary culture for 3~5d. There were many dead cells in neurospheres by continued culture without passage. The label rate of DAPI was 100%; the growth curve of labeled cells was similar to the control group. DAPI fluorescence also could be detected 1 month after labeled. Conclusion: It was demonstrated that the cultured cells belong to neural stem cells. The introduced method is simple, operative and reliable one used tostudy neural stem cells. Our study also showed that the neural stem cells should be cultured with high density and passaged in time. Cells among neurospheres are heterogeneous. DAPI can be used as an effective marker in vitro. Part 2: The structure analysis of in vitro cultured neural stem cell clusters Objective: Observe the inner structure of cultured rat neural stem cell clusters, and analyze its component. Methods: 1 Dissociation and culture of neural stem cells Methods was same as the one described above. 2 Cells collection The cell clusters of the 3rd passage cells were centrifuged and rinsed with PBS for 2 times. The clusters were fixed in 4% paraformaldehyde at 4℃for 30min. The collected clusters were embedded in wax as routine method. The paraffin sections were cut with Leica microtome in 5μm thickness. 3 Light microscopy The paraffin sections were used in HE staining, TUNEL labeling and immunocytochemistry. TUNEL was done to detect apoptosis. Immunocytochemistry was carried out to detect nestin (special marker for neural stem cell), GFAP (special marker for astrocytes) and NSE (special marker for neurons) antigen in those cells. 4 Scanning electron microscopy Plated the 3rd passage cells on PLL coated cover glasses and cultured them in DMEM containing 5% FBS for 2h. After rinsed with PBS, the cells were fixed in 1% glutaraldehyde at 4℃for 1h. Then the cells were dehydrated with graded ethanol series, spurted with gold and observed under Hitachi S-3500 scanning electron microscope. 5 Semi-thin section and transmission electron microscopy Collected neural stem cell clusters, fixed in 1% glutaraldehyde at 4℃for48h. Postfixed in 1% osmium tetroxide for 1h, and dehydrated with graded ethanol and acetone series, then embedded in epoxy resin 812. Semi-thin sections was stained with toluidine blue and examined under a light microscope; ultrathin sections were double stained with uranium acetate and lead citrate, and observed under Hitachi H-7500 transmission electron microscope. Result: 1 Observation with light microscope Floating neurospheres showed a round or oval morphology and were consisted of several to hundreds cells. Neurospheres had a noncompact structure with large intercellular spaces filled with amorphous structures. It was noted that there were healthy cells and dead cells in the cell clusters. The healthy cells surrounded the dead cells to form a typical nest-like stucture. The healthy cells were consisted of two cell types, small cells and large cells. Small round cells showed a dark stained nucleus and little cytoplasm, the ratio of nucleus vs cytoplasm is high. Large cells showed a light stained large nucleus, plentiful cytoplasm, and long processes, the ratio of nucleus vs cytoplasm is low. Apoptotic cells in the clusters were detected by TUNEL technique, and the apoptotic rate is 18.2±2.6% . 2 Observation with scanning electron microscope A layer of small round cells, similar in size, with smooth surface, located on the outer edge of these neural stem cell clusters. The processes of these small round cells were stretching from the cellular bodies and adherent to clusters. Among the interspaces of small round cells, some flat cells were noticed which adhere to the adjacent small round cells. 3 Observation with transmission electron microscope Two subpopulations of cells were distinguished: light cells and dark cells, which showed a variable appearance in both morphology and size. Light cells showed lower electron density and had less cytoplasm than dark cells. The ratio of nucleus vs cytoplasm was high in light cells, but low in dark cells. The organelles in light cells were less than that of dark cells. Moreover, light cellsshowed intermediate filaments (10~12 nm thick) that were randomly distributed and arranged in bundles, whereas dark cells did not have filaments bundles. Compared with light cells, the dark cells showed more phagosomes and lamellar membrane structures. The other ultrastructural features were similar for both cell types, such as the adherens junctions were seen between adjacent healthy cells; desmosomes, gap junctions, tight junctions, and synapses were not observed. Amorphous structures, and transect of tube-like processes coated by intact membrane or incomplete membrane could be observed in the intercellular spaces. Apoptotic or necrotic cells were surrounded by healthy cells'processes, which formed the nest-like structure. 