Thompson Carol L, Bowes Rickman Catherine, Shaw Steven J, Ebright Jessica N, Kelly Una, Sancar Aziz, Rickman Dennis W
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Invest Ophthalmol Vis Sci. 2003 Oct;44(10):4515-21. doi: 10.1167/iovs.03-0303.
To analyze the patterns of expression of the cryptochromes, CRY1 and CRY2, in the human retina and to correlate expression of these putative blue-light receptors with nonvisual photoreceptor localization.
CRY1 and CRY2 mRNA expression was analyzed in 4-mm diameter punches of macula and midperipheral human retina by quantitative RT-PCR. CRY2 protein expression was examined by immunohistochemistry in cross sections of human retina, and its subcellular localization was determined by immunoblot analysis of fractionated human retinal extracts.
CRY2 mRNA was 11 times more abundant than CRY1 throughout adult human retina. CRY2 immunoreactivity was detected in most cells in the ganglion cell layer (GCL) and in a subset of cells in the inner nuclear layer (INL) in both the macula and periphery. Immunoperoxidase staining further revealed that CRY2 was localized throughout the cytoplasm of cells in the GCL as well as within nuclei. This intracellular localization of CRY2 was confirmed by immunoblot analysis of fractionated human retinal extracts.
Photopigments governing circadian photoreception have been localized to the inner retina. The relative abundance of CRY2 transcripts, coupled with CRY2 localization to the inner retina, supports a photoreceptive role for CRY2 in human retina. Furthermore, the discovery that CRY2 is also localized within the cytoplasm of some cells in the GCL, suggests it may perform a function separate from its known nuclear role in the transcriptional feedback loop underlying the molecular circadian clock.
分析隐花色素CRY1和CRY2在人类视网膜中的表达模式，并将这些假定的蓝光受体的表达与非视觉光感受器定位相关联。
通过定量逆转录聚合酶链反应（RT-PCR）分析黄斑和中周部人类视网膜直径4毫米的组织块中CRY1和CRY2 mRNA的表达。通过免疫组织化学检查人类视网膜横切片中CRY2蛋白的表达，并通过对分级分离的人类视网膜提取物进行免疫印迹分析来确定其亚细胞定位。
在整个人类成年视网膜中，CRY2 mRNA的丰度比CRY1高11倍。在黄斑和周边区域的神经节细胞层（GCL）中的大多数细胞以及内核层（INL）中的一部分细胞中检测到CRY2免疫反应性。免疫过氧化物酶染色进一步显示，CRY2定位于GCL细胞的整个细胞质以及细胞核内。通过对分级分离的人类视网膜提取物进行免疫印迹分析证实了CRY2的这种细胞内定位。
控制昼夜节律光感受的光色素已定位于视网膜内层。CRY2转录本的相对丰度，以及CRY2定位于视网膜内层，支持CRY2在人类视网膜中具有光感受作用。此外，CRY2也定位于GCL中一些细胞的细胞质内这一发现表明，它可能执行与其在分子昼夜节律钟的转录反馈环中已知的核作用不同的功能。
