IGF-1激活20S蛋白酶体β5亚单位活性对N2a细胞活力的影响  

Effect of IGF-1 on N2a cell viability through activating 20S proteasome subunit beta type-5 activity

作　　者：王必慧[1] 曹静[1] 牛晓洁[1] 王婷玉 陆利[1] Wang Bihui;Cao Jing;Niu Xiaojie;Wang Tingyu;Lu Li(Department of Analomy,Shanxi Medical Universily,Talyuan 030001,China;The Scond Clinical School,Shanxi Medical Universily,Talyuan 030001,China)

机构地区：[1]山西医科大学人体解剂学教研室,太原030001 [2]山西医科大学第二临床医学院,太原030001

出　　处：《神经解剖学杂志》2020年第2期155-160,共6页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81571381);山西医科大学大学生创新项目(20160232)。

摘　　要：目的:探索胰岛素样生长因子(IGF-1)对蛋白酶体的作用,并观察其对小鼠神经母细胞瘤来源的N2a细胞活力的影响,寻找维持细胞活力的有效手段。方法:体外培养N2a细胞加入不同浓度的IGF-1后,分析N2a细胞肽基谷氨酰肽水解酶样(PGPH-L)、胰蛋白酶样(T-L)和糜蛋白酶样(CT-L)三种蛋白酶体活性的变化,real time RT-PCR比较分别具有PGPH-L活性、T-L活性和CT-L活性的20S蛋白酶体β1亚单位(PSMBI)、20S蛋白酶体β2亚单位(PSMB2)、205蛋白酶体β5亚单位(PSMB5)mRNA水平的变化。双荧光素酶实验检测IGF-1对PSMB5转录活性的影响。CCK8实验和Hoechst 3342/PI染色实验检测IGF-1和PSMB5对N2a细胞活性和凋亡的影响。结果:应用不同浓度的ICF-I干预N2a细胞后,PGPH-L活性和CT-L活性均显著增高,其中CT-L活性升高最显著,50 ng/ml和100 ng/ml ICF-1干预后,CT-L活性分别是对照组的1.32倍(P<0.05)和1.49倍(P<0.01),但是IGF-1干预后T-L活性无显著变化;real time RT-PCR结果显示应用100 ng/ml ICF-1作用后,PSMB5和PSMBI的mRNA表达水平显著上升(P<0.05);双荧光素酶结果显示IGF-1能够上调N2a细胞PSMB5转录活性;CCK8实验和Hoechst 3342/PI染色结果表明IGF-1干预使N2a细胞活力增强(P<0.01),调亡细胞减少;相反,敲低PSMBS后,N2a细胞活力减弱(P<0.05),调亡细胞增多;使用IGF-I干预PSMB5-siRNA组,发现PSMB5-iRNA组细胞活力较NC-siRNA组降低(P<0.05)、凋亡细胞增加。结论:ICF-1通过激活CT-L蛋白酶体活性,提高N2a细胞的活力。Objective.To crplore the ffct of ICF 1 on proteacome and viability of mouce neuroblactoma derived N2a cells,and to find the effective way 10 maintain the vitality of cells.Methods:After adding different concentrations of ICF-1 into N2a celle which wor crulured in ritrn,the changes nf the PCPH-I,T-I.and CT-1.protenlytir aetivity of N2a cells were analyzed.Subunitsβl,β2 andβ5 possess different proteolytic activities-PGPH-L,T-L,CT-L,respeetively.The mRNA levels of PSMBI(encoding theβl subunit),PSMB2(encoding theβ2 subunit)and PSMB5(encoding theβ5 subunit)were evaluated using real time RT-PCR.The ffect of IGF-1 on PSMB5 transeriptional activ-ity was examined by Dual-Luciferase assay.CCK-8 assay and Hoechst 3342/PI staining experiment were conducted to tost the efet nf 1CF-1 and PSMRS on the vitality and apoptosis of N2a cells.Results.Interfered hy different concentra-tions of IGF-1,PGPH-L and CT-L activities of N2a cells were increased significantly,especially CT-L activity of cells.The CT-L activity in 50 ng/ml and 100 ng/ml IGF-1 groups were 1.32 times(P<0.05)and 1.49 times(P<0.01)of the control group,respectively.However,the T-L activity of the IGF-1 group was no significant change.Real time RT-PCR results showed that PSMB5 and PSMBI mRNA expression increased significantly at 100 ng/ml IGF-1 interven-tion(P<0.05).The Dual-Luciferase assay showed that ICF-1 inereased the transcriptional activity of PSMB5 in N2a cells,similarly,CCK8 and Hoechs133342/PI staining showed that the vitality of N2a cells was increased(P<0.01)and the apoptosis was decreased in IGF-1 group.After knocking down PSMB5,the activity of N2a cells was weakened and apoptosis was increased.After ICF-I intervention in PSMB5-siRNA group,PSMB5-siRNA group showed deereased.cell viability(P<0.05)and increased cell apoptosis,suggesting that ICF-1 can promote the survival of N2a cells by activating PSMB5.Conclusion:ICF-1 enhances the viability of N2a cell by activating CT-L activity.

关 键 词：蛋白酶体 IGF-1 细胞活力 N2A细胞 

分 类 号：R741[医药卫生—神经病学与精神病学]