Study The Cultivation/proliferation/immigration And Differentiation Of Neural Stem Cells

Part â…  The cultivation/differentiation and verification of neural stem cellsObjective Approach the neural stem cell cultural method in vitro from mouse corpora striata and the verification of neural stem cell. Method Adopt common health adult SD mouse from animal center of The PLA University, when mouse pregnant about 13-14 days, adopt the embryonal corpora striata, made mono-cell suspension, count cell in micro and culture cell in culture plate, divide into two groups, add DMEM-F12 in control group and add EGF/bFGF and DMEM-F12 together in experiment group. Result Observe the growth of neural stem cells every day in inverted microscope, 24h later, in experiment group, we can observe many living cell homogeneous distribution and many coUagenoblast sink in the base of culture flask, part of cells grow floating in culture fluid, cell appear conjugation usually and some cells begin to division. 72h later, cell aggregation and the cell colony consist of about 4-6 cells; 5 days later, cell colony is consisted of about ten to hundred cells, we can observe lots of cell colony. The cell sphere growth floating and arrange tight among cells, some cell colony appear cellular necrosis because of alimentary deficiency. In control group, we can not observe cell proliferate. Made floating cell sphere into mono-cell suspension and differentiate according sink nature. We find the mono-cell Nestin~+ and it can differentiate into nerve and horizontal cell. Conclusion The cell sphere can proliferate self and differentiate into nuron and horizontal, so the cell sphere is neural stem cell. Part â…¡ The action of EGF and bFGF on the proliferation of neural stem cellsObjective Approach the proliferate effect of neural stem cell acted by EGF and bFGF. Method Made cell sphere into mono-cell suspension, add dispase during dispersive process. The method is to obtain neural cell spheres that growth good in culture flask first\digest them for 2 hours\then blow lightly and get out supernatant after sink nature and obtain mono-cell suspension. Divide into 4 groups, Add EGF in experiment group 1; add bFGF in experiment group 2; add EGF and bFGF together in experiment group 3; add base serum-free medium only in control group. Count cell amount of cell spheres in micro After cultured for 3 days and measure the diameter of cell spheres at 7 days. Result Cultured for 3 days, in EGF and EGF+bFGF experiment group, we can observe budding and proliferation of neural stem cell in inverted microscope; In EGF experiment group, under the double 20 field of vision, we can observe about 4.3 cell spheres and every cell spheres is consist of 4 cells usually. In EGF+bFGF experiment group, under the double 20 field of vision, we can observe about 7 cell spheres and every cell spheres is consist of 16 cells usually. In control group, we do not observe budding and proliferation of neural stem cell. Cultured for 7 days, In EGF experiment group, cell sphere is consist of about 40 cells, the diameter of cell sphere is about 27um; but in EGF+bFGF experiment group, cell sphere is consist of about 100 cells, the diameter of cell sphere is about 35um; the cell conjugation between cells is looser than the cell conjugation in primary cultured; the cause is related with dispase during digestion. Conclusion EGF can excite neural stem cells proliferate lonely; bFGF can not excite neural stem cells proliferate lonely but can enhance the proliferation function of EGF.Part III The action of polylysine on the neural stem cell proliferation and differentiationObjective Observe the effection of polylysine on the neural stem cell proliferation and differentiation. Method Passage mono-cell in culture plate contain polylysine or not; divided into experiment group and control group, add polylysine in experiment. Result In experiment group, cells is divided into two layers; In adherence layer, cultured for 2 days, we can observe many cell colony that consist of about 16 cells, some cells migrate from cell spheres and differentiate into neuron and horizontal cell; In micro, cell that have one or two long ecptoma is neuron, cell that have many long coarse ecptomas is horizontal cell or oligodendroglial cell; Neural stem cells proliferate continuely from 3 to 5 days. Cultured for six days, the adherence cell sphere is consist of about hundred cells and the diameter of cell sphere is about 42um. In floating layer, we can observe cell budding and cell proliferation in second day, the amount of cell sphere is consist of about 2 to 4 cells. Cultured continue, in fifth day, the amount of cell sphere is about 10-16 cells. Cultured for 6 days, cell spheres's amount is about 40 cells and the diameter of cell sphere is about 25 um. In control group, cell growth floating and no adherence cell sphere. Conclusion The proliferation function of Neural stem cells in adherence layer is more enhancer than Neural stem cells in floating layer. Remove EGF from culture fluid, cell sphere begin to differentiate into horizontal cell, so polylysine can induce neural stem cell differentiate into horizontal cell.Part IV Approach the migration of neural stem cellsObjective Approach the transport phenomena of adherence and floating neural stem cells. Method According observe the adherence neural stem cell sphere in part ITT and the floating cell sphere acted in EGF for 14-17 days. Result In subcultivation, we can observe amount of adherence cell spheres , cell migrate from center to circumference and have evident chemotaxis. Between the cell spheres, we can observe cell migrate along ecptoma, if the distance is long, the ecptoma can interconnect and enhance the distance, so neural stem cell can migrate longer. In floating cell spheres, cell proliferate under the action of EGF, in forteenth day, cell sphere's amout is about one hundred. Cell sphere stop proliferating and at this time, ecptoma growth among cell spheres, the amout of ecptoma is increased gradually; From micro, we can observe cell migrate along the ecptoma; if the distance between cell sphere is exceed 5mm, some neural stem cells that migrate first will proliferate in new condition and then made cell migrate again, so the distance of floating neural stem cell migrate is enhanced obviously. Conclusion We duplicate the tangent migration model in vitro and find EGF can induce neural stem cell migrate in special time.