PS1 Modulating Autophagy Of The Model Of AD Induced By β Amyloid In PC12 Cells

The two major pathological features of Alzheimer’s disease are extracellular beta-amyloid abnormal sedimentary formation of senile plaque and intracellular excessive phosphorylated Tau proteins,however,its mechanism is not fully clear.Autophagy in neurodegenerative diseases,including AD,plays a central role in the process of development.Failures of autophagy formations and the fusion of lysosome and antophagosome are the two performance of autophagy in AD.In the early stages of AD,abnormal function of APP and A beta protein triggers the increased autophagy vesicles,protection of cells,autophagy activation can promote gamma-secretase compounds from the connotation of the body or endoplasmic reticulum moved to autophagy body membrane,Aβ continues to increase in autophagy vesicles,Aβfurther lead to increased autophagy activity,The production of Aβ is more than the degradation of Aβ,the accumulation of intracellular Aβ further destroys the autophagy system,further cause Aβ abnormal deposition,cause the swelling of synapses,loss of function,eventually lead to neuronal dysfunction induced cell death,form A vicious circle.To solve this problem,our study used A mixture of Aβ42 and Aβ40 oligomers,A model of AD induced by Aβ in PC12 cells in vitro,which can imitate the pathological process of patients with AD,and discuss the mechanism of autophagy functions,the result of successful AD early cell model is established,by adjusting the quantity of Aβ42 in the mixture,and the proportion of Aβ40,found that Aβ42 does not cause the damage of cells in the certain concentration range,based on the mechanism of autophagy activation,provides A new clinical early prevention and treatment of AD.The general research content are as follows:1.The model of Alzheimer’s disease induced by Aβ in PC12 cells Objective: Establishing the model of Alzheimer’s disease,which can imitate the pathological process of AD in vivo.Methods: Using different proportion of Aβ42 with Aβ40 oligomers in PC12 cells.CD spectrum analysis the structures of Aβ42.CCK 8 detect cell survival,and flow cytometry detect ell apoptosis.Western blot detect expression of apoptosis related protein of Caspase 3.Results: CD spectra analysis found that the quantity of toxicity structure of layer structure is highest after 24 hours of aggregation of Aβ.CCK 8 detection found that,in the certain concentration range,as Aβ42 / Aβ40 ratio increased,the activity of cells increased.Flow cytometry measure cell apoptosis rate has no obvious change,Western blot detecting apoptosis related proteins of Caspase 3 have no obvious change.Conclusion: The successful construction of the model of AD in PC12 cells.2.The reaearch of PS1 induced by Aβ in PC12 cells.Objective: To explore the change of PS1 induced by different proportion of Aβ42with Aβ40 in PC12 cells.Methods: Using ELISA method to detect the contents of Aβ42 protein,Western blot detection the expression of PS1,immunohistochemical method to detect the expression of PS1.Results: The method of ELISA test results that in the certain concentration range,at the phase of the same quantity of the total oligomers,when the ratio of Aβ42 / Aβ40happened micro changes,the expression of Aβ42 increased significantly,the greater of the ratio of Aβ42/ Aβ40,the expression quantity of Aβ42 is higher,when the content of Aβ42 is same,just the pure Aβ42 oligomers,than the mixture with A Aβ40caused Aβ42 accumulation,and has A dose dependent.Western blot detect the expression of PS1:found that:,the different proportion of Aβ42/ Aβ40 in the oligomers,the expression of PS1 had no statistical difference.When the content of Aβ42 were same,whether it is simply formation of Aβ42,or a mixture with Aβ40there is no difference of the expression of PS1.Conclusion:1.Microchanges of Aβ42/ Aβ40 in oligomers can cause significant buildup of Aβ42,The abnormal deposition of Aβ is much related with the percentage of Aβ42,and has a dose dependent.Aβ40 can inhibit the abnormal deposition of Aβ,protecting cells from injury.2.The abnormal deposition of Aβ induced by Aβ42 through the changes of the structure and function of PS1,and don’t change the content of intracellular PS1.3.PS1 modulating autophage induced by Aβ in PC12 cells.Objective: To explore autophagy induced by Aβ in PC12 cells.Methods: Western blot method to detect the expression of autophagy related proteins of Beclin 1 and p62,immunohistochemical detecting the expression of autophagy related proteins Beclin 1 and p62.Immunofluorescence microscopy detect the accumulations of