一种用于APP和APLP2 mRNA的新型定量溶液杂交-RNase保护分析方法。

Johnston J A, Norgren S, Annerén G, Cowburn R F, Lannfelt L

Department of Clinical Neuroscience and Family Medicine, Karolinska Institute, Novun KFC, Huddinge, Sweden.

Brain Res Mol Brain Res. 1996 Dec 31;43(1-2):77-84. doi: 10.1016/s0169-328x(96)00160-x.

Amyloid precursor protein (APP) and amyloid precursor-like protein 2 (APLP2) are members of a multigene family of proteins implicated in the pathogenesis of Alzheimer's disease. We describe the development of an RNA-RNA solution hybridisation-RNase protection assay to quantify APP mRNA. APP mRNA splice forms containing the Kunitz-type protease inhibitor (KPI) insert, and APLP2 mRNA in total nucleic acid extracts from a range of tissue types. Solution hybridisation-RNase protection assay enables absolute quantification of target mRNA, by conversion of the hybridisation signal to pg mRNA using a standard curve. The assay is sensitive, capable of detecting 1 pg target mRNA, and reproducible, with an inter-assay variability of less than 10% and an intra-assay variability of 3-4%. We quantified APP and APLP2 mRNA in cell lines and post-mortem human brain tissue samples. To test whether we could detect physiological differences in APP mRNA levels, a fibroblast cell line with a paternal chromosome 21 deletion of the region including the APP gene was analysed and found to express half as much APP mRNA as control fibroblasts. In addition, a reversible, approx. 30% increase in APP mRNA levels was detected in human lymphoblastoid cell lines following heat shock, a physical stimulus previously shown to increase APP expression. Regional differences in the expression of APP and APLP2 were seen in human post-mortem cerebral cortex and cerebellum. Levels of APP and APLP2 mRNA were highest in the temporal cortex, slightly lower in frontal and occipital cortices, and lowest in the cerebellum. The highest proportion of KPI-containing APP was seen in the frontal and temporal cortices. The ratio of APP:APLP2 mRNA was 1:0.3 in the cortical tissue and 1:0.8 in the cerebellum. In conclusion, quantitative solution hybridisation-RNase protection assay of total APP. APP KPI and APLP2 mRNA provides a new tool to improve the resolution of studies of potentially subtle alterations in the expression of these genes in both cell culture model systems and Alzheimer's disease post-mortem human brain tissue.

淀粉样前体蛋白（APP）和淀粉样前体样蛋白2（APLP2）是与阿尔茨海默病发病机制相关的多基因家族蛋白成员。我们描述了一种用于定量APP mRNA的RNA - RNA溶液杂交 - RNase保护测定法的开发。该方法可检测一系列组织类型的总核酸提取物中含有Kunitz型蛋白酶抑制剂（KPI）插入片段的APP mRNA剪接形式以及APLP2 mRNA。溶液杂交 - RNase保护测定法通过使用标准曲线将杂交信号转换为pg mRNA，能够对目标mRNA进行绝对定量。该测定法灵敏，能够检测到1 pg目标mRNA，并且具有可重复性，测定间变异小于10%，测定内变异为3 - 4%。我们对细胞系和死后人类脑组织样本中的APP和APLP2 mRNA进行了定量。为了测试我们是否能够检测到APP mRNA水平的生理差异，我们分析了一个父本21号染色体上包括APP基因区域缺失的成纤维细胞系，发现其表达的APP mRNA量仅为对照成纤维细胞的一半。此外，在人淋巴母细胞系中，热休克（一种先前已证明可增加APP表达的物理刺激）后检测到APP mRNA水平可逆性地增加了约30%。在人类死后大脑皮层和小脑中观察到APP和APLP2表达的区域差异。APP和APLP2 mRNA水平在颞叶皮层最高，在额叶和枕叶皮层略低，在小脑中最低。含KPI的APP比例在额叶和颞叶皮层最高。皮层组织中APP:APLP2 mRNA的比例为1:0.3，小脑中为1:0.8。总之，对总APP、APP KPI和APLP2 mRNA进行定量溶液杂交 - RNase保护测定，为提高在细胞培养模型系统和阿尔茨海默病死后人类脑组织中这些基因表达潜在细微变化研究的分辨率提供了一种新工具。