Ye H, Yang J, Hernandez M R
Schepens Eye Research Institute, Boston, MA 02114.
Exp Eye Res. 1994 Jan;58(1):53-63. doi: 10.1006/exer.1994.1194.
Previous studies using immunocytochemistry and electron microscopy have localized collagen type III (CIII) in the human optic nerve head (ONH). CIII is present in the core of the cribriform plates of the lamina cribrosa and increases with age. In this study, in situ hybridization was used to localize mRNA for CIII in specific cells in human fetal and adult ONH to determine its cellular origin and to provide temporal and regional information on the synthesis of CIII in the ONH. Human ONH from donors with no history of eye disease (16-24 weeks gestation and 6-75 years) were fixed, embedded and sectioned serially. Sections were hybridized with antisense riboprobe for CIII and controls with sense riboprobe and processed for ISH. Immunoperoxidase staining with antibodies against GFAP and von Willebrand factor was used to detect astrocytes and vascular endothelial cells respectively. Fetal ONH: CIII mRNA was localized in most blood vessels throughout the ONH in sections hybridized with antisense probe. The label was localized to endothelial cells of small vessels. Endothelial cells of central retinal vessels were not labeled. In the lamina cribrosa, CIII mRNA was localized to small vessels, but not to astrocytes. Fibroblasts of the peripapillary sclera were labelled with antisense probe. Young-Adult ONH: In the cribriform plates, lamina cribrosa cells hybridized the probe at all ages. Few astrocytes hybridized the probe. Some endothelial cells of small vessels were also labeled. In the insertion region, most vessels were labeled in young ONH, but very few, if any, in old adults. The apparent intensity of hybridization signal associated with blood vessels in adult ONH is markedly reduced with age. No hybridization was observed inside of nerve bundles consistent with the absence of mRNA inside axon and CIII in this location. Localization of mRNA for CIII to blood vessels in fetal lamina cribrosa suggests a vascular origin for CIII in the plates, and perhaps, for lamina cribrosa cells. In young and old lamina cribrosa, localization of CIII mRNA suggests lifelong synthesis of this protein in agreement with age-related increase of CIII in this tissue.
以往利用免疫细胞化学和电子显微镜技术的研究已将III型胶原（CIII）定位于人视神经乳头（ONH）。CIII存在于筛板的核心部位，且随年龄增长而增加。在本研究中，采用原位杂交技术将CIII的mRNA定位于人胎儿和成人ONH的特定细胞中，以确定其细胞来源，并提供ONH中CIII合成的时间和区域信息。取自无眼部疾病史供体（妊娠16 - 24周和6 - 75岁）的人ONH被固定、包埋并连续切片。切片与CIII的反义核糖探针杂交，用正义核糖探针作为对照，并进行原位杂交处理。分别用抗GFAP和血管性血友病因子的抗体进行免疫过氧化物酶染色以检测星形胶质细胞和血管内皮细胞。胎儿ONH：在与反义探针杂交的切片中，CIII mRNA定位于整个ONH的大多数血管中。标记定位于小血管的内皮细胞。视网膜中央血管的内皮细胞未被标记。在筛板中，CIII mRNA定位于小血管，但不定位于星形胶质细胞。视乳头周围巩膜的成纤维细胞用反义探针标记。青年 - 成人ONH：在筛板中，筛板细胞在所有年龄段均与探针杂交。很少有星形胶质细胞与探针杂交。一些小血管的内皮细胞也被标记。在插入区域，青年ONH中的大多数血管被标记，但在老年人中极少（如果有的话）被标记。成人ONH中与血管相关的杂交信号的表观强度随年龄显著降低。在神经束内部未观察到杂交，这与该位置轴突内不存在mRNA以及CIII一致。胎儿筛板中CIII mRNA定位于血管表明筛板中CIII可能起源于血管，也许筛板细胞也是如此。在青年和老年筛板中，CIII mRNA的定位表明该蛋白终身合成，这与该组织中CIII随年龄增加的情况一致。
