Role Of Nuclear Factor Kappa B In The Proliferation Of Neural Stem Cells

ObjectiveIschemic cerebrovascular disease and brain injury have become one of the main dangerous for the mankind health and lives. The main pathological lesions is the losses and the injury of neuron,which causes the losses of neural function.But in 1990s of last century, the discoveries of neural neogenesis and neural stem cells broght the hope for repairing the central nervous system damage. Neural stem cells have the capacity of self-renewal and multi-differentiation. In Many pathological conditions, the endogenous neural stem cells have the capacity of proliferation to migrate and to differentiate into multiple cell types such as glial cells and neurons, in order to repair the central nervous system damage.But the research find that the capacity of proliferation is limited. and easily differentiate into glial cells..The surrounding plays a crucial role in control of neural stem cell proliferation and differentiation. Thus,how to regulate neural stem cell proliferation and the direction of differentiation is the problem needs to solve for the neural stem cells applys to treat the central nervous system diseases. Recent studies find that nuclear factor-kappa B signal pathway is correlated closely with the generation and developing in the central nervous system diseases. and likely participate regulating the neural stem cell proliferation and differentiation. However, the mechanism is not clear.In the experiments, we observed that the nuclear factor-kappa B signal pathway activating stimuli such as tumor necrosis factor and the repressors such as PDTC have effect on the neural stem cells proliferative situation so as to approach the Role of nuclear factor kappa B in the proliferation of neural stem cells.In same, we explored the effects of lanthanum chloride on the neural stem cells proliferative situation so as to furtherly know the redulation mechanisms of the proliferation and differentiation of neural stem cells.And offer the experimental rationale that the transplant of neural stem cells treat central nervous system disease.Methods1.Isolation,culture and identification of mouse neural stem cells: Mouse neural stem cells were cultured by Neural stem cell suspension method to the third passage,and identified by immunofluorescence with nestin.2.Experiment group:NSCs applied to experiment were the third passage cells and were randomly divided into the control group,TNFgroup and PDTCgroup,which were cultured in common NSCs medium , in NSCs medium supplemented with 10ng/ml TNF-αand in NSCs medium supplemented with 0.1mM PDTC for thirty minuters,four hours ,seventy-two hours, respectively. Anothor Lanthanum chloride group were cultured in NSCs medium supplemented with 2.5μmol/L LaCl3.3.Observation index and method:To detect NF-κB activity situationin of neural stem cells (that is distribution change of p65)by Immunocytochemistry and Western Blotting technique. To detect the related cell proliferation index (including CyclinD1 and Ki-67) by Immunocytochemistry and observe the proliferation situation of the neural stem cells by neurosphere counting and neurosphere volume measurement.Results1.Growth situation and identification findings of mouse neural stem cells:In primary culture, the cells proliferated into the clusters and continued to increase in size, forming small, uniform neurosphere after 2 days incubation, they will detach from the surface and floated in suspension. 5 to 7 days later, neurospheres reach a diameter hindering sufficient central nutrient supply, passaging of cells is required. Immunofluorescence analysis showed that a large number of nestin positive cells resided in neurospheres.2. NF-κB activity in neural stem cells: Immunofluorescence analysis showed that TNF group remarkblely activated the translocation of NF-κB /p65 from cytoplasm to nuclear ,while PDTC group did not occurre and expressed p65 in cytoplasm. Western Blotting analysis showed that a higher expression of p65 in TNF group compared with control group, while the P65 protein content in PDTC group is mostly none.The findings shows that TNF activates NF-κB in mouse neural stem cells.3 The proliferation situation of Neural stem cell: neurosphere counting shows that TNFgroup and 2.5μmol/L Lanthanum chloride group were 74.56±2.6 piece/bot and 62.32±2.8 piece/bot ,significantly increased more than control group (42.28±3.5 piece/bot),P<0.05;and neurosphere volume measurement shows that TNFgroup and 2.5μmol/L Lanthanum chloride group were(0.82±0.16)×106(μm3)and(0.66±0.12)×10(6μm3), significantly increased larger than control group(0.26±0.08)×10(6μm3),P<0.05. Immunofluorescence analysis showed that the percentage of CyclinD1 positive cells were(68.6±4.2)% and (38.8±3.7)%in TNF group and control group were remarkblely higher than PDTC group,which was (24.2±2.8)%,p<0.01.while the percentage of ki-67 positive cells were(48.2±3.6)% and (35.2±4.7)%in TNF group and control group were remarkblely higher than PDTC group,which was (18.7±1.8)%,P<0.01。The upper results show that TNF activates NF-κB in mouse neural stem cells and significantly activated NSC proliferation,while PDTC inhibits NF-κB in mouse neural stem cells and significantly inhibited NSC proliferation 2.5μmol/L LaCl3.can promote the proliferation of mouse neural stem cells.Conclusions1. The activation or inhibition of the NF-κB can activate or inhibit neural stem cells proliferation and NF-κB is a crucial regulator of NSCs proliferation2. 2.5μmol/L Lanthanum chloride can activate neural stem cells proliferation...