CAJ | 학술논문

目的探讨α-突触核蛋白(α-synuclein)的小泛素样修饰蛋白(small ubiquitin-like modifier,SUMO)-1对α-synuclein基因过表达或突变诱导的细胞包涵体形成及细胞凋亡的影响.方法构建野生型(WT)、A53T突变型和缺失SUMO-1互作氨基酸的K96R突变型、K96R-A53T突变型α-synuclein真核表达质粒.应用脂质体介导转染方式将构建好的质粒转入HEK293细胞；48 h后Hoechst 33258染色,采用Axiovert200型倒置荧光显微镜观察对细胞的影响,应用四甲基偶氮唑盐检测细胞活力,Annexin V-PE流式细胞仪检测细胞凋亡.结果将构建所得真核表达质粒经双酶切鉴定及DNA测序证实；将WT型、A53T型、K96R型、K96R-A53T型α-synuclein真核表达质粒转染HEK293细胞,48 h后伊红染色显示转染野生型α-synuclein- pEGFP组及A53T突变型组某些细胞胞浆内出现圆形的嗜酸性小体(类Lewy小体)形成,分别占细胞总数的9.4％和11.7％,转染K96R及K96R-A53T融合表达载体组细胞内嗜酸性小体形成比率为10.2％和9.8％,两两相比差异无统计学意义(P＞0.05).Hoechst染色结果显示,WT组、A53T组细胞核变大,出现着色不均,染色质聚集成斑点状,未见核浓缩或核碎裂.K96R组及K96R-A53T组胞核着色基本均匀.采用四甲基偶氮唑盐法结果显示转染空质粒组细胞活力为96.2％,转染WT组、A53T组细胞活力下降至53.4％及56.1％,转染K96R组及K96R-A53T组细胞活力为72.3％和69.8％；转染48 h后,WT组、A53T组细胞凋亡率分别为32.2％和34.1％,转染K96R组及K96R-A53T突变组细胞凋亡率下降为19.4％和20.3％,两两相比差异有统计学意义(P＜0.05).结论 SUMO-1对α-synuclein基因过度表达及α-synuclein基因致病突变A53T诱导的细胞包涵体的形成无明显影响；SUMO-1可加强α-synuclein基因过度表达及α-synuclein基因致病突变A53T诱导的细胞毒性及细胞凋亡,提示SUMO-1参与了细胞凋亡过程.
목적 탐토α-돌촉핵단백(α-synuclein)적소범소양수식단백(small ubiquitin-like modifier,SUMO)-1대α-synuclein기인과표체혹돌변유도적세포포함체형성급세포조망적영향.방법 구건야생형(WT)、A53T돌변형화결실SUMO-1호작안기산적K96R돌변형、K96R-A53T돌변형α-synuclein진핵표체질립.응용지질체개도전염방식장구건호적질립전입HEK293세포；48 h후Hoechst 33258염색,채용Axiovert200형도치형광현미경관찰대세포적영향,응용사갑기우담서염검측세포활력,Annexin V-PE류식세포의검측세포조망.결과 장구건소득진핵표체질립경쌍매절감정급DNA측서증실；장WT형、A53T형、K96R형、K96R-A53T형α-synuclein진핵표체질립전염HEK293세포,48 h후이홍염색현시전염야생형α-synuclein- pEGFP조급A53T돌변형조모사세포포장내출현원형적기산성소체(류Lewy소체)형성,분별점세포총수적9.4％화11.7％,전염K96R급K96R-A53T융합표체재체조세포내기산성소체형성비솔위10.2％화9.8％,량량상비차이무통계학의의(P＞0.05).Hoechst염색결과현시,WT조、A53T조세포핵변대,출현착색불균,염색질취집성반점상,미견핵농축혹핵쇄렬.K96R조급K96R-A53T조포핵착색기본균균.채용사갑기우담서염법결과현시전염공질립조세포활력위96.2％,전염WT조、A53T조세포활력하강지53.4％급56.1％,전염K96R조급K96R-A53T조세포활력위72.3％화69.8％；전염48 h후,WT조、A53T조세포조망솔분별위32.2％화34.1％,전염K96R조급K96R-A53T돌변조세포조망솔하강위19.4％화20.3％,량량상비차이유통계학의의(P＜0.05).결론 SUMO-1대α-synuclein기인과도표체급α-synuclein기인치병돌변A53T유도적세포포함체적형성무명현영향；SUMO-1가가강α-synuclein기인과도표체급α-synuclein기인치병돌변A53T유도적세포독성급세포조망,제시SUMO-1삼여료세포조망과정.
Objective To investigate the effect of small ubiquitin-like modifier(SUMO-1) modification on the formation of Lewy body-like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of α-synuclein.Methods cDNA encoding the human α-synuclein without the stop codon was cloned into a pGEM T-easy vector.Restriction enzyme mapping and DNA sequencing were performed to analyze the plasmid,which was then subcloned into a pEGFP-N1 vector.The recombinant plasmid α-synuclein-pEGFP was transfected into HEK293 cells by lipofectamin method.Inclusions in the cultured cells were identified with HE staining.Apoptosis of the HEK293 cell was measured by Hoechst 33258 staining,MTT and Annexin V-PE flow cytometry.Results The Lewy body-like inclusions were found in cytoplasm of cultured cells.Hoechst staining showed that the nuclei of cells were enlarged in the wild-type and A53T mutation groups 48 h after transfection,chromatin were accumulated and appeared spot-like.The nucleus stain was equitable in the K96R and K96R-A53T groups.MTT assay showed that the viability of cells transfected with empty plasmid was 96.2％,but it dropped to 53.4％ and 56.1％ in cells transfected with wild-type α-synuclein-pEGFP and A53T mutant group,respectively.The viability was 72.3％ and 69.8％ in cells transfected with K96R and K96R-A53T,respectively (P＜0.05).Forty-eight hours after transfection,the apoptosis rate was 3.9％ in empty plasmid group,32.2％ and 34.1％ in cells transfected with wild-type and mutant α-synuclein-pEGFP,19.4％ and 20.3％ in the K96R and K96R-A53T transfected cells.There was significant difference between the two groups (P＜ 0.05).Conclusion SUMO-1 modification did not have influence on the Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro,but had a toxic effect which could increase the apoptosis induced by wild-type overexpression and mutation of α-synuclein.