| Research BackgroundAlzheimer's syndrome is a progressive development of chronic neurodegenerative diseases,which mainly characterized by progressive memory impairment,cognitive dysfunction,and language disorders.It is the most common type of senile dementia.According to statistics,the incidence rate of Chinese people over 65 years old is 5.6%,and the elderly people over 85 years old is as high as 20%.At present,there are several hypotheses in medicine to explore the pathogenesis of AD,including the mitochondrial cascade hypothesis,the Tau protein hypothesis,the A? toxicity hypothesis,and the vascular hypothesis.The mitochondrial cascade hypothesis suggests that more reactive oxygen species(ROS)produced in cells can damage mitochondria and neurons.The Tau protein hypothesis suggests that Tau protein hyper-phosphorylation is one of the pathogenesis of AD.The A? toxicity hypothesis suggests that A? has neurotoxic,and forms A? senile plaques when accumulated in a large amount in cells,which promotes neuronal cell damage or death.The A? toxicity hypothesis has a certain correlation with the mitochondrial cascade hypothesis.The vascular hypothesis is that,due to the increase in age and various vascular diseases in the brain,insufficient oxygen supply in the brain is likely to cause AD.Although these hypotheses have certain theoretical support,they are not sufficient to fully explain the pathogenesis of AD.In recent years,the fiery research of long-chain non-coding RNA(lnc RNA)has pointed out another direction for the study of the pathogenesis of AD.There is evidence suggested that lnc RNAs such as BACE1-AS,51 A and NDM29 directly or indirectly increase the formation of A? and affect the ratio of A? x-42/x-40,which may be related to the pathogenesis of AD.Our study first constructed a cellular model of AD,in which SH-SY5 Y cells were induced by A?1-40,allowing cells to exhibit a more pronounced AD phenotype.Based on this,the aim of this study was to investigate the role of lnc RNA 17 A in AD phenotype cells and its potential regulatory mechanisms.Chapter OneObjective: To determine whether A?1-40 has an effect on the viability of SHSY5 Y cells,and secondly to detected the effect of A?1-40 on the expression of lnc RNA 17 A in SH-SY5 Y cells.Finally,to examined the effect of cell viability and apoptosis,autophagy,the expression of neuroectodermal stem cell marker Nestin,and the effects of migration and invasion after overexpression and knockdown lnc RNA 17 A combined with interfered with A?1-40.Methods: The m RNA expression of Tau and p-Tau was detected by q RT-PCR method to verify the successful construction of AD model cells.MTT assay was used to detect the activity of SH-SY5 Y cells induced by A?1-40.The knockdown and overexpression lnc RNA 17 A cell lines were constructed by transfection of si RNA and pc DNA3.1(+)-lnc RNA 17 A plasmid,respectively.q RT-PCR was used to detect the expression of lnc RNA 17 A m RNA in SH-SY5 Y cells induced by A?1-40.The cell apoptosis of different groups were detected by flow cytometry.Fluorescence technique was used to detect the autophagy marker LC3 B to detect the degree of autophagy and the expression of neuroectodermal stem cell marker Nestin.Finally,the changes of cell migration and invasion ability were detected by transwell assay.Results: 1.Compared with the control group,the expression of total Tau and pTau in A?1-40 treated SH-SY5 Y cells was significantly increased,indicating that the AD model cells were successfully constructed.A?1-40 can significantly inhibit cell viability in a dose-dependently manner.2.A?1-40 can induce the expression levels of lnc RNA 17 A in SH-SY5 Y cells.3.Overexpression of lnc RNA 17 A further inhibits the activity of SH-SY5 Y cells induced by A?1-40,promotes apoptosis,inhibits autophagy,inhibits the expression of intracellular neuroectodermal stem cell marker Nestin,and promotes cell migration and invasion.4.Knockdown of lnc RNA 17 A