Hernandez M R, Ye H, Roy S
Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114.
Exp Eye Res. 1994 Jul;59(1):41-51. doi: 10.1006/exer.1994.1079.
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.
以往研究已证实原发性开角型青光眼（POAG）患者视神经乳头处基底膜增生。我们使用针对人IV型胶原（基底膜的一种普遍成分）的放射性标记核糖探针进行原位杂交（ISH），以鉴定POAG患者视神经乳头中积极合成基底膜的细胞。此外，为了检测并进一步表征IV型胶原mRNA转录本，我们对从POAG患者个体视神经乳头以及年龄匹配的正常对照中提取的总RNA进行逆转录聚合酶链反应（RT-PCR）。ISH结果表明，在POAG中，板层前区的许多星形胶质细胞表达IV型胶原mRNA。筛板细胞以及受压筛板中的星形胶质细胞与探针杂交。在年龄匹配的正常对照的视神经乳头中，很少有星形胶质细胞和筛板细胞与探针杂交。通过琼脂糖电泳检测到IV型胶原和作为参照基因的甘油醛-3-磷酸脱氢酶（G3PDH）的RT-PCR产物为预期大小的单一条带，并使用正常和POAG样本中的特异性cDNA探针通过Southern杂交进行了阳性鉴定。在RT-PCR实验中未观察到其他产物（条带），表明总RNA提取物中没有基因组DNA污染。没有额外条带表明，至少在本研究使用的十个样本中，任何扩增序列中都没有可变剪接的RNA产物。使用光密度测定法的半定量分析显示，POAG样本中IV型胶原PCR产物增加了两倍。POAG样本和正常样本之间G3PDH PCR产物水平未检测到差异。本研究提供了证据，表明POAG患者视神经乳头处IV型胶原在mRNA水平上生物合成增加。这种现象是对眼压升高的反应还是对轴突丧失的修复机制，仍有待确定。
