小泛素样修饰蛋白-1对α-突触核蛋白基因突变或过表达诱导的细胞包涵体形成及细胞凋亡的影响

The effect of small ubiquitin-like modifier-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

目的探讨α-突触核蛋白（ocsynuclein）的小泛素样修饰蛋白（small ubiquitin-like modifier，SUMO）-1对α-synuelein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ccsynuclein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；48h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，AnnexinV-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ccsynuclein真核表达质粒转染HEK293细胞，48h后伊红染色显示转染野生型。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〉O．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48h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。synuelein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。

Objective To investigate the effect of small ubiquitin-like modifier（SUM（Yl） 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, 32.20% and 34.1% in cells transfeeted 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.