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Expression of the fetal HLA-G molecule by ocular surface epithelium is enhanced when expanded on amniotic membranes

当在羊膜上扩张时，眼表上皮的胎儿 HLA-G 分子的表达增强

基本信息

批准号：
15591879
负责人：
SHIMAZAKI Jun
金额：
$ 2.24万
依托单位：
TOKYO DENTAL COLLEGE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

PURPOSE. To investigate the expression and function of HLA-G in limbal and conjunctival epithelial cells when expanded on amniotic membrane(AM). METHODS. A conjunctival epithelial cell line (CCL20.2) was used to screen mRNA expression of HLA molecules when cultured with or without amniotic membrane. Expression of HLA-G mRNA in primary cultured limbal and conjunctival epithelial cells was measured by RT-PCR and real-time PCR. Immunocytochemical staining and western blot analysis was done to confirm HLA-G protein expression. The HLA class I negative K-562 human erythroleukaemia cell line was transfected with limbal epithelium-derived HLA-G mRNA for functional analysis using an NK cell-lysis assay.RESULTS. Microarray analysis revealed increased expression of HLA-G, and decreased expression of other HLA genes when CCL20.2 cells were cultured on AM rather than plastic. Limbal and conjunctival epithelial cells were also shown to express HLA-G by RT-PCR, immunocytology and western blots. Semi-quantitative real-time PCR showed a significant increase in HLA-G expression in conjunctival epithelial cells when cultured on AM. Although expression of HLA-G in limbal epithelial cells did not change with AM substrate alone, a more than 2-fold greater upregulation was observed following the addition of IFN-γ when cultured on AM. NK cell-induced cytolysis of K-562 cells was slightly inhibited by transfection of HLA-G.CONCLUSION. Conjuctival and corneal epithelial cells express functional HLA-G when expanded ex vivo, which is enhanced by using an AM substrate and by IFN-γ stimulation.

目的。目的：研究人白细胞抗原-G(HLAG)在羊膜(AM)上扩增后在角膜缘和结膜上皮细胞中的表达及功能。方法：研究方法。以结膜上皮细胞系CCL20.2为研究对象，检测有无羊膜培养条件下人类白细胞抗原分子的表达。采用逆转录聚合酶链式反应和实时荧光定量聚合酶链式反应检测原代培养的角膜缘和结膜上皮细胞中人类白细胞抗原G基因的表达。免疫细胞化学染色和免疫印迹分析证实HLAG蛋白的表达。结果：基因芯片分析显示，当CCL20.2细胞在AM而不是塑料上培养时，HL A-G的表达增加，其他基因的表达降低。RT-PCR、免疫细胞学和免疫印迹显示角膜缘和结膜上皮细胞表达人类白细胞抗原-G。半定量实时荧光定量聚合酶链式反应显示AM培养的结膜上皮细胞中HLAG的表达显著增加虽然单独的AM底物对角膜缘上皮细胞HLAG的表达没有影响，但加入干扰素-γ后，在AM上培养的角膜缘上皮细胞中HLAG的表达增加了2倍以上结论体外扩增的结膜和角膜上皮细胞表达功能性的HLAG，AM底物和干扰素-γ刺激可增强其表达。