Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: evidence for K+ secretion by duct cells.
Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: evidence for K+ secretion by duct cells.
复制标题
使用激光捕获显微切割收集的大鼠泪腺导管细胞中的基因表达：导管细胞分泌 K 的证据。
DOI：
10.1167/iovs.05-0363
复制
发表时间：
2006
影响因子：
4.4
通讯作者：
Webb,CraigP
中科院分区：
文献类型：
--
作者：
Ubels,JohnL;Hoffman,HollyM;Srikanth,Sujata;Resau,JamesH;Webb,CraigP
目的.为了比较激光捕获显微切割（LCM）后泪腺导管和腺泡细胞的基因表达谱，并确定与K+分泌相关的分子网络，测试导管细胞负责泪液中高K+水平的假设。对5只大鼠的泪腺冷冻切片进行LCM，以分离导管和腺泡细胞的纯样品。提取RNA，扩增，逆转录，并与大鼠cDNA微阵列杂交。用内部软件扫描并分析来自五只动物的导管和腺泡的配对阵列。基因表达与荧光抗体和共聚焦显微镜证实。使用与离子转运相关的基因本体搜索导管和腺泡中表达的10，294个基因的列表。从导管中表达的55个基因的列表中，选择了一组假设参与K+和Cl−从基底侧向顶端转运的基因，通过免疫荧光和共聚焦显微镜进行验证。该分析证实了感兴趣的基因的翻译，并表明NKCC 1，Na+，K+-ATP酶和M3胆碱能受体表达的导管细胞的基底外侧膜上，而KCC 1，IK钙1，CFTR，和ClC 3的顶部定位。激光捕获显微切割结合基因表达分析提供了一个很好的方法来研究泪腺导管细胞，在分子水平上知之甚少。如在一个拟议的模型中所示，泪腺导管膜上的转运蛋白和通道的极化表达与导管细胞分泌相对较高的K+的泪液的假设是一致的。
purpose. To compare gene expression profiles of lacrimal gland duct and acinar cells after laser capture microdissection (LCM) and identify molecular networks related to K+ secretion, testing the hypothesis that duct cells are responsible for high K+ levels in tears.methods. Frozen sections of lacrimal glands from five rats were subjected to LCM to isolate pure samples of duct and acinar cells. RNA was extracted, amplified, reverse transcribed, and hybridized to rat cDNA microarrays. Paired arrays from ducts and acini of the five animals were scanned and analyzed with in-house software. Gene expression was confirmed with fluorescent antibodies and confocal microscopy.results. A list of 10,294 genes expressed in ducts and acini was searched using gene ontologies related to ion transport. From a list of 55 genes that were expressed in ducts, a panel of genes hypothesized to be involved in basolateral-to-apical transport of K+ and Cl− was chosen for validation by immunofluorescence and confocal microscopy. This analysis confirmed translation of the genes of interest and showed that NKCC1, Na+, K+-ATPase and the M3 cholinergic receptor are expressed on the basolateral membrane of duct cells, whereas KCC1, IK Ca 1, CFTR, and ClC3 are apically localized.conclusions. Laser capture microdissection in conjunction with gene expression analysis provides an excellent approach for studying lacrimal gland duct cells about which relatively little is known at the molecular level. As demonstrated in a proposed model, the polarized expression of transporters and channels on lacrimal gland duct membranes is consistent with the hypothesis that duct cells secrete the relatively high K+ in lacrimal fluid.