COLLAGEN TYPE-IV GENE-EXPRESSION IN HUMAN OPTIC-NERVE HEADS WITH PRIMARY OPEN-ANGLE GLAUCOMA
COLLAGEN TYPE-IV GENE-EXPRESSION IN HUMAN OPTIC-NERVE HEADS WITH PRIMARY OPEN-ANGLE GLAUCOMA
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DOI：
10.1006/exer.1994.1079
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发表时间：
1994-07-01
影响因子：
3.4
通讯作者：
ROY, S
中科院分区：
文献类型：
--
作者：
HERNANDEZ, MR;YE, HQ;ROY, S
以往的研究表明原发性开角型青光眼（POAG）视乳头基底膜增生。我们使用放射性标记的核糖核酸探针特异性的人IV型胶原蛋白，基底膜的一个普遍存在的组成部分，在原位杂交（ISH），以确定细胞积极合成基底膜在POAG的视神经乳头。此外，为了检测和进一步表征IV型胶原mRNA的转录，我们使用逆转录-聚合酶链反应（RT-PCR）从POAG和年龄匹配的正常对照组的视神经头中提取的总RNA，ISH结果表明，在POAG中，许多星形胶质细胞在前板层区域表达IV型胶原mRNA。筛板细胞和受压筛板中的星形胶质细胞与探针杂交。少数星形胶质细胞和筛板细胞杂交的探针在正常年龄匹配的对照组的视神经乳头。胶原IV和甘油醛-3-脱氢酶（G3 PDH），一个参考基因的RT-PCR产物，通过琼脂糖电泳检测到预期大小的单一条带，并通过Southern杂交使用特定的cDNA探针在正常和POAG样本中进行了积极的鉴定。在RT-PCR实验中没有观察到额外的产物（条带），表明在总RNA提取物中没有基因组DNA污染。缺乏额外的条带表明，至少在本研究中使用的10个样品中，在任何扩增序列中均不存在选择性剪接的RNA产物。使用光密度法的半定量分析显示，POAG样本中IV型胶原蛋白PCR增加了两倍。POAG与正常对照组G3 PDH PCR产物的表达水平无明显差异，提示POAG视神经乳头组织中IV型胶原的合成在mRNA水平上增加。这种现象是否代表了对眼内压升高的反应或对轴突损失的修复机制仍有待确定。
Previous studies have demonstrated proliferation of basement membranes in the optic nerve head in primary open angle glaucoma (POAG). We used in situ hybridization (ISH) of a radiolabeled riboprobe specific for human collagen IV, a ubiquitous component of basement membranes, to identify cells actively synthesizing basement membranes in the optic nerve head in POAG. In addition, to detect and further characterize the collagen IV mRNA transcripts, we used reverse transcriptase-polymerase chain reaction (RT-PCR) in total RNA extracted from individual optic nerve heads with POAG and from age-matched normal controls.ISH results demonstrate that, in POAG, numerous astrocytes in the prelaminar region expressed collagen IV mRNA. Lamina cribrosa cells and astrocytes in the compressed lamina cribrosa hybridized the probe. Few astrocytes and lamina cribrosa cells hybridized the probe in the optic nerve head of normal age-matched controls. RT-PCR products for collagen IV and for glyceraldehyde-3-dehydrogenase (G3PDH), a reference gene, were detected by agarose electrophoresis as single bands of the expected sizes and positively identified by Southern hybridization using specific cDNA probes in normal and POAG samples. No additional products (bands) were observed in RT-PCR experiments, indicating that there was no genomic DNA contamination in the total RNA extract. The lack of additional bands suggests that, at least in the ten samples used in this study, there were no alternatively spliced RNA products in any of the amplified sequences. Semi-quantitative analyses using densitometry showed a two-fold increase in collagen type IV PCR present in POAG samples. No differences were detected in levels of G3PDH PCR products between POAG and normal samples.This investigation provides evidence of increased biosynthesis of collagen type IV at the mRNA level in optic nerve heads with POAG. Whether this phenomenon represents a response to elevated intraocular pressure or a reparative mechanism to the loss of axons remains to be determined.