哈尔滨商业大学学报（自然科学版）
哈爾濱商業大學學報（自然科學版）
합이빈상업대학학보（자연과학판）
JOURNAL OF HARBIN UNIVERSITY OF COMMERCE(NATURAL SCIENCES EDITION)
2015年
1期
4-8
,共5页
王可心%李煦照%徐红英%张宁%刘树民
王可心%李煦照%徐紅英%張寧%劉樹民
왕가심%리후조%서홍영%장저%류수민
a-突触核蛋白%慢病毒表达载体%人神经元母细胞瘤细胞%帕金森病
a-突觸覈蛋白%慢病毒錶達載體%人神經元母細胞瘤細胞%帕金森病
a-돌촉핵단백%만병독표체재체%인신경원모세포류세포%파금삼병
a-Synuclein%lentiviral expression vector%human neuroblastoma cell neurons%Parkinson’ s disease
为检测a－突触核蛋白（a－Synuclein）在人神经元母细胞瘤细胞（SH－SY5Y）中的表达，构建人野生型（WT）和A53T突变型a－Synuclein质粒（pEGFP－SNCA－WT和pEGFP－SNCA－A53T），经酶切鉴定无误后转染SH－SY5Y细胞，并对转染完成的细胞进行嘌呤霉素筛选，然后通过PCR法扩增转染后SH－SY5Y细胞的a－Synuclein片段，并对其进行测序，检测目的基因，运用Western blot法检测转染后SH－SY5Y细胞中a－Synuclein的表达．酶切鉴定结果表明pEGFP－SNCA－WT和pEGFP－SNCA－A53T质粒构建成功，嘌呤霉素筛选和转基因细胞a－Synuclein片段测序结果显示慢病毒表达载体能成功的整合到SH－SY5Y细胞基因组中，Western blot结果表明，转染后的SH－SY5Y细胞能成功的过表达a－Synuclein．a－Synuclein慢病毒表达载体构建成功，并且SH－SY5Y细胞作为宿主细胞能够表达a－Synuclein．为今后帕金森病（ PD）体外模型的建立及帕金森病发病机制的研究奠定了基础．
為檢測a－突觸覈蛋白（a－Synuclein）在人神經元母細胞瘤細胞（SH－SY5Y）中的錶達，構建人野生型（WT）和A53T突變型a－Synuclein質粒（pEGFP－SNCA－WT和pEGFP－SNCA－A53T），經酶切鑒定無誤後轉染SH－SY5Y細胞，併對轉染完成的細胞進行嘌呤黴素篩選，然後通過PCR法擴增轉染後SH－SY5Y細胞的a－Synuclein片段，併對其進行測序，檢測目的基因，運用Western blot法檢測轉染後SH－SY5Y細胞中a－Synuclein的錶達．酶切鑒定結果錶明pEGFP－SNCA－WT和pEGFP－SNCA－A53T質粒構建成功，嘌呤黴素篩選和轉基因細胞a－Synuclein片段測序結果顯示慢病毒錶達載體能成功的整閤到SH－SY5Y細胞基因組中，Western blot結果錶明，轉染後的SH－SY5Y細胞能成功的過錶達a－Synuclein．a－Synuclein慢病毒錶達載體構建成功，併且SH－SY5Y細胞作為宿主細胞能夠錶達a－Synuclein．為今後帕金森病（ PD）體外模型的建立及帕金森病髮病機製的研究奠定瞭基礎．
위검측a－돌촉핵단백（a－Synuclein）재인신경원모세포류세포（SH－SY5Y）중적표체，구건인야생형（WT）화A53T돌변형a－Synuclein질립（pEGFP－SNCA－WT화pEGFP－SNCA－A53T），경매절감정무오후전염SH－SY5Y세포，병대전염완성적세포진행표령매소사선，연후통과PCR법확증전염후SH－SY5Y세포적a－Synuclein편단，병대기진행측서，검측목적기인，운용Western blot법검측전염후SH－SY5Y세포중a－Synuclein적표체．매절감정결과표명pEGFP－SNCA－WT화pEGFP－SNCA－A53T질립구건성공，표령매소사선화전기인세포a－Synuclein편단측서결과현시만병독표체재체능성공적정합도SH－SY5Y세포기인조중，Western blot결과표명，전염후적SH－SY5Y세포능성공적과표체a－Synuclein．a－Synuclein만병독표체재체구건성공，병차SH－SY5Y세포작위숙주세포능구표체a－Synuclein．위금후파금삼병（ PD）체외모형적건립급파금삼병발병궤제적연구전정료기출．
To detect the expression of α-Synuclein in SH-SY5Y cells, the Wild-Type ( WT) or A53T mutantα-Synuclein lentiviral expression vector ( pEGFP-SNCA-WT and pEGFP-SNCA-A53T) was constructed, the orientation of which was confirmed by restric-tion analysis and was transfected into SHSY5Y cells, respectively.The individual stably transfected colony was subsequently selected in the presence of puromycin, and the gene from transfected SHSY5Y cells was PCR amplified and confirmed by restriction analysis and DNA sequencing.Restriction endonuclease results showed that pEGFP-SNCA -WT and pEGFP-SNCA-A53T plasmids were constructed successfully.The results from antibiotic selection and DNA sequencing showed that the lentiviral vector can be integrated into the ge-nome of SH-SY5Y cell successfully.Western blot assay showed that a-Synuclein could be overexpressed in the transfected SHSY5Y cells successfully.Wild-Type or A53T mutant a -Synuclein lentiviral expression vector was constructed successfully and SH-SY5Y can ex-press a-Synuclein as host cells.This paper lay the foundation for the establishment of Par-kinson’ s disease ( PD) model in vitro and the research on the pathogenesis of PD.