autophagosome.Results: Western blot method to detect the expression of autophagy related protein of Beclin 1 and p62,the results showed that:in the certain concentration range,the micro changes of Aβ42 / Aβ40 can itroduc the much changes of the expression of Beclin 1,shows that Aβ42 can activate autophagy,and cause the strength of autophagy is associated with the ratio of Aβ42,has a dose dependent.When the ratio of Aβ42 increases to A certain concentration,the expression of Beclin 1 significantly reduced,autophagy is restrained,suggests the activation of autophagy induced by Aβ42 effectively within the certain concentration range,when the concentration is too large,cell autophagy is abate,cause cellular damage.In the mixture of oligomers,when the ratio of Aβ42 / Aβ40 changes,the expression of p62 is different.When the quantity of Aβ oligomers is certain,with the proportion of Aβ42 increasing,the expression of p62 is reduced,statistically significant difference,suggests the activation of autophagy is associated with the ratio of Aβ42,the greater of the ratio,the greater of the autophagy.When the content of Aβ42 is same,Aβ 42 / Aβ40 ratio is different,the expression of p62,shows that the strength of the activation of autophagy is associated with the ratio of Aβ42,and has a dose dependent.Immunohistochemical staining results shown the positive cells of Beclin 1 and p62 in the cytoplasm is pale yellow to brown granules.Immunofluorescence autophagosome accumulation within the cell,and as the ratio of Aβ42/ Aβ40,autophagosome to accumulate.Conclusion:1.In a certain concentration range,the expression of related autophagy protein of Beclin1 increased,autophagy is activated,when the concentration of Aβ42 reach to 10umol/L,the expression of Beclin1 decreased,autophagy is inhibited.2.In a certain concentration range,the expression of p62 decreased,autophagy is activated.3,Aβ42 inducing autophagy occurs with Aβ42 was significantly associated with the ratio of Aβ42/ Aβ40,with A dose dependent.4.The different expression of PS1,Beclin 1,p62 in PC12 cells induced by Aβ and 3-MAObjective: To study the autophagy in PC12 cells induced by Aβ and 3-MA.Methods: Western blot method to detect the expressions of PS1,autophagy related protein Beclin 1 and p62,immunohistochemical detect the expression and positioning of PS1,autophagy related protein Beclin 1 and p62,immunofluorescence microscopy detect the accumulations of autophagosome.Results: Western blot test detect the expression of PS1 in PC12 cells induce by Aβand 3-MA.Inducing by 3-MA,the expression of PS1 decreased,the higher of the ratio of Aβ42/ Aβ40the more obvious the decreases of PS1.When the quantity of Aβoligomers is certain,Aβ42 for 4 umol/L,the expression of PS1 drops obviously,when Aβ42 is 10 umol/L,the more significant different of the expression of PS1,This industrates that the autophagy inhibitor 3-MA can reduce the expression of PS1,and when Aβ42/ Aβ40 increasing,PS1 expression to reduce more obvious.When the content of Aβ42 is same,the ratio of Aβ42 increases,the low expression of PS1 tend to be more significant,the difference has statistical significance,and has a dose dependent.After 3-MA stimulation,the expression of Beclin1 increase,and the greater of the ratio of Aβ42/ Aβ40,the greater the expression of Beclin1 significantly.When the quantity of total oligomers is certain,as A beta 42 / A beta ratio 40 small changes,thhe micro changes of Aβ42/ Aβ40,can inducing the expression of Beclin1 increasing significantly,it induce that Aβ42 can activate the expression of Beclin 1.When the content of Aβ42 is certain,the groups icduce by 3-MA,the expression of Beclin 1 increased.At the small proportion of Aβ42,the increase of the expression of Beclin 1 has no obvious difference,as Aβ42 ratio increasing,the expression of Beclin 1 increased significantly,and is associated with Aβ42 / Aβ40 ratio.Western blot test showed that 3-MA reduced the expression of p62.When the quantity of the total oligomers is certain,the changes of Aβ 42/Aβ40 ratio affect the p62 has no significant difference.The group of 10 umol/L of Aβ42 set down is not obvious compared with other groups.Therefore,Aβ42 can stimulate autophage.Immunofluorescence autophagosome accumulation within the cell,and as the ratio of Aβ42/ Aβ40,autophagosome to accumulate.Conclusion:1.In the certain concentration range,3-MA increased the expression of autophagy related protein of Beclin 1,decreasing the expression of p62.2.3-MA decreased the expression of PS1.3.The mechanism of Aβ42 activating autophagy is related to the failure of autophagy-lysosome function modulated by PS1.