Fang Bo-yan, Jia Jian-ping
Department of Neurology, Xuanwu Hospital, Capital University of Medical Sciences, Beijing 100053, China.
Zhonghua Yi Xue Za Zhi. 2007 Jan 30;87(5):336-40.
To study the effects of two novel presenilin-1 (PS1) mutations, V97L and A136G, discovered in 2 families of Alzheimer's disease (AD) in China, on human neuroblastoma cells and the protective role of insulin-like growth factor-1 (IGF-1).
Two mutation type (MT) PS-1 genes with the mutational sites V97L and A136G were established. These 2 MT genes, wild type (WT) PS-1 gene, and mock were stably transfected into the human neuroblastoma cells of the line SH-SY5Y. The 4 kinds of transfected cells were divided into 3 subgroups: (1) control subgroup, cultured in normal medium with serum, (2) serum deprivation subgroup, cultured in the medium without serum, and (3) serum deprivation + IGF-1 subgroup, cultured in serum deprivation medium plus IGF-1. Twenty-four hours later MTT colorimetry was used to calculate the cell survival rate. Hoechst33258 method and phosphatidylinositol/annexin V-FITC double staining flow cytometry were used to observe the cell apoptosis. The glucose uptake was measured. Western blotting was used to analyze the protein expression of glucose transporter protein (GLUT)-1 in the cell membrane.
Different tests all showed that the cell survival rates of the V97L and A136G serum deprivation subgroups were both significantly lower than those of the corresponding WT and Mock subgroups (P < 0.01, P < 0.05), the cell survival rates of the all serum deprivation + IGF-1 subgroups were significantly higher than those of the corresponding serum deprivation subgroups (all P < 0.01); The glucose uptake rate was not significantly different among different subgroups, but IGF-1 addition made the glucose uptake increase by about 25% (all P > 0.05). The GLUT1 expression in the cell membrane of the serum deprivation + IGF-I subgroups increased by 15% - 20% respectively, however, without significant differences.
Enhanced sensitivity to trophic withdrawal in the cells with the two Chinese PS1 mutations most likely contributes to the neuron loss in AD. The sensitivity to apoptosis is not caused by impaired glucose metabolism, but IGF-1 still can protect cells from serum deprivation that compensates the negative effect through enhancing glucose transport and mitochondria activities.
研究在中国两个阿尔茨海默病（AD）家系中发现的两种新型早老素-1（PS1）突变V97L和A136G对人神经母细胞瘤细胞的影响以及胰岛素样生长因子-1（IGF-1）的保护作用。
构建具有V97L和A136G突变位点的两种突变型（MT）PS-1基因。将这两种MT基因、野生型（WT）PS-1基因和空载体稳定转染至SH-SY5Y人神经母细胞瘤细胞系。将4种转染细胞分为3个亚组：（1）对照亚组，在含血清的正常培养基中培养；（2）血清剥夺亚组，在无血清培养基中培养；（3）血清剥夺+IGF-1亚组，在血清剥夺培养基中加IGF-1培养。24小时后，采用MTT比色法计算细胞存活率。采用Hoechst33258法和磷脂酰肌醇/膜联蛋白V-FITC双染流式细胞术观察细胞凋亡情况。检测葡萄糖摄取量。采用蛋白质印迹法分析细胞膜上葡萄糖转运蛋白（GLUT）-1的蛋白表达。
不同检测均显示，V97L和A136G血清剥夺亚组的细胞存活率均显著低于相应的WT和空载体亚组（P<0.01，P<0.05），所有血清剥夺+IGF-1亚组的细胞存活率均显著高于相应的血清剥夺亚组（均P<0.01）；不同亚组间葡萄糖摄取率差异无统计学意义，但添加IGF-1使葡萄糖摄取量增加约25%（均P>0.05）。血清剥夺+IGF-I亚组细胞膜上GLUT1表达分别增加15%-20%，但差异无统计学意义。
具有这两种中国PS1突变的细胞对营养物质剥夺的敏感性增强很可能导致AD中的神经元丢失。对凋亡的敏感性并非由糖代谢受损引起，但IGF-1仍可保护细胞免受血清剥夺，通过增强葡萄糖转运和线粒体活性来补偿负面影响。