4 Immunocytochemistry There were many nestin-positive cells in the clusters. The nestin-postive rate was more than 95%. GFAP-positive cells showed large bodies and variform processes interweaved with each other, which formed the scaffold of clusters. The NSE-positive cells were relatively fewer than others. Conclusion: Apoptotic or necrotic cells in neural stem cell clusters were surrounded by healthy cells'processes, which formed the nest-like structure. Healthy cells could be divided into two subpopulations: small round cells showed electron-lucent, nestin-positive strongly, might belong to naive neural stem cells; and the cells with large processes, showed electron-dense, nestin-positive weakly, might belong to pre-differentiating or differentiating cells. GFAP-positive cells formed the scaffold of clusters, which attached by other cells. Adherens junctions existed between cells. Neural stem cells might have phagocytic capability. Part 3 Directed differentiating of neural stem cells into neurons in vitro Objective: Find an effective method to induce neural stem cells directed differentiating into neurons. Methods: 1 Dissociation and culture of neural stem cellsMethods were same as the one described above. 2 Induced differentiation Neural stem cells were cultureed on poly-L-lysine (PLL) coated glass. The exprements were divided into 4 groups: neurospheres cultured in condition medium; the cells digested from the neurospheres were cultured in condition medium; neurospheres cultured in medium containing 10% FBS; the cells digested from the neurospheres were cultured in medium containing 10% FBS. Condition medium was composed of serum-free DMEM/F12 medium, B27, 10ng/ml GDNF, and 1% FBS. 3 Observation with inverted microscope Examining the numbers of spreading cells, the length and spreading field of their processes. 4 Nissle staining The differentiating cells were harvested at 5d and 10d after induction, and stained with toluidine blue. 5 Immunocytochemistry The differentiating cells were harvested at 5d and 10d after induction. Immunocytochemistry was carried out to detect nestin, GFAP and NSE of those cells. In the 10d group, spreading cells in 10 high visual fields of each group were counted and the percentage of NSE positive cells was calculated. t test was used to verify the difference between groups. Results: 1 Neurospheres groups 1.1 Observation with inverted microscope: ①Condition medium group: Neurospheres adhered to the glass at 1~2d after cultured in this medium. Cells begun to spread from the attached neurospheres, after cultured for 2~3d. Then, the spreading cells became more and more, their processes and the extending field were increasing. Those earlier spreading cells showed long radial thin processes, those later spreading cells migrating along those processes. Along with the distance from neurospheres, the shape of those spreading cells changed from round to polygonal. Those polygonal cells showed neuronsmorphological character. The interweaved processes of those spreading cells were stretching between the layered cells. Cells density decreased from neurospheres to marginal zone. It was found that neurospheres with large size could not fully spread and still had undifferatiated round cells within them. ②10% FBS group: The number and field of cells spreading from neurospheres were less than condition medium group. Spreading cells distributing irregularly. 1.2 Nissle staining: After cultured in condition medium for 10d, the number and field of spreading cells containing Nissle bodies was more than that cultured for 5d. Those cells showed clear Nissle bodies in cytoplasm, and a light stained nucleus with obvious nucleolus. The nucleus of those migrating cells located on the edge of cytoplasm, reverse to the migrating direction. Taking one with another, The number of cells containing Nissle bodies in 10% FBS group was much less than that in condition medium group. 