Chen Tao, Liao Xiao-ping, Wen Guo-qiang, Long Zhi-gang, Ouyang Feng, Deng Yi-dong, Guo Min
Department of Neurology, Hainan Provincial People's Hospital, Haikou, Hainan, People's Republic of China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2011 Oct;28(5):511-6. doi: 10.3760/cma.j.issn.1003-9406.2011.05.008.
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 alpha-synuclein.
cDNA encoding the human alpha-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 alpha-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.
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 alpha-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 alpha-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).
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 alpha-synuclein.
研究小泛素样修饰物（SUMO-1）修饰对α-突触核蛋白过表达及突变诱导的HEK293细胞胞质中路易体样包涵体形成及细胞凋亡的影响。
将编码无终止密码子的人α-突触核蛋白的cDNA克隆到pGEM T-easy载体中。进行限制性酶切图谱分析和DNA测序以分析质粒，然后将其亚克隆到pEGFP-N1载体中。通过脂质体转染法将重组质粒α-突触核蛋白-pEGFP转染到HEK293细胞中。用苏木精-伊红染色鉴定培养细胞中的包涵体。通过Hoechst 33258染色、MTT法和膜联蛋白V-PE流式细胞术检测HEK293细胞的凋亡情况。
在培养细胞的胞质中发现了路易体样包涵体。Hoechst染色显示，转染后48小时，野生型和A53T突变组细胞的细胞核增大，染色质聚集并呈点状。K96R和K96R-A53T组细胞核染色均匀。MTT法检测显示，转染空质粒的细胞活力为96.2%，而转染野生型α-突触核蛋白-pEGFP和A53T突变组的细胞活力分别降至53.4%和56.1%。转染K96R和K96R-A53T的细胞活力分别为72.3%和69.8%（P<0.05）。转染后48小时，空质粒组的凋亡率为3.9%，转染野生型和突变型α-突触核蛋白-pEGFP的细胞凋亡率分别为32.2%和34.1%，转染K96R和K96R-A53T的细胞凋亡率分别为19.4%和20.3%。两组间差异有统计学意义（P<0.05）。
SUMO-1修饰对体外培养的HEK293细胞胞质中路易体样包涵体的形成无影响，但具有毒性作用，可增加野生型α-突触核蛋白过表达及突变诱导的细胞凋亡。
