【作者】 李娜；

【导师】 顾平；

【作者基本信息】 河北医科大学 ， 神经病学， 2018， 硕士

【摘要】 目的:帕金森病(Parkinson’s disease,PD)是常发于中老年人的中枢神经系统变性疾病,主要临床表现为震颤、强直、运动弛缓、姿势平衡障碍等运动症状,此外,常伴发抑郁、焦虑、便秘、睡眠障碍、认知功能减退等非运动症状。自噬是依赖溶酶体途径对胞质蛋白和细胞器进行降解的一种过程,是细胞进行自我保护的一种重要机制,自噬发生在基础生理状态下,参与细胞的基本生理过程,但也可以由不同种类的应激所诱发,然而过度的自噬会导致机能紊乱。研究表明,自噬失调与帕金森病的发病机制有关。艾司西酞普兰(escitalopram,ESC)是新型的选择性5-羟色胺再摄取抑制剂(Selective Serotonin Reuptake Inhibitors,SSRIs),在临床用于广泛性焦虑、抑郁症的治疗,并有一定的神经保护作用,目前研究发现ESC可以在海马初级神经元细胞中激活泛素介导的自噬-溶酶体降解途。本研究旨在探讨ESC对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)处理的PC12细胞的保护作用及可能的作用机制。方法:根据研究目的将ESC(终浓度5μM、10μM、20μM、40μM、60μM、80μM)或/和MPTP(终浓度500μM、750μM、1000μM、1250μM、1500μM)加入到培养的PC12细胞中,以及在应用1m M的3-MA后通过MTS检测细胞存活率,Hoechst33258染色用荧光显微镜观察细胞凋亡情况,Western blot检测自噬标记物LC3B-Ⅱ、LC3B-Ⅰ和凋亡蛋白Bcl-2、Bax的表达情况。结果:1 MTS检测各组PC12细胞存活率1.1 MPTP对PC12细胞存活率的影响用500μM、750μM、1000μM、1250μM、1500μM的MPTP处理PC12细胞24h、48h、72h,PC12细胞的存活率呈现出剂量依赖性和时间依赖性。随着MPTP浓度的升高及时间的延长,PC12细胞存活率显著下降。1.2 ESC对PC12细胞存活率的影响用5μM、10μM、20μM、40μM、60μM、80μM的ESC处理PC12细胞,处理24h时,细胞存活率较CON组无统计学差异(P>0.05),虽然没有统计学差异,但可以看出细胞存活率有先上升后下降的趋势,在10μM时上升最明显。处理48h时,80μM的ESC对PC12细胞具有损伤作用,细胞存活率下降约14.3%,较CON组有统计学差异(P<0.05),其余浓度的ESC对PC12细胞存活率较CON组无统计学差异(P>0.05)。处理72h时,5μM、10μM和20μM的ESC增加细胞存活率,以10μM时增长最明显,增长约12%,60μM和80μM的ESC降低细胞存活率,分别下降了约18.7%和32.9%,与CON组比有统计学差异(P<0.05),40μM的ESC对PC12细胞存活率较CON组无显著差异(P>0.05)。所以大剂量的ESC也会引起细胞凋亡。1.3 ESC预处理对MPTP处理的PC12细胞存活率的影响用10μM、20μM的ESC预处理PC12细胞1h,再加1000μM的MPTP继续处理24h,以及5μM、10μM的ESC预处理PC12细胞1h,再加1000μM的MPTP继续处理72h,与MPTP组相比均能显著增加细胞存活率(P<0.05)。1.4自噬抑制剂3-MA对各组细胞存活率的影响ESC+MPTP+3-MA组细胞存活率(65.23±2.96%)较ESC+MPTP组细胞存活率(73.18±2.27%)降低,差异有统计学意义(P<0.05)。应用3-MA能显著抑制ESC预处理对PC12细胞的神经保护作用。2 Hoechst33258染色检测细胞凋亡情况Hoechst33258染色发现1000μM的MPTP组PC12细胞核明显固缩,细胞核呈现亮蓝色荧光。10μM的ECS组PC12细胞核较CON组未见明显改变,ESC预处理组较MPTP组能显著减少细胞核固缩的现象,ESC预处理可以降低细胞凋亡率,且差异有统计学意义(P<0.05)。3 Western blot检测LC3B-Ⅱ/Ⅰ及Bcl-2/Bax蛋白表达情况Western blot检测研究发现MPTP组LC3B-Ⅱ/Ⅰ水平明显增加,Bcl-2/Bax较CON组显著下降,10μM的ESC预处理后LC3B-Ⅱ/Ⅰ水平较MPTP组升高,而Bcl-2/Bax显著下降,应用自噬抑制剂3-MA后,LC3B-Ⅱ/Ⅰ水平和Bcl-2/Bax水平均出现下降。说明ESC通过PI3K/Akt/Bcl-2途径对MPTP处理的PC12细胞发挥神经保护作用。结论:1.一定浓度的ESC处理适宜的时间能够促进细胞存活。2.ESC预处理可以增加PC12细胞存活率,降低MPTP处理的PC12细胞的凋亡率,表明了ESC有一定的神经保护作用。3.ESC通过PI3K/Akt/Bcl-2途径对MPTP处理的PC12细胞发挥神经保护作用,自噬抑制剂3-MA抑制PI3K/Akt/Bcl-2途径,抑制保护作用,推测保护机制可能部分与细胞自噬有关。更多还原

