Construction Of Screening Technique Promoting Neuro-differentiation Induced By Active Compounds And Correlation Research On Neuro-differentiation And Expression Of Tubulins

Chapter one:Construction of screening technique which according to the activity of promoting P19 embryonal carcinoma differentiate into neuron-like cells.1.Application research of P19 embryonal carcinoma stablely transfected by Tubulin alphaâ… to promote differentiation of neuron-like cells.Objective:Establishment of P19 embryonal carcinoma stablely transfected by Tubulin alphaâ… , to construct cell model which induced the differentiation of neuron-like cells in vitro,in order to screen active compounds in high performance.Method:Drawing assistance from basic ion liposome transfection,stablely transfect Tα1-PGL4.17[luc2/Neo]Vector into P19 cells,to go through the screening of G418,transfer of culture positive clone.When neuro-differentiation to promote the expression of Neo,detecting fluorescence with luciferase kit,fluorescence intensity compare with the quantity of neuron-like cells is direct ratio.Control by DMSO,RA different concentration induce the differentiation of neuron-like cells.After 2 days adherence culture in the 96-wells plates,we detect the fluorescence with luciferase kit,overview whether the transfection cells could be used to screen the activity of considerable compound.Result:The differentiated phenotype after adherence culture RA compare with DMSO and blank is much better.But experimental data is not stable,the fluorescent numerical value of the cells which not be stablely transfected is zero;but RA compare with DMSO and blank is lower,and to follow the increasing of adherence culture time,fluorescence have the tendency of degrade gradually.Conclusion: Establishment of P19 embryonal carcinoma stablely transfected by Tubulin alphaâ… initially,but using it to screen active compounds is not ideal enough,so,we should establish much better estimated system.2.Application researeh of P19 cells FM1-43FX fluorescent staining method used for evaluating differentiation of neuron-like cells.Objective:Exploring P19 cells FM1-43FX fluorescent staining method used for the differentiation of neuron-like cells,in order to construct the cell model of inducing the differentiation of neuron-like cells used for screening compounds in high performance.Method:According to FM1-43FX fluorescent dye could combine to cell membrane,through high kalium ion inducing nerve cell action potential,following Ves reuptake process into the cells,so,we can use it staining nerve cell specially,fluorescence intensity compare with the quantity of neuron-like cells differentiating from P19 cells is direct ratio.Cells differentiated by RA was used as positive control group,while Cells differentiated by DMSO was used as negative control group,constructing the estimated method refering to estimate the activity of compounds inducing the differentiation of neuron-like cells.To install 6 different concentration in the 96-wells plates,adherent culture 2,4,6 and 8 days,to part explore the best time and concentration of RA.In addition,MTT assay estimate cell viability in order to avoid false positive.Result:After P19 cells adherent culture 4 days,the fluorescence intensity between DMSO and RA exist statistics disparity,but can't be observed conspicuous neurite in phase-contrast microscope.After adherent culture 6 days,RA group we can observe neuron-like cells,but FM1-43FX fluorescent staining image can't be displayed completely. After adherent culture 6 days,not also the fluorescence intensity increasing,but also we can observe conspicuous neurite in phase-contrast microscope and fluorescent staining image,to hint can be used for screening.Conclusion:FM1-43FX fluorescent staining method could be used for staining neuron-like cells specially.After P19 cells adherent culture 8 days,the fluorescence intensity is the largest,and the fluorescence value with one accord to the phenotype of neuron-like cells.Our research successfully construct primary screening method which can induce neurogenesis in high performance.Chapter two:Active screening of molecules in flavanoids library inducing neurogenesis.1.P19 cells fluorescent staining method used for primary screen for neurogenesis.Objective:To direct differentiate Embryonal carcinoma cells(P19 cells) into neuron-like cells in vitro.Use fluorescent staining method,MTT assays,to screen 30 flavanoids.Methods: