【摘要】 目的构建分泌表达神经保护短肽NAP的重组慢病毒载体,观察重组慢病毒对APPsw转基因人神经母细胞瘤细胞(SK-N-SH)的保护作用。方法采用PCR方法克隆融合基因NT4-NAP cDNA;磷酸钙沉淀法包装重组慢病毒Lent/NT4-NAP;平行包装含有报告基因EGFP的报告病毒;SK-N-SH细胞分为正常对照组、单纯APPsw转基因组、空慢病毒载体+APPsw转基因组和NT4-NAP慢病毒+APPsw转基因组,在倒置相差显微镜下观察形态学改变;MTT检测细胞增殖活力;AnnexinV/PI法流式细胞术检测各组细胞凋亡和坏死情况。结果基因测序证明克隆的NT4-NAP cDNA与实验设计完全一致;FACS检测重组病毒滴度为2×106U/L;单纯APPsw转基因组,大量细胞出现凋亡及坏死(与正常对照组比较P<0.01);NT4-NAP慢病毒+APPsw转基因组的细胞数量和活力明显较单纯APPsw转基因细胞改善(P<0.01);FACS检测无论凋亡还是坏死细胞的比率保护组都明显低于加害组(P<0.01)。结论成功构建了能够分泌表达NAP的重组慢病毒载体;重组病毒能够高效转染人神经母细胞瘤细胞;重组病毒表达的蛋白能对AD细胞模型起到很好的保护作用。更多还原

【Abstract】 Objective To construct the lentiviral vector expressing and secreting NAP (NAPVSIPQ), a neuroprotective short peptide, and to observe its protective effect on amyloid precursor protein gene (APPsw) transgene SK-N-SH. Methods The cDNA encoding NT4-NAP was cloned by PCR. The recombinant lentiviral Lent/NT4-NAP was packaged by calcium phosphate precipitation. The report virus Lent/EGFP was constructed by the same way. SK-N-SH cells were divided into normal control group, APPsw transfected group, lentiviral vector+APPsw group, NT4-NAP+APPsw group to observe the morphological changes under inverted phase contrast microscope. The cellular proliferation activity and apoptosis were detected by MTT and Annexin V/PI flow cytometry respectively.Results The cloned NT4-NAP cDNA was fully coincidence with the designed experiment verified by gene sequencing. The titer of Lent/EGFP was 2×106 U/L detected by FACS. APPsw exhibited cytotoxicity effects and apoptosis and necrosis in Alzheimer disease (AD) cellular model (compare with those in control group, P<0.01). The cellular number and activity in NT4-NAP Lent+APPsw group were improved than those in APPsw group ( P<0.01). The Lent/NT4-NAP significantly decreased neurotoxicity of Aβ and the rate of apoptosis and necrosis in vitro(P<0.01).Conclusions The recombinant Lent/NT4-NAP secreting NAP is successfully constructed, which could transfect effectively SK-N-SH cells. The protein expressed by recombinant Lent/NT4-NAP could play the protective role in AD cellular model.更多还原