1.3 Immunocytochemistry: Nestin-positive cells were less and stained weaker in spreading cells than in neurospheres. On 5d after inducement, GFAP-positive cells with radial long thin processes spreaded fast, which seemed play a role in leading other cells to migrate from the neurospheres. A few NSE-positive cells could be seen in this group. On 10d after inducement, most of the spreading cells were the NSE-positive. Those cells attached to the interweaved long thin processes of GFAP-positive cells. The staining of NSE-positive cells with large size and long processes were thicker than the cells with little size and small processes. In conclusion, the number of NSE-positive cells in 10% FBS group (38.1±5.8%) was much less than the cells in condition medium group (79.4±8.0%); Whereas the number of GFAP-positive cells in 10% GS group was much more than in condition medium group. t test showed p<0.001. 2 Cells digested from the neurospheres group 2.1 Observation with inverted microscope: ①Condition medium group: Cells adhered to the glass very fast, dispersed evenly. The processes of those cells were much more than the above group. Two types of cells could be seen:glial cells and differentiating neural stem cells. Glial cells'processes grew fast, interweaved long thin processes were streching between the stratified cells and forming the scaffold for other cells to attachment. The differentiating neural stem cells adhered to the long thin processes of glial cells. As the culture time prolonged, the shape of differentiating cells turned from round into polygonal and showed the morphological character of neurons. Processes of those differentiating cells emerged gradually and became long and long, untill they finally differentiated into mature neurons. ②10% FBS group: The neural stem cells mainly differentiated into glial cells. The number of neurons was fewer. 2.2 Nissle staining: A great deal of cells containing Nissle bodies, different in size, could be observed in condition medium cultured group. Cells with small size and less process might be differentiating neurons; wherase cells with large size and many long processes might be mature neurons. The number of cells containing Nissle bodies in 10% FBS group was much less than those in condition medium group. 2.3 Immunocytochemistry: Nestin-positive cells stained weaker in large cells than in small cells. Interweaved long thin processes of GFAP-positive cells formed the scaffold and the NSE-positive cells adhered to that. The staining of NSE-positive cells, with large size and long processes was thicker than those with little size and small processes. In conclusion, the percentage of NSE-positive cells in 10% FBS group (31.9±4.2%) was much less than in condition medium group (79.4 ±8.0%); Whereas the percentage of GFAP-positive cells in 10% FBS group was much more than in condition medium group. t test showed p<0.001. Conclusion: PLL could provide adherence matrix for in vitro cultured cells, which might be suitable for processes extending. GDNF and low concentration of FBS in the condition medium could induce neural stem cells differentiating into neurons effectively. The cells cultured after digested would be benefit to the neural stem cells differentiation into neurons. During differentiating period of neural stem cells, astrocytes were showngrowing fast, which seemed to play a role in leading other cells to migrat from the neurospheres. Part 4 The DNA topoisomerase Ⅱexpression in neural stem cells cultured in vitro Objective: Detect the expression of DNA topoisomerase Ⅱ(Topo Ⅱ) mRNA and protein in neural stem cells during the proliferation and during the directed differentiation into neurons. Methods: 1 Groups ⑴Directed differentiation into neurons group: Collected cells digisted from neurospheres, after cultured in condition medium for 6~7d, when the neural stem cells were differentiating into neurons. ⑵Control Group: Collected the neurospheres in proliferate stage. 2 Immunocytochemistry Detecting the expression of TopoⅡαand PCNA protein in two groups. 3 Western blotting Detecting the expression of TopoⅡαandβprotein in two groups. 4 RT-PCR Detecting the expression of TopoⅡαandβmRNA in two groups. 5 Results analysis Using t test to compare the difference between each coupled groups. Results: 1 Immunocytochemistry ⑴There were many dark stained TopoⅡα-positive cells in neurospheres, the positive rate was 61.4±5.7%, the average densities was 0.731±0.021. There were some light stained TopoⅡα-positive cells when the cells differentiating from neural stem cells into neurons, the positive rate was 11.6±2.9%, the average densities was 0.398±0.027. t test showed p<0.001. ⑵PCNA-positive cells in neurospheres were mainly the small cells, the...
|
PDF Full Text Request