摘要
目的观察人载脂蛋白E(apoE)在神经母细胞瘤细胞SK-N-SH内表达的分布情况及对细胞生长的影响。方法构建含apoEcDNA和绿色荧光蛋白(GFP)基因的真核表达载体,转染至SK-N-SH细胞,经G418结合荧光动态监测筛选稳定表达人apoE基因的细胞克隆,荧光显微镜下观察GFP-apoE融合蛋白在SK-N-SH细胞中的表达。采用MTT法和流式细胞术观察apoE3、apoE4对细胞增殖和细胞周期的影响。结果经酶切和DNA序列测定证实目的基因已插入重组质粒,经G418筛选得到的SK-N-SH细胞克隆中存在apoE基因表达,其表达分布与GFP不同,呈点状分布。转染apoE3的细胞增殖速度加快,G0G1期细胞比例降低,S期细胞增加;而转染apoE4的细胞则相反,表现为增殖速度减缓,G0G1期细胞比例增加,S期细胞减少。结论神经母细胞SK-N-SH细胞内apoE的表达对其生长具有表型特异性。通过建立稳定表达不同多态性的apoE的细胞克隆,为进一步研究apoE在神经细胞中的生物学功能以及apoE在阿尔茨海默病发病机制中的作用奠定了基础。
Objective To inve human neuroblastoma SK-N-SH cDNA fragments of the full lengt pEGFP/apoE4, were tr detected by fluorescence expression on growth fe ansfected stigate the subcellular localization of human apolipoprotein E （apoE） in and the isoform-specific effects on the survival of SK-N SH. Methods h apoE fused with green fluorescent protein （GFP） gene, pEGFP/apoE3 into SK-N-SH stably. The expression of GFP/apoE fusion proteins microscopy atures and MTT me cell analysis and sequencing showed that cycle the tar thods and flow progression of get gene was cytometry were used to measure the effects of a the the The cells. Results Enzyme digestion cloned into recombinant vector correctly expression of GFP-apoE fusion protein in the SK-N-SH cells was identified and the localization of the protein was different from GFP. This fusion protein was localized mainly in the endosome/lysosome nuc cells as detected by fluorescence microscopy. Cells transfected with apoE3 resulting in increased proliferatio Stable fusion leus of n rate, a percentage decrease of cell populations at GOG1 phases and increase of cell populations at S phases. In contrast, the proliferation GOG1 phases an rate of cells transfected with apoE4 were markedly reduced, along with increased populations at d decreased at S phases. Conclusions Intracellular expression of apoE exerted isoform-specific effects on the growth of SK-N-SH ceils. The positive SK-N-SH cell expressing human apoE gene stably may be a promising cell model for studying the biological function of the apoE gene and the role of apoE in the pathogenesis of Alzheimer disease.
出处
《中国神经免疫学和神经病学杂志》
CAS
2008年第6期442-445,476,共5页
Chinese Journal of Neuroimmunology and Neurology
基金
南京医科大学科技发展基金重点资助项目(06NMUZ007)
