Endogenous Elevation of Homocysteine Induces Retinal Neuron Death in the Cystathionine-b-Synthase Mutant Mice

The effects of endogenous elevation of homocysteine on retinal gene expression were screened by microarray analysis, which was performed in triplicate (i.e., three different paired RNA samples) with RNA isolated from neural retinas of cbs+/- HM at 5-weeks after a methionine diet and age-matched wildtype mice. The microarray chips used in this study permitted the screening of 36,212 genes. Of particular interest were those genes in cbs+/- HM mice whose expression levels changed by at least a factor of 2 (increased or decreased expression) compared with the wild-type in each of the replicate experiments. Using these selection criteria, we identified 1219 genes. They were categorized into six functional groups: (1) proapoptosis, (2) antiapoptosis, (3) cell cycle, (4) antioxidant, (5) calcium signaling, and (6) axon growth/guidance, with the remaining genes with marked changes in expression placed in a miscellaneous group. After categorization, relevant genes were further narrowed by SAM analysis; q < 0.06 was considered significant. Microarrays were performed in triplicate as biological repeats and reverse fluoro (i.e., three different paired RNA samples) with RNA isolated from neural retinas of cbs+/- HM at 5-weeks after a methionine diet and age-matched wildtype mice after a methionine diet.

本研究通过微阵列分析（microarray analysis）筛选同型半胱氨酸（homocysteine）内源性升高对视网膜基因表达的影响。实验设置三次生物学重复，即采用三份独立配对的RNA样本，样本提取自蛋氨酸饮食（methionine diet）干预5周后的cbs+/- HM小鼠以及同周龄野生型小鼠的神经视网膜组织。本研究使用的微阵列芯片可覆盖36212个基因的检测。本研究重点关注在三次重复实验中，相较于野生型小鼠，cbs+/- HM小鼠中表达量变化至少2倍（上调或下调）的基因。按上述筛选标准，本研究共鉴定出1219个差异表达基因，并将其划分为6个功能类别：(1) 促凋亡（proapoptosis）、(2) 抗凋亡（antiapoptosis）、(3) 细胞周期（cell cycle）、(4) 抗氧化（antioxidant）、(5) 钙信号通路（calcium signaling）以及(6) 轴突生长/导向（axon growth/guidance），其余表达存在显著变化的基因归入其他组。完成分类后，通过SAM分析对相关基因进行进一步筛选，将校正P值q<0.06视为具有统计学显著性。本实验同时设置三次生物学重复并开展反向荧光标记实验，即采用三份独立配对的RNA样本，样本提取自蛋氨酸饮食干预5周后的cbs+/- HM小鼠以及同期接受蛋氨酸饮食的同周龄野生型小鼠的神经视网膜组织。