Isolation And Culture Of Human Embryo Neural Stem Cells And The Study Of Inducing Nsc Differentiate Into Dopaminergic Neurons

Background: Stem cell technique has gained great progress in recent years, it has become the hot spot of biology and medical domain in the world. In 1999 and 2000, the investigation of stem cell had continuously been commented the most important investigation domain by 《Science》.Stem cell investigation provide a ideal study model for the earlier development of embryo ,the differentiation of cell and constitution, the expression and regulation of gene. At the same time, it also hews out a well prospect for the clinical cellular therapy and constitution engineering of homo sapiens diseases. In recent years, the rapid development of the study of NSC had open up a new path way for the therapy of central nervous system diseases. Parkinson's disease (PD) is the most common neurodegenerative disorder among the old people, which affects approximately 1% of the age of 50 years and seriously affects the health of individuals. Thereby, considering the growing number of the elderly, PD forming an increasing economic burden for society. Pathologically, PD experience a progressive loss of dopaminergic neurons within the sustantia nigra and a reduction of dopamine in the striatum. There is no effective treatment up to date. Though dopamine replacement therapy using the dopamine precursor, levodopa or dopamine receptor agonists can alleviate symptoms, they could not mitigate progression of the disease. Fetal transplantation is a new therapeuticstrategy for PD. Several studies have shown that fetal dopaminergic transplants in an animal model of PD ameliorate deficits induced by lesioning the dopaminergic nigrostriatal neurons. Clinical trials of human fetal transplantation as a therapy for PD have been attempted in several centers with encouraging results. However, widespread application is severely limited by the moral and practical problems of obtaining sufficient donor tissue from human embryos, the longterm therapeutic effect is inconsistent, and the rejection in host brain is also can not be neglected. Recently, some researchers attempted to treat PD by combining gene transfer and brain transplantation; Some reseachers tried to induce embryonic stem cells to differentiate into neural stem cells(NSC) and induce NSC to differentiate into dopaminergic neurons. These cells could be used as a cell replacement material. Although more experiments have been done, there is no breakthrough finding in this field. At present, the related studies are main about rodent NSC .Theory study must service clinical application, on the base of this starting point, we proceed this study and intended to find out a good way to induce NSC to differentiate into dopaminergic neurons.Objective: To establish a system for isolating and cultivating human embryo neural stem cells, on this base, we go further to study the biology character of the NSC and to explore a suitable method for the induction of NSC into the dopaminergic neurons, so we can provide enough donator for cell transplant of PD. Since CNS stem cells have an extended self-renewal capacity and are multipotential,their isolation from human tissue would provide a renewable source of neurons, astrocytes, and oligodendrocytes,significantly reducing the need for fetal tissue in experimental and clinical neural transplantation.Material and Methods:part one: Isolating and cultivating human embryo CNS hippocampus neural stem cells by the technique of serum free cultivation combined with growth factor(EGF and bFGF).The hippocampus tissue was taken from the clinicle aborted foetus(gestational age range from 8~16 week). After the NSChad grown into neurosphere, we passaged and freezed it. The multipotential of NSC was also be certified by immunocytochemistry method, the special antibodies matching neural stem cell(Nestin),neuron(NSE),astrocyte(GFAP) and oligodendrocyte(GC) were applied to identify the primary > the passaged neural stem cells and the three cell types differented from NSC in vitro.Part two: To compare the differentiation result of NSC between the groups incubated in serum-free medium with different additional components. The proportions of neuron and glial cells of each group was compared. After the NSC having differentiated for 5 days NesthuNSE^ GFAP and GC were stained by the method of immunocytochemistry, then the percentage of various kinds of cells were calculated and the statistics analysis was proceeded. According to the difference of additional components in basic medium(DMEM/F-12+B27),the cells were divided into five groups:A used basic medium only;B added with 5% fetal bovine serum;C added with human embryo striatal extracts(50ul/ml);D added with IL-6(20ng/ml).Part three: To innducing human embryo NSC differentiate into dopaminergic neurons.The inducer we used were:?human embryo striatal extracts;(2)Vc;(3)IL-l(ou p); (4)The NSC going to be induced were divided into five groups: group A was the control group(no inducer in the basic medium); group B added with human embryo striatal extracts(50ul /ml);C added with Vc (20ug/ml) ;D added with IL-l(ou |3) (both were lOOpg/ml); E added with all of the above inducers simultaneously. At the 7th day after the differentiation, the cells growed on the cover slip were taken out of the cell culture plate for immunocytochemistry identification. The anti-TH antibody labeled with fluorescent second antibody was used in immunocytochemistry identification. The TH positive cells(dopaminergic neurons) will be be stained, the percentage of dopaminergic neurons will be caculated in each group. The expression of TH mRNA also had been evaluated by the method of RT-PCR, at last statistics analysis was proceeded to contrast the induction results of each group. Results:part one: The results of immunocytochemistry showed.either the primary or the passaged neurosphere were nestin-positive, the results of immunocytochemistry the cells differentiated from them expressed NSE> GFAPand GC.Part two: NSE+ rates of group D were highest, averaged to 29.72%. There was significant difference between D and other groups (P<0.05) . Although NSE+ rates of group C were higher than A, there was no significant difference between them (P>0.05) . NSE+rates of group B were lower than A, there was significant difference between them (P<0.05 ) .GFAP+ rates of group B (66.52±2.87%)was higher than other groups, there was significant difference between them (P<0.05) . but there was no significant difference between the other groups.There was no significant difference of all the groups about GC+ rates.Part three: TH+ cells rates of group E was highest, averaged to 26.72±0.54%, there was significant difference (P<0.05) . TH+ cells rates of group B> C and D were also higher than A(the control group), there was significant difference between them (PO.05) . TH+ cells rates of group C was higher than group B and D, there was also significant difference between them (P<0.05) .The results of RT-PCR showed: The expression of TH-mRNA was not obviously in the control group, but each of the induce groups has a strong expression of TH-mRNA, and the expression of TH-mRNA in the E group was strongest. The tite of OD of each group has been analysised by image analysis system and been given statistical analysis, there was significant difference between A group and BXnD>E group(P<0.05), there was significant difference between E group and other groups also (P<0.05) . From stronger to feeble, The sequence about the expression intensity of TH-mRNA was E, C, D, B, A in order.Conclusion:part one:. We have established an effective method for culturing human embryo neural stem cells in vitro.The primary cultured and passaged human...