Ubels John L, Hoffman Holly M, Srikanth Sujata, Resau James H, Webb Craig P
Department of Biology, Calvin College, Grand Rapids, Michigan 49546, USA.
Invest Ophthalmol Vis Sci. 2006 May;47(5):1876-85. doi: 10.1167/iovs.05-0363.
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.
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.
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.
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.
比较激光捕获显微切割（LCM）后泪腺导管细胞和腺泡细胞的基因表达谱，识别与钾离子分泌相关的分子网络，验证泪腺导管细胞导致泪液中高钾水平的假说。
对5只大鼠的泪腺冰冻切片进行LCM，以分离出纯净的导管细胞和腺泡细胞样本。提取RNA、进行扩增、逆转录，并与大鼠cDNA微阵列杂交。对5只动物的导管和腺泡的配对阵列进行扫描，并使用内部软件进行分析。通过荧光抗体和共聚焦显微镜确认基因表达。
利用与离子转运相关的基因本体，搜索了在导管和腺泡中表达的10294个基因列表。从在导管中表达的55个基因列表中，选择一组假定参与钾离子和氯离子从基底外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。该分析证实了相关基因的翻译，并表明NKCC1、钠钾ATP酶和M3胆碱能受体在导管细胞的基底外侧膜上表达，而KCC1、IK(Ca)1、CFTR和ClC3定位于顶端。
激光捕获显微切割结合基因表达分析为研究泪腺导管细胞提供了一种很好的方法，目前在分子水平上对泪腺导管细胞的了解相对较少。如一个提出的模型所示，泪腺导管膜上转运体和通道的极化表达与导管细胞分泌泪液中相对较高钾离子的假说一致。