flavanoids were divided into three groups(a,b,c).Every group contains 10 compounds.Cells differentiated by RA(5×10^-7mol/L) was used as positive control group,while Cells differentiated by DMSO was used as negative control group.P19 cells were cultured in medium with the density of 1×10⁵ cells/ml.We added candidate compounds in the suspension culture medium for 4 days. Suspension medium was 90%α-MEM and 10%fetal bovine serum.Collect aggregate embryonic bodys and trysined EBs with 0.25%trypsinase.Then culture the cells with the density of 1×10⁶ cells/ml in the 96-wells plates.The medium was 95%α-MEM and 5%bovine serum.Change the medium every 2 or 3 days.Use FM1-43FX fluorescent staining method to evaluate.MTT assay was used to evaluate cell proliferation rate to avoid false positive results.Results:1a,4a,5a,3'a,4'a,5'a,3b,5c could induce cells to neuron-like cells,fluorescence intensity have statistics disparity(P＜0.05,P＜0.01).MTT assay indicates that these 8 compounds didn't suppress cell proliferation.Conclusion:(1) The Phenotype of induced cells was coninsistence with fluorescence intensity.(2) 1a,4a,5a,3'a,4'a,5'a,3b,5c could induce P19 cells into neuron-like cells,and they didn't have cytotoxicity.(3)a' group compounds to show 8 position substitution structure-function relationship, following the prolong of 8 position substitution,fluorescence intensity is increased,to hint the direct ratio relationship between the carbon atom quantity of 8 position substitution and the activity of neuro-differentiation.2.Embryonic stem cells used for phenotypic screen for neurogenesisObjective:Using the classical EB 4-/4+ method,to further demonstrate the neurogenesis activity of active compound in primary screen,in order to discover promote neural regeneration lead compound.Methods:To go through primary screen,8 compounds have positive result,according to EB 4-/4+ method further evaluate the effect of inducing ES cells differentiate into neuron-like cells. Phase-contrast microscope initially evaluate neuron which have neurite and cell body,when the length of neurite is more than 3 times comparing to the length of cell body,we see it as neuron.Then, we use Immunocytochemical staining and flow cytometry to confirm the positive cells.Results:To go through the analysis of phase-contrast microscope and Immunocytochemical staining,8 compounds have positive result in primary screen could promote ES cellsβ-Tubulinâ…¢positive expression,among compound 4a have the most effect;2 compounds 1c,5'c have no activity in primary screen almost noβ-Tubulinâ…¢positive expression.The result of ES cells Immunocytochemical staining essential anastomosis with P19 cells screening result,flow cytometry to show:The positive result of negative control group DMSO is altus a little,but compare to positive control group RA have certain disparity,The result of compound essential anastomosis with Immunocytochemical staining.Conclusion:(1) To go through secondary screen on ES cells, compounds 4a,5c have better effect of neurogenesis,but compound 5'c almost have no effect.(2) Analysis the structure-function relationship of compound to show:1,The liposolubility is larger,the activity of neurogenesis is stronger;2,The replacement of 4'will to weaken the activity of neurogenesis.Chapter three:Correlation research between the differentiation of neuron-like cells induced by flavanoids and the expression of tubulins. Objective:The research is try to find whether the compound 4a could induce ES cells to differentiate to neuron-like cells by up-regulating the cystoskeleton proteins specially expressed by neuron cell:microtubule-associated protein2(MAP2),neurofilament protein(NEFM),β-Tubutinâ…¢.Method:Using the classical EB 4-/4+ method which could simulate the embryonic development in vivo.The ES cells form the embryonic bodies,then adherence and differentiate to neuron-like cells.Western blot could evaluate the level of expression of MAP2,NEFM andβ-Tubulinâ…¢as the indicatrix of the promotion of ES cells to neuron cells by drugs.Result:in the negative control group,DMSO and positive control group,RA,the expression of this three proteins are time dependent.In the group of compound 4a,NEFM andβ-Tubulinâ…¢show the same tendency of time dependence,while MAP2 didn't show a definite tendency.Conclusion:compound 4a may promote the differentiation of ES cells to neuron-like cells by up-regulating the cystoskeleton proteins: NEFM andβ-Tubulinâ…¢.