嘌呤霉素敏感的氨肽酶对β-淀粉样肽诱导神经细胞凋亡的影响

发布时间：2018-02-28 21:30

本文关键词： 嘌呤霉素敏感的氨肽酶(PSA/NPEPPS) 神经细胞 Aβ25-35 细胞凋亡 caspase-3　出处：《山东大学》2013年硕士论文　论文类型：学位论文

【摘要】：背景阿尔茨海默病(Alzheimer's Disease,AD),又称老年痴呆,是老年人群痴呆的最常见、最重要的类型。临床主要表现为进行性的记忆力减退,认知功能障碍等症状。其患病率随年龄增加而增高,随着我国及世界人口结构日益趋向老龄化,AD的发病率逐年上升,严重危害老年人的身心健康和生活质量,给病人造成巨大的痛苦,给家庭和社会带来沉重的负担,已成为严重的社会问题,引起了普遍关注。阿尔茨海默病发病机制尚不明确,目前对其治疗也没有行之有效的方法。研究显示疾病的发生与其病理学改变有关,其特征性病理改变主要表现为：神经元细胞内神经纤维缠结(neurofibrillary tangles,NFT)、细胞外老年斑或神经炎性斑(senile plaque,SP)的形成及神经细胞的过度凋亡。神经纤维缠结主要由高度磷酸化的Tau蛋白沉积所致：老年斑则主要是由β淀粉样肽(βamyloid peptide, Aβ)沉积引起的,并进一步引起神经细胞凋亡嘌呤霉素敏感的氨肽酶(puromycin-sensitive aminopeptidase PSA/NPEPPS)是一种广泛分布的金属氨肽酶,在脑中含量很丰富。Cons tam等研究发现PSA具有蛋白水解作用,在有丝分裂期与纺锤体微管的形成有关,并且PSA的抑制剂能引起细胞的凋亡。此外PSA对于细胞信号转导、蛋白质转运、主要组织相容性复合物I(MHC I)的处理、细胞周期调节、胚胎发育都有重要作用。近年来研究发现PSA可以降解Tau蛋白,改善AD的症状,为以Tau蛋白为靶标治疗AD提供了新思路——Tau蛋白降解剂。作为AD病理学改变之一的Aβ沉积,与Tau蛋白异常过度磷酸化及神经细胞的凋亡密切相关,但是Aβ与PSA之间的关系却很少有人研究。本研究试图探讨Aβ诱导的神经细胞凋亡与PSA之间的关系。目的在SH-SY5Y细胞内沉默PSA表达、PC12细胞内上调PSA表达的基础上,加入Aβ25-35诱导细胞凋亡,研究PSA对Aβ诱导的神经细胞凋亡的影响,进一步探讨PSA作为一种潜在的AD治疗药物的分子机制。方法 1.采用MTT法、Hoechst染色检测不同浓度(0、10、20、30μmol/L)的Aβ25-35对SH-SY5Y、PC12细胞的影响：MTT法检测对细胞生长的影响；Hoechst染色检测对细胞核形态的影响,从而确定最佳浓度的Aβ25-35来诱导细胞的凋亡。 2.采用脂质体转染法在SH-SY5Y细胞中瞬时转染PSA-siRNA,在PC12细胞中瞬时转染PSA重组质粒,转染24h后,加20μmol/L的Aβ25-35处理细胞。SH-SY5Y细胞分组为：A：NC组,B:NC+Aβ25-35组,C:PSA-siRNA组,D:PSA-siRNA+Aβ25-35组； PC12细胞分组为： E：空质粒转染组,F：空质粒+Aβ25-35组,G：PSA过表达组,H：PSA过表达+Aβ25-35组。加入Aβ25-35作用24h后收集各组细胞,(a)进行流式细胞术检测细胞凋亡率；(b)Western blot检测PSA、caspase-3蛋白的表达情况；(c)酶标仪检测caspase-3活性。结果 1.Aβ25-35抑制SH-SY5Y细胞、PC12细胞增殖,细胞核发生细胞凋亡的形态学改变,确定20μmol/L的Aβ25-35作用24h进行实验； 2.在SH-SY5Y细胞中,沉默PSA表达能使Aβ25-35诱导的细胞凋亡率升高,促进caspase-3酶原激活,caspase-3活性升高。反之,在PC12细胞过表达PSA能使Aβ25-35诱导的细胞凋亡率下降,抑制caspase-3酶原激活,caspase一3活性降低。结论 Aβ25-35能抑制神经细胞生长,引起神经细胞凋亡,最佳作用浓度为20μmol/L:PSA能抑制Aβ25-35诱导的神经细胞凋亡,对神经元具有保护作用,其机制可能与抑制caspase-3通路的激活有关。
[Abstract]:Background Alzheimer ' s disease ( AD ) , also known as senile dementia , is the most common and most important type of dementia in the elderly . The prevalence of AD increases with age . With the aging of the population structure of our country and the world , the incidence of AD rises year by year . The pathogenesis of Alzheimer ' s disease is not clear , and there is no effective method for the treatment of Alzheimer ' s disease . The study shows that the occurrence of the disease is related to its pathological changes . The characteristic pathological changes are mainly caused by the formation of neurofibrotangles ( NFT ) , extracellular senile plaques or neuroinflammatory plaques ( SP ) and the excessive apoptosis of nerve cells . Puromycin - sensitive PSA / NPEPPS is widely distributed in the brain . It has been found that PSA has proteolytic action , which is related to the formation of spindle microtubules during mitosis . In recent years , it has been found that PSA can degrade Tau protein and improve AD . In recent years , it has been found that PSA can degrade Tau protein and improve AD . Purpose To investigate the effect of PSA on the apoptosis of PC12 cells induced by A尾25 - 35 and to further study the molecular mechanism of PSA as a potential drug for AD treatment . method 1 . The effects of A尾25 - 35 of different concentrations ( 0 , 10 , 20 , 30 渭mol / L ) on cell growth were determined by MTT assay . 2 . The PSA - siRNA was transiently transfected into the cells by liposome transfection . The cells were transiently transfected with PSA - siRNA in PC12 cells . After 24 h of transfection , the cells were treated with 20 渭mol / L of A尾25 - 35 . The groups were : A : NC group , B : NC + A尾25 - 35 group , C : PSA - siRNA group , D : PSA - siRNA + A尾25 - 35 group ; PC12 cells were grouped into E : empty plasmid transfection group , F : empty plasmid + A尾25 - 35 group , G : PSA overexpression group , H : PSA overexpression + A尾25 - 35 group . After 24 h of the addition of A尾25 - 35 , group cells were collected , ( a ) flow cytometry was performed to detect cell apoptosis rate ; ( b ) Western blot was used to detect the expression of PSA and caspase - 3 protein ; ( c ) caspase - 3 activity was detected by microplate reader . Results 1.A尾25 - 35 inhibited the proliferation of PC12 cells , the morphological changes of cell apoptosis in PC12 cells , and determined that 20 渭mol / L of A尾25 - 35 was used for 24h . 2 . The apoptosis rate of A尾25 - 35 induced by A尾25 - 35 was increased and caspase - 3 activity was increased . On the contrary , the apoptosis rate induced by A尾25 - 35 was decreased and caspase - 3 activity was inhibited and caspase - 3 activity was decreased in PC12 cells . Conclusion A尾25 - 35 can inhibit the growth of nerve cells , induce apoptosis of nerve cells , and the optimal concentration is 20 渭mol / L : PSA can inhibit the apoptosis of nerve cells induced by A尾25 - 35 . It has protective effect on neurons , and its mechanism may be related to the inhibition of caspase - 3 pathway activation .

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

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