【Abstract】 Objective: Parkinson’s disease(PD)is a degenerative disease of the central nervous system(CNS)that often occurs in middle-aged and elderly people,the main clinical manifestations are tremor,ankylosis,motor relaxation,postural balance disorder and other motor symptoms.In addition,it is often accompanied by some non-motor symptoms such as depression,anxiety,constipation,sleep disorders,cognitive impairment.Autophagy is a process that depends lysosomal pathway for degradation of cytoplasmic proteins and organelles,it is an important mechanism of self protection.Autophagy occurs in the basic physiological state and participates in the basic physiological process of the cell,but it can also be induced by different types of stress.However,excessive autophagy can lead to dysfunction.Studys have shown that autophagy disorder is associated with the pathogenesis PD.Escitalopram(ESC)is a selective serotonin reuptake inhibitor(SSRI),it is used in the treatment of generalized anxiety and depression,it also shows neuroprotective effect.Studys have shown that ESC can activate ubiquitin mediated autophagy lysosomal degradation pathway in hippocampal primary neuron cells.This study aimed to investigate the ESC on the1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-treated PC12 cells and its mechanism.Method: According to the purpose of the study,ESC(final concentration5μM,10μM,20μM,40μM,60μM,80μM)or / and MPTP(final concentration500μM,750μM,1000μM,1250μM,1500μM)were added to the PC12 cells.After 1m M 3-MA was used,MTS were used to detect cell viability,Hoechst33258 staining for the apoptosis,Western blot were used to detectthe expression of autophagy marker LC3B-Ⅰ,LC3B-Ⅱ and apoptotic protein Bcl-2,Bax.Result:1 MTS was used to detect the cell viability of PC12 cells.1.1 The effect of MPTP on the cell viability of PC12 cells.PC12 cell were treated with the MPTP 500μM,750μM,1000μM,1250μM,1500μM for 24 h,48h,72 h,the cell viability of PC12 cells showed a dose-dependent and time-dependent manner,with the increase of MPTP concentration and the prolongation of time,the cell viability of PC12 cells decreased significantly.1.2 The effect of ESC on the cell viability of PC12 cells.PC12 cells were treated with the ESC 5μM,10μM,20μM,40μM,60μM,80μM for 24 h,there was no statistical difference in cell viability compared with CON group(P>0.05).Although there is no statistical difference,we can see that the trend of cell survival is first rising and then decreasing,and the increase is most obvious at 10μM.For 48 h,80μM ESC had damaged the PC12 cells,and the cell viability decreased by about 14.3% compared to CON group,the difference was statistically significant(P<0.05).There was no significant difference in the cell viability of PC12 cells between the other concentration of ESC and CON group(P>0.05).For 72 h,5μM,10μM,20μM ESC increased cell viability,especially at 10μM,increased about 12%,while60μM,80μM ESC decreased cell viability,respectively by 18.7% 和 32.9%compared to CON group,the difference was statistically significant(P<0.05).40μM ESC was no statistical difference in cell viability compared with CON group(P>0.05).Therefore,large dose of ESC can also cause cell apoptosis.1.3 ESC pretreatment on the cell viability of PC12 cells.PC12 cells were pretreated with 10μM and 20μM ESC for 1h,followed by MPTP 1000μM for another 24 h,or 5μM and 10μM ESC for 1h,followed by MPTP 1000μM for another 72 h,the cell viability was significantly increased compared with MPTP group.1.4 Effects of autophagy inhibitor 3-MA on cell viability in each group.The autophagy inhibitor 3-MA could significantly inhibit the neuroprotective effect of ESC pretreatment on PC12 cells.The cell viability of ESC+MPTP+3-MA group(65.23 + 2.96%)was significantly different from ESC+MPTP group(73.18 + 2.27%)(P<0.05).2 Detection of cell apoptosis by Hoechst33258 staining.Hoechst33258 staining showed that the nucleus of PC12 in 1000μM MPTP group was obviously constricted,and the nucleus showed bright blue fluorescence.There was no obvious change in the nucleus of PC12 in 10μM ESC group compared with CON group.ESC pretreatment group could significantly reduce the rate of apoptosis compared with MPTP group,the difference was statistically significant(P<0.05).3 Western blot detection of the expression of LC3B-Ⅱ/Ⅰand Bcl-2/Bax protein.Western blot detection showed that the level of LC3B-Ⅱ/Ⅰ in MPTP group was significantly higher than that in CON group,after 10 μM ESC pretreatmentand,the level of LC3B-Ⅱ/Ⅰ higher than that in MPTP group,this phenomenon was reversed by the application of autophagy inhibitor3-MA,both the level of LC3B-Ⅱ/Ⅰ and Bcl-2/Bax were all decreased.Conclusion:1.The proper time of ESC treatment at a certain concentration can promote cell viability.2.ESC pretreatment can increase the cell viability of PC12 cells and decrease the apoptosis rate of PC12 cells treated with MPTP,which indicates that ESC has a certain neuroprotective effect.3.ESC plays a neuroprotective role on PC12 cells mediated by MPTP via PI3K/AKT/Bcl-2 pathway.Autophagy inhibitor 3-MA inhibits PI3K/AKT/Bcl-2 pathway and inhibits the protective effect.So we speculated that the protective mechanism may be partly related to autophagy.更多还原