The Analysis of Differentially Expressed Gene by DNA Micro Array

我们对黄斑变性食蟹猴合并玻璃疱疹进行了组织学、分子生物学和遗传学分析。此前在筑波国家灵长类中心发现的这一猴子家族，通过对视网膜的观察和视网膜切片的荧光成像，简要地描述了这一家族的特征。对60只15~24岁繁殖群体的猕猴进行了眼底镜检查。在10只猴子中观察到黄斑变性。取黄斑视网膜制作组织切片，HE、PAS、LFB、MT染色，光镜下观察。用透射电子显微镜观察玻璃体的超微结构。病猴视网膜色素上皮(RPE)内可见色素沉着、部分空泡化、自体荧光增强和胞浆内PAS阳性颗粒。然而，在神经…观察到最小的组织学异常。更多的血管和脉络膜。这些观察结果表明，脂褐素颗粒在RPE中高度积聚。使用Clontech公司的塑料DNA阵列，分别用Cy3和Cy5标记从患病和未患病的猴视网膜提取的RNA，进行分子生物学分析。在所有8,000个基因归一化后，对两个样本进行差异表达基因的比较。14个基因上调2.5~7.6倍，5个基因下调2.4~3.8倍。在上调的基因中，三磷酸腺苷合成酶、H+转运、线粒体F0复合体、亚基f、异构体2与正常相比增加了7.6倍以上。淋巴管内皮细胞透明质酸受体显着下降3.8倍。同时进行连锁分析和候选基因分析，以确定致病基因。克隆了常染色体显性遗传性Stargardt样黄斑营养不良(STGD3)基因ELOVL4的猴子同源基因，以确定黄斑变性猴子是否存在任何可能的突变。采用基因组DNA聚合酶链式反应和5‘/3’末端快速扩增技术，测定了猴ELOVL4基因的全序列。实时荧光定量聚合酶链式反应检测到ELOVL4 RNA在视网膜和胸腺中大量表达。用抗人ELOVL4多肽多克隆抗体对猴视网膜ELOVL4蛋白进行免疫组织化学分析。该基因未发现突变。较少

We have conducted histology, molecular biology, and genetic analysis of macular degeneration cynomolgus monkey with drusen. This monkey family previously discovered in National Primate Center in Tsukuba has been briefly characterized by observation of the retina and fluorescence imaging of the retina section. Sixty monkeys aged 15 to 24 years in propagation colonies were examined by fundus scope. Macular degeneration was observed in ten monkeys. Histological sections prepared from excised macular retina were stained by HE (hematoxylin and eosin), PAS (Periodic Acid Sciff), LFB (luxol fast blue) and MT (masson trichrome) and observed under light microscopy. Ultrastructure of drusen was examined by transmission electron microscopy. Hyper-pigmentation, partial vacuolation, increased autofluorescence, and PAS-positive granules in cell cytosol were observed in retinal pigment epithelium (RPE) of these affected monkey retina. However, minimum histological abnormality was observed in neural r … More etina and choroid. These observations indicate that lipofuscin granules are highly accumulated in RPE.. Molecular biology analysis using Clontech Inc, plastic DNA array with 8,000 genes were performed by labeling RNA extracted from affected and non-affected monkey retina by Cy3 and Cy5 respectively. After the normalization of all 8,000 genes, two samples were compared for differentially expressed gene. Fourteen genes were up-regulated by, 2.5 to 7.6-fold, while, five genes were down-regulated by 2.4 to 3.8-fold. Among the up-regulated genes, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit f, isoform 2 gave more than 7.6-fold compare to normal. Lymphatic vessel endothelial hyaluronan receptor gave significant drop of 3.8-fold. Linkage analysis and candidate gene approach was performed in parallel to determine the gene responsible for the disease. The monkey homologue of the ELOVL4, gene responsible for autosomal dominant Stargardt-like macular dystrophy (STGD3) was cloned to determine any possible mutation in macular degeneration monkey. PCR of genomic DNA and 5'/3'-rapid amplification of cDNA ends were carried out to determine the complete sequence of the monkey ELOVL4 cDNA and the gene. ELOVL4 RNA was abundantly expressed in retina and thymus determined by real time quantitative PCR. Immunohistochemical analysis was performed using anti-human ELOVL4 peptide polyclonal antibody to localize ELOVL4 protein in monkey retina. No mutation was found in this gene. Less