Buono R J, Lang R K
Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, 1020 Locust St., Philadelphia, PA, 19107, USA.
Exp Eye Res. 1999 Dec;69(6):685-93. doi: 10.1006/exer.1999.0745.
In order to elucidate mechanisms controlling hypoxia induced gene repression in the retina, we studied expression of the lactate dehydrogenase gene LDH-B. RT/PCR was used to isolate cDNA fragments of the LDH-A and LDH-B genes from both rat and chick. Northern analysis was used to measure LDH-B mRNA expression and in situ hybridization to localize LDH-B mRNA during development of the avascular chick retina. Primary retinal cultures of rat and chick were subjected to hypoxia and reperfusion paradigms, and levels of LDH-A and LDH-B mRNA expression were measured by Northern and semi-quantitative RT/PCR analyses. Northern analysis demonstrated that LDH-B mRNA is developmentally regulated in the chick retina according to a time course that correlates with oxygen availability. In developing chick retina in situ hybridization localized LDH-B mRNA to aerobic regions of the retina, coincident with the distribution of active mitochondria. Northern and semi-quantitative RT/PCR analyses demonstrated that LDH-B mRNA levels decrease in primary rat and chick retinal cells in response to hypoxia. LDH-B mRNA increased to control levels in retinal cells exposed to hypoxia then reperfused with oxygen, while the opposite was true for LDH-A, an hypoxia inducible gene. These data demonstrate that LDH-B gene repression after hypoxia and reactivation after oxygen reperfusion occurs in both vascular and avascular retinal cells. Oxygen regulated expression of LDH-B is opposite and complementary to that of LDH-A and provides a system to elucidate the mechanisms underlying hypoxic gene repression and reactivation after reperfusion. Mechanisms controlling gene repression and reactivation based on oxygen availability are likely to be critically involved in ischemic damage and neovascular retinopathy.
为了阐明视网膜中控制缺氧诱导基因抑制的机制，我们研究了乳酸脱氢酶基因LDH-B的表达。采用RT/PCR从大鼠和鸡中分离LDH-A和LDH-B基因的cDNA片段。利用Northern分析来测量LDH-B mRNA的表达，并通过原位杂交在无血管鸡视网膜发育过程中定位LDH-B mRNA。对大鼠和鸡的原代视网膜培养物进行缺氧和再灌注实验，通过Northern和半定量RT/PCR分析来测量LDH-A和LDH-B mRNA的表达水平。Northern分析表明，在鸡视网膜中，LDH-B mRNA的表达根据与氧供应相关的时间进程受到发育调控。在发育中的鸡视网膜中，原位杂交将LDH-B mRNA定位到视网膜的需氧区域，与活跃线粒体的分布一致。Northern和半定量RT/PCR分析表明，原代大鼠和鸡视网膜细胞在缺氧时LDH-B mRNA水平降低。暴露于缺氧然后再灌注氧气的视网膜细胞中，LDH-B mRNA增加到对照水平，而缺氧诱导基因LDH-A则相反。这些数据表明，缺氧后LDH-B基因的抑制以及氧再灌注后的重新激活在血管性和无血管性视网膜细胞中均会发生。氧对LDH-B表达的调控与LDH-A相反且互补，为阐明缺氧基因抑制和再灌注后重新激活的潜在机制提供了一个系统。基于氧供应控制基因抑制和重新激活的机制可能在缺血性损伤和新生血管性视网膜病变中起关键作用。
