Effect And Mechanism On Neural Progenitor Cells Differentiation And Synapse By Astrocyte-conditioned Medium In AD Cell Model

ObjectivesTo set up the Alzheimer's Disease( AD) cell model with Aβ_1-40 - induced PC12 cells apoptosis.MethodsPC12 cells which were induced by Aβ_1-40 with different concentration were devided into four groups ransdomly: control group, 5ug/ml Aβ_1-40 group, 10ug/ml Aβ_1-40 group, 20ug/ml Aβ_1-40 group. PC12 cells in each group were incubated with Aβ_1-40 at different times. Cell survival rates was detected by MTT assay .nucleus morphous by acridine orange fluorescence staining,apoptosis rates by flow cytometry and ultrastructures analyzed by transmission electron microscopy.ResultsAfter PC12 incubated with Aβ_1-40, typical change of apoptosis morphology were seen. Cell survival rates and apoptosis rates showed dose - dependent and time -dependent . At 6h, apoptosis rates and cell death rates of PC12 cells induced by 10ug/ml Aβ_1-40 were higher and lower.ConlusionFrom time of apoptosis high - peak and cell death rates , Typical apoptosisof PC12 cells induced by lOug/mlAPj _A0 were used as a good Alzheimerss Disease (AD) cell model.SECOND: Changs of neurotrophins gene byastrocyte and BDNF protein in astrocyte - conditiondemedium in AD cell modelAbstract ObjectiveTo observe the change of neurotrophins gene expressed by astrocyte in AD cell model with Af^.^ -induced PC12 cells apoptosis,including BDNFmRNA, NGFmRNA and NT - 3mRNA. To detect the change of BDNF protein in astrocyte - conditioned medium in AD cell model.MethodAfter removing the Afi,,^ medium,10 ug/ml Afjj..^ -induced PC 12 cellswere divided into two sections. Cell apoptosis rates of one section were measured by Flowcytomerry. Another section were incubated with primary cultured astro-cytes from cortical tissues of newborn Rats. The change of BDNF mRNAxNGF mRNA and NT - 3 mRNA were detected by RT - PCR . The astrocyte cell apoptosis rates were measured by Flowcytometry. BDNF protein was measured by ELASA.ResultsRT - PCR: BDNFmRNA difference between A group and B group wasn 1 statistically significant (P > 0. 05 ). Contrast with other groups , BDNFmRNA in C3 group was remarkable, difference being statistically significant (P<0.05). NGFmRNA was expressed in each group, but the differences among all groups werent statistically significant ( P > 0. 05 ). NT - 3 mRNA wasnt seen in every group. ELASA: Contrast with other intergroups , BDNF heigh amplitude/d between C2 group C3 group was remarkable, difference being statistically significant (P <0.05). Contrast with A group,B group,Cl group and C2 group, BDNF total protein/2d in C3 group was remarkable, the difference being statistically significant ( P < 0. 05 ). But differences among C3 group, C4 group and C5 group werent statistically significant (P >0.05).CondussionRefering to the results of PC 12 cell apoptosis rates in the study, we supposed that the up - regulation of BDNFmRNA and BDNFprotein by astrocyye were related to A.^l₄Q - induced PC12 cells apoptosis , which was neurotrophy and repair mechanism of astrocyte in AD. The astrocy from mainly expressed BDNFmRNA, but no change of NGFmRNA and no expression of NT -3mRNA . We thought that astrocyte from newborn Rats cortical tissue selectively expressed neurotrophins genes after incubating with PC 12 cells which were injuried by A(3, .^ neurotoxicity.THIRD: Effect and mechanism on neural progenitor cellsdifferentiation and synapse by astrocyte - conditionedmedium in AD cell modelAbstract ObjectivesTo observe the neuron differentiatin g rates , synapse, the expression of two kinds of synaptic proteins and Trk B of neural progenitor cells induced by astrocyte - conditioned medium in AD cell model. To investigate whether ERK signal transduction pathway was involved in this process.MethodsNeural progenitor cells from embryonic rats cortical tissue were induced by astrocyte - conditioned medium ( A group C5 group) in last experiment mixed with DMEM/F12 medium according to the proportion of 1:3. The induced cells were detected and counted by immunofuorescent techenique. The expression of synaptophysin and growth — associated protein — 43 were detected by Laser co -focalization scanning microscope. Maturate synapse number was observed by transmission electron microscope (TEM). The expression of TrkB , p - ERK and ERK were detected by Western Blot.ResultsContrast with I group [ ACM + NPCgroup ] , II group [ ACM ( PC 12) + NPC-group] , I group [ ACM ( Aï¿¡ - PC12 Oh) + NPC group ] and IV group [ ACM (Aï¿¡ -PC12 4h) +NPC group ] and Wgroup(NPC group) .neuron differentiation rates, synaptophysin and growth - associated protein - 43 , maturate synapse number, the expression of TrkB , p - ERK in Vgroup [ ACM ( A(3 - PC 126h) + NPC group] were remarkable,difference being stastically significant! P < 0. 05). The differences of above indexes among V group [ ACM (A(3 - PC12 6h) + NPC group] .VIgroup [AC M( A(3 -PC12 12h) +NPC group] and VII group [ ACM( Ap - PC 12 24h) + NPC group] werent stasticallly significant (P > 0. 05).ConclusionIn AD cell model, astrocyte from newborn rat cortical tissue conduced to induce the neurons differentiation of and synapse formation of neural progenitor cells. Moreover, BDNF -Trk B - ERK signal transduction were involved in this process which increased the possibility of neural stem cell therapy in AD.