Gao Hong, Frost Michael R, Siegwart John T, Norton Thomas T
Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294-4390, USA.
Mol Vis. 2011 Apr 12;17:903-19.
To increase our understanding of the mechanisms that remodel the sclera during the development of lens-induced myopia, when the sclera responds to putative "go" signals of retinal origin, and during recovery from lens-induced myopia, when the sclera responds to retinally-derived "stop" signals.
Seven groups of tree shrews were used to examine mRNA levels during minus lens compensation and recovery. Starting 24 days after eye opening (days of visual experience [VE]) lens compensation animals wore a monocular -5D lens for 1, 4, or 11 days. Recovery animals wore the -5D lens for 11 days, which was then removed for 1 or 4 days. Normal animals were examined at 24 and 38 days of VE. All groups contained 8 animals. Scleral mRNA levels were examined in the treated and contralateral control eyes with quantitative real-time polymerase chain reaction (qPCR) for 27 genes divided into four categories: 1) signaling molecules, 2) matricellular proteins, 3) metalloproteinases (MPs) and tissue inhibitors of metalloproteinases (TIMPs), and 4) cell adhesion and other proteins. Four groups (n=5 per group) were used to examine protein levels. One group wore a -5D lens for 4 days. A second group recovered for 4 days after 11 days of -5D lens treatment. Two groups were used to examine age-matched normal protein levels at 28 and 39 days of VE. The levels of six scleral proteins that showed differential mRNA expression were examined with quantitative western blots.
Nineteen of the genes showed differential (treated eye versus control eye) expression of mRNA levels in at least one group of animals. Which genes showed differential expression differed after 1 and 4 days of compensation and after 1 or 4 days of recovery. The mRNA level for one gene, a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), was upregulated in the treated eyes after 1 day of compensation. After 4 days, transforming growth factor beta receptor 3 (TGFBR3), transforming growth factor-beta-induced protein ig-h3 (TGFBI), and matrix metalloproteinase 14 (MMP14) mRNA levels were upregulated. Downregulated were mRNA levels for transforming growth factor beta-1 (TGFB1), transforming growth factor beta-2 (TGFB2), thrombospondin 1 (THBS1), tenascin (TNC), osteonectin (SPARC), osteopontin (SPP1), tissue inhibitor of metalloproteinases 3 (TIMP3), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5). After 11 days of lens wear, there was no differential expression. During recovery, after 1 day, treated-eye mRNA downregulation was found for TGFB2, TGFBR1, TGFBR2, TGFBR3, SPARC, ADAMTS1, ADAMTS5, syndecan 4 (SDC4), and collagen type VI, alpha 1 (COL6A1). After 4 days, TGFB1, TGFB2, TGFB3, THBS2, and TIMP3 mRNA levels were upregulated in the recovering eye. Significant downregulation, relative to normal eyes, was found in both the control and treated eyes for most genes after 1 day of compensation; a similar decrease was found, compared to lens-compensated eyes, after one day of recovery. Protein levels for THBS1 showed positive correlation with the differential mRNA levels and TGFBR3 showed a negative correlation. No differential protein expression was found for TGFB2, TGFBI, MMP14, and TIMP3.
The different patterns of differential mRNA expression during minus lens compensation (hyperopia) and recovery (myopia) show that scleral fibroblasts distinguish between "go" and "stop" conditions. There is evidence of binocular global downregulation of genes at the start of both lens wear and recovery. As additional information accumulates about changes in gene expression that occur during compensation and recovery the "signature" of differential changes may help us to understand in more detail how the sclera responds in "go" and "stop" conditions.
加深我们对晶状体诱导性近视发展过程中巩膜重塑机制的理解，此时巩膜对视网膜来源的假定“启动”信号作出反应；以及在从晶状体诱导性近视恢复过程中巩膜重塑机制的理解，此时巩膜对视网膜来源的“停止”信号作出反应。
使用七组树鼩来检测在负透镜补偿和恢复过程中的mRNA水平。在睁眼后24天（视觉经验天数[VE]）开始，透镜补偿组动物单眼佩戴-5D透镜1、4或11天。恢复组动物佩戴-5D透镜11天，然后取下透镜1或4天。正常组动物在VE的24天和38天进行检查。所有组均包含8只动物。使用定量实时聚合酶链反应（qPCR）检测处理眼和对侧对照眼中27个基因的巩膜mRNA水平，这些基因分为四类：1）信号分子，2）基质细胞蛋白，3）金属蛋白酶（MPs）和金属蛋白酶组织抑制剂（TIMPs），4）细胞黏附蛋白和其他蛋白。四组（每组n = 5）用于检测蛋白水平。一组佩戴-5D透镜4天。第二组在-5D透镜处理11天后恢复4天。两组用于检测VE 28天和39天年龄匹配的正常蛋白水平。使用定量蛋白质印迹法检测六种显示出差异mRNA表达的巩膜蛋白水平。
19个基因在至少一组动物中显示出mRNA水平的差异表达（处理眼与对照眼相比）。在补偿1天和4天后以及恢复1天或4天后，显示差异表达的基因有所不同。补偿1天后，处理眼中一个基因（含血小板反应蛋白基序的解聚素和金属蛋白酶1（ADAMTS1））的mRNA水平上调。4天后，转化生长因子β受体3（TGFBR3）、转化生长因子β诱导蛋白ig-h3（TGFBI）和基质金属蛋白酶14（MMP14）的mRNA水平上调。转化生长因子β-1（TGFB1）、转化生长因子β-2（TGFB2）、血小板反应蛋白1（THBS1）、腱生蛋白（TNC）、骨连接蛋白（SPARC）、骨桥蛋白（SPP1）、金属蛋白酶组织抑制剂3（TIMP3）和含血小板反应蛋白基序的解聚素和金属蛋白酶5（ADAMTS5）的mRNA水平下调。佩戴透镜11天后，未发现差异表达。在恢复过程中，1天后，处理眼中TGFB2、TGFBR1、TGFBR2、TGFBR3、SPARC、ADAMTS1、ADAMTS5、多配体蛋白聚糖4（SDC4）和VI型胶原α-1（COL6A1）的mRNA下调。4天后，恢复眼中TGFB1、TGFB2、TGFB3、THBS2和TIMP3的mRNA水平上调。补偿1天后，相对于正常眼，大多数基因在对照眼和处理眼中均显著下调；与透镜补偿眼相比，恢复1天后也发现了类似的下降。THBS1的蛋白水平与差异mRNA水平呈正相关，TGFBR3呈负相关。未发现TGFB2、TGFBI、MMP14和TIMP3的差异蛋白表达。
负透镜补偿（远视）和恢复（近视）过程中差异mRNA表达的不同模式表明，巩膜成纤维细胞能够区分“启动（go）”和“停止（stop）”状态。有证据表明在佩戴透镜和恢复开始时，基因存在双眼整体下调。随着关于补偿和恢复过程中发生的基因表达变化的更多信息不断积累，差异变化的“特征（signature）”可能有助于我们更详细地了解巩膜在“启动”和“停止”状态下的反应。
