Ocular Surface Research Center, Department of Neurosciences, Ophthalmology, and Genetics, University of Genoa, viale Benedetto XV 5, Genoa, Italy.
Exp Eye Res. 2010 Oct;91(4):524-9. doi: 10.1016/j.exer.2010.07.008. Epub 2010 Jul 21.
The aim of our project was to test the hypothesis that patients with dry eye have a significant degree of inflammation and lymphocyte infiltration in conjunctival epithelium by using flow cytometry analysis of cells stored in cell culture medium. Impression cytology specimens were collected in 15 normal subjects and 15 dry eye patients. Samples collected from the right eye were placed in Phosphate Buffered Saline containing 0.05% paraformaldehyde (PFA), and samples from the left eye in cell culture medium containing 10% foetal calf serum (FCS). Phenotypic analysis was performed on cells derived from 21 dry eye patients and 16 healthy controls. The cells collected in FCS were stained for the expression of CK19, CD3, CD4, CD8, CD56, CD19, CD20, CD14 and HLA-DR, and analyzed by flow cytometry. FCS samples contained a statistically increased number of cells when compared to PFA samples. No statistically significant differences were present in the number of CD45+CK19- cells, CD3+ and CD4+T cells, B and NK cells in dry eye patients compared to healthy controls. In the dry eye group there was a significant difference in the CD4/CD8 ratio respect to what observed in normal subjects, and an increased number of CD14+ cells. HLA-DR expression was increased only in CK19+ conjunctival epithelial cells of dry eye patients. This study indicates that immune cells isolated from the superficial layer of the conjunctiva may play a pivotal role in the pathogenesis of dry eye, and that a new method of preservation of impression cytology samples can enhance flow cytometry analysis of epithelial and immune cells of the conjunctiva.
我们项目的目的是通过使用流式细胞术分析储存在细胞培养液中的细胞来检验这一假设，即干眼症患者的结膜上皮存在显著程度的炎症和淋巴细胞浸润。我们收集了 15 名正常受试者和 15 名干眼症患者的印迹细胞学标本。右眼的样本置于含有 0.05%多聚甲醛（PFA）的磷酸盐缓冲液（Phosphate Buffered Saline）中，左眼的样本置于含有 10%胎牛血清（Fetal Calf Serum，FCS）的细胞培养液中。我们对 21 名干眼症患者和 16 名健康对照者的细胞进行了表型分析。在 FCS 中收集的细胞被用于表达 CK19、CD3、CD4、CD8、CD56、CD19、CD20、CD14 和 HLA-DR 的染色，并通过流式细胞术进行分析。与 PFA 样本相比，FCS 样本中细胞数量明显增加。与健康对照组相比，干眼症患者的 CD45+CK19-细胞、CD3+和 CD4+T 细胞、B 细胞和 NK 细胞数量无统计学差异。在干眼症组中，与正常组相比，CD4/CD8 比值存在显著差异，且 CD14+细胞数量增加。仅在干眼症患者的 CK19+结膜上皮细胞中观察到 HLA-DR 表达增加。本研究表明，从结膜浅层分离的免疫细胞可能在干眼症的发病机制中发挥关键作用，并且印迹细胞学标本的新保存方法可以增强结膜上皮和免疫细胞的流式细胞术分析。
