[The wild-type alpha-synuclein over-expression to induce the protein aberrant aggregation of alpha-synuclein in HEK293 cells in vitro].
OBJECTIVE
To investigate over-expression of wild-type alpha-synuclein inducing the aberrant aggregation of alpha-synuclein in HEK293 cell in vitro.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Chen
Tao
T
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R.China.
Tang
Bei-sha
BS
Liao
Xiao-ping
XP
Yan
Xin-xiang
XX
Zhang
Ru-xu
RX
Zhang
Yu-hu
YH
Tang
Jian-guang
JG
Cao
Li
L
Guo
Ji-feng
JF
Li
Jing
J
D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D002479:Inclusion Bodies / Q000378:metabolism; D016631:Lewy Bodies / Q000378:metabolism*; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism*; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
D051844:alpha-Synuclein