Chen Tao, Tang Bei-sha, Liao Xiao-ping, Yan Xin-xiang, Zhang Ru-xu, Zhang Yu-hu, Tang Jian-guang, Cao Li, Guo Ji-feng, Li Jing
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R.China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2006 Feb;23(1):19-22.
To investigate over-expression of wild-type alpha-synuclein inducing the aberrant aggregation of alpha-synuclein in HEK293 cell in vitro.
The cDNA encoding the human alpha-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 alpha-synuclein cDNA fragment into pEGFP-N1 vector. The recombinant plasmids alpha-synuclein-pEGFP were transfected into HEK293 cells by lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence, anti-alpha-synuclein immunocytochemistry. The inclusions in the cultured cells were identified with HE staining.
The restriction enzyme map suggested that eukaryotic expression vector for human wild-type alpha-synuclein gene was constructed successfully. By EGFP fluorescence, anti-alpha-synuclein immunocytochemistry, it could be observed that the alpha-synuclein protein could aggregate in cytoplasm and the Lewy body-like inclusions found in cytoplasm of cultured cells.
The over-expression of wild-type alpha-synuclein can induce protein aberrant aggregation and Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro.
研究野生型α-突触核蛋白过表达在体外诱导HEK293细胞中α-突触核蛋白异常聚集的情况。
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。通过酶切图谱和DNA测序分析并确定重组质粒，然后将α-突触核蛋白cDNA片段亚克隆到pEGFP-N1载体中。用脂质体2000将重组质粒α-突触核蛋白-pEGFP转染到HEK293细胞中。通过EGFP荧光、抗α-突触核蛋白免疫细胞化学检测α-突触核蛋白的异常聚集。用苏木精-伊红染色鉴定培养细胞中的包涵体。
酶切图谱表明成功构建了人野生型α-突触核蛋白基因的真核表达载体。通过EGFP荧光、抗α-突触核蛋白免疫细胞化学观察到，α-突触核蛋白可在细胞质中聚集，且在培养细胞的细胞质中发现了路易体样包涵体。
野生型α-突触核蛋白过表达可在体外诱导HEK293细胞细胞质中蛋白质异常聚集和路易体样包涵体形成。
