摘要
目的观察α-synuclein过度表达诱导HEK293细胞α-synuclein蛋白病理性积聚的作用。方法将消除终止密码子α-synuclein基因扩增产物连入pGEMT-easy载体,DNA测序证实后亚克隆入增强型绿色荧光蛋白pEGFP-N1质粒,通过限制性内切酶酶切鉴定重组质粒。采用脂质2000将野生型α-synuclein-EGFP真核表达载体转染HEK293细胞,48h后采用Axiovert200型倒置荧光显微镜、荧光免疫细胞化学观察其在细胞内的表达,HE染色观察细胞胞浆内嗜酸性小体的形成。结果消除终止密码子的扩增序列经测序证实并亚克隆入pEGFP-N1质粒后,限制性内切酶酶切鉴定质粒大小、酶切位点上均符合实验设计。将野生型α-synuclein-EGFP真核表达载体转染HEK293细胞,48h后荧光显微镜下观察细胞在波长488nm蓝色激发光下发出明亮的绿色荧光,其中某些细胞出现绿色荧光物质积聚;免疫荧光细胞化学检测,EGFP阳性细胞同时也表现为a-synuclein免疫反应阳性,某些细胞胞浆内可见α-synuclein免疫阳性产物聚集;HE结果发现一些细胞胞浆内出现圆形的嗜酸性小体形成,约占细胞总数的10.8%。结论野生型α-synuclein基因过表达可诱导α-synuclein蛋白在HEK293细胞内积聚,胞浆内嗜酸性小体形成。
Objective To investigate over-expression of wild-type αv-synuclein inducing the aberrant aggregation of α-synuclein in HEK293 cell in vitro. Methods The cDNA encoding the human α-synuclein without the stop code was cloned into PGEM T-easy vector. Using enzyme map and DNA sequencing analyzed and determined the recombinant plasmid, and then sub-clone the α-synuclein cDNA fragment into pEGFP-N1 vector. The recombinant plasmids α-synucleinpEGFP were transfected into HEK293 cells by lipofectamin 2000. The aberrant aggregation of α-synuclein was measured by EGFP fluorescence, anti-α-synuclein immunocytochemistry. The inclusions in the cultured cells were identified with HE staining. Results The restriction enzyme map suggested that eukaryotic expression vector for htanan wild-type α- synuclein gene was constructed successfully. By EGFP fluorescence, anti-α-synuclein immunocytochemistry, it could be observed that the α-synuclein protein could aggregate in cytoplasm and the Lewy body-like inclusions found in cytoplasm of cultured cells. Conclusion The over-expression of wild-type α-synudein can induce protein aberrant aggregation and Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2006年第1期19-22,共4页
Chinese Journal of Medical Genetics
基金
国家863高科技研究发展计划项目(2004AA227040
2002BA711A07)
"十五"国家科技攻关计划项目(2004BA720A03)
国家自然科学基金项目(30370515)
高等学校博士学科点专项科研基金项目(20020533024)~~