increases the activity of SH-SY5 Y cells induced by A?1-40,inhibits apoptosis,promotes autophagy,promotes expression of intracellular neuroectodermal stem cell marker Nestin,and inhibits cell migration and Invasion.Conclusion: A?1-40 can significantly inhibit cell activity,promote cell apoptosis,inhibit autophagy,inhibit the expression of endogenous neuroectodermal stem cell marker Nestin,and promote cell migration and invasion.Knockdown of lnc RNA 17 A increased cell activity of SH-SY5 Y cells induced by A?1-40,inhibits apoptosis,promotes autophagy,promotes expression of the neuroectodermal stem cell marker Nestin,and inhibits cell migration and invasion.Chapter TwoObjective: The purpose of this part was to investigate the effects of A?1-40 and lnc RNA 17 A on the activity of succinate dehydrogenase and Na+-K+-ATPase,as both are important markers of mitochondrial activity,so these two changes can reflect the effects of A?1-40 and lnc RNA 17 A on mitochondrial activity.Methods: Extract mitochondria from cells according to the instructions and determine the activity of succinate dehydrogenase and Na+-K+-ATPase.Results: 1.The activity of succinate dehydrogenase and Na+-K+-ATPase in A?1-40 group was significantly inhibited.2.After overexpression of lnc RNA 17 A,the succinate dehydrogenase and Na+-K+-ATPase of A?1-40-induced damage of SH-SY5 Y cells were further inhibited compared with A?1-40 group.3.After knockdown of lnc RNA 17 A,the succinate dehydrogenase and Na+-K+-ATPase activities of A?1-40-induced damage of SH-SY5 Y cells were increased compared with the A?1-40 group.Conclusion: A?1-40 can inhibit the activity of mitochondria in cells,but after knocking down lnc RNA 17 A,the enzyme activity of mitochondria in SH-SY5 Y cells induced by A?1-40 is restored.Chapter ThreeObjective: To investigate the effects of A?1-40 and lnc RNA 17 A on ROS,MMP,SOD and MDA in SH-SY5 Y cells.These substances indirectly indicate the degree of damage of mitochondria,which can reflect the response of cells to A?1-40 and lnc RNA 17 A.Methods: The changes of each component were detected by the corresponding kit in this section.Results: 1.A?1-40 can induce increase the content of intracellular ROS,MMP and MDA,while the content of SOD decreases.2.After overexpression of lnc RNA 17 A,the content of ROS,MMP and MDA in A?1-40-induced damage of SH-SY5 Y cells was further increased,while the content of SOD was further decreased.3.After knocking down lnc RNA 17 A,the content of ROS,MMP and MDA in A?1-40-induced damage of SH-SY5 Y cells decreased,while the content of SOD increased.Conclusion: A?1-40 can stimulate the increase of cellular reactive oxygen species and other factors,and activate oxidative stress to cause cell damage.After knockdown lnc RNA 17 A,all indicators improved,indicating that down-regulation of lnc RNA 17 A expression can improve some of the effects of A?1-40.Chapter FourObjective: To investigate the effect of A?1-40 on the expression of GABABR2 in SH-SY5 Y cells and to investigate whether the expression of lnc RNA 17 A can affect the expression of GABABR2.GABABR2 under different treatment conditions.Methods: q RT-PCR and western blotting were used to detect the expression ofResults: 1.Compared with the blank control group,the expression of GABABR2 protein and m RNA decreased after A?1-40 treatment.2.Compared with the A?1-40 group,the expression of GABABR2 protein and m RNA in A?1-40-induced damage of SH-SY5 Y cells was further decreased after overexpression of lnc RNA 17 A.3.Compared with the A?1-40 group,the expression of GABABR2 protein and m RNA in A?1-40-induced damage of SH-SY5 Y cells was increased after knockdown lnc RNA 17 A.Conclusion:A?1-40 can inhibit the expression of GABABR2 in cells.After knockdown lnc RNA 17 A,GAABABR2 expression increased,indicating that downregulation of lnc RNA 17 A expression can improve some of the effects of A?1-40. |
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