摘要
背景Myocilin蛋白的错误折叠是原发性开角型青光眼（POAG）的重要发病机制，GRP78能将错误折叠的蛋白质转移到内质网外，以保持应激状态下内质网蛋白质合成功能的正常运转，但二者的相互关系及其在POAG发病中的作用机制仍未阐明。目的检测体外培养的POAG患者小梁细胞内GRP78与Myolicin蛋白的共定位表达。方法在手术中获取POAG患者的小梁网组织，采用组织块培养法分离和培养小梁细胞，采用免疫化学染色法检测培养的细胞中特征性因子的表达，并将其形态学特征与健康供体眼培养的小梁细胞进行比较。将体外培养的小梁细胞分为衣霉素（Tm）组、十字孢碱（STS）组和正常对照组，分别在培养液中加入含有1μmol／L Tm、0．1μmol／LSTS和不含药物的培养液培养细胞24h，采用免疫荧光法检测各组培养的细胞中GRP78与Myocilin蛋白的共表达情况。结果培养的原代细胞多呈扁平星形或不规则形，可见细胞突起，细胞核呈椭圆形，细胞体较大，细胞质丰富，可见大量黑色吞噬颗粒。POAG来源小梁细胞培养3～7代生长良好，细胞中层黏连蛋白（LN）、纤维连接蛋白（FN）、神经烯醇化酶（NSE）和波形蛋白（Vimentin）均呈阳性表达。免疫荧光染色结果显示，Tm组、STS组和正常对照组细胞的细胞质中均可见GRP78和Myocilin阳性表达，分别为红色和绿色荧光，正常对照组中人来源小梁细胞的周边部可见GRP78和Myocilin的不完全共表达，POAG来源小梁细胞周边部GRP78和Myocilin呈完全共表达；Tm组和STS组中不同来源的小梁细胞中GRP78和Myocilin均呈完全共表达，Tm组中不同来源小梁细胞均出现细胞核周围GRP78蛋白聚集现象。结论POAG来源小梁细胞中GRP78蛋白与Myocilin蛋白共表达，提示二者在POAG的发病和进展过程中可能存在相互作用。GRP78可能保护小梁细胞免受内质网应激途径诱导的�
Background Misfolding of Myocilin protein plays an important role in the pathogenesis of primary open angle glaucoma （POAG）. GRP78 can transfer the misfolded proteins out endocytoplasmic reticulum and keep the protein synthesis function of endoplasmic reticulum. However the relationship between GRP78 and Myocilin in the pathogenesis of POAG has not been elucidated. Objective This study was to investigate and analyze the co-expressions of GRP78 and Myolicin in trabeeular meshwork cells （TMCs） of POAG. Methods The trabeeular meshwork tissues were obtained during the surgery of POAG and healthy donor for the isolated and in vitro cuhure of TMCs,and the morphology of the cells was compared between POAG-derived TMCs and donor-derived TMCs. The expression of specific factors for TMCs were detected by immunochemistry. The ceils were divided into normal control group, tunicamycin （Tm） -treated group and staurosporine （STS） -treated group, and the cells were cultured by regular medium,the 1μmol/L Tm medium or 0. 1μmol/L STS medium for 24 hours respectively. The co-expression of GRP78 and Myocilin in the cells were detected using immunofluoreseence assay. This study was approved by Ethics Committee of Xi＇an No. 4 Hospital, and written informed consent was obtained from each patient before surgery. Results Primarily cultured cells showed the fiat star-like and irregular appearance with 3-5 processes,round nuclei,abundant cytoplasm and black engulf particles. The 3-7 generations of POAG-derived cells grew well, showing positive expressions of laminin （LN） ,fibronectin （FN） , neuronal specific enolase （NSE） and Vimentin. Immunofluorescence assay showed positive expressions of GRP78 and Myocilin in the cells of the Tm group,STS group and normal control group,with the reddish and green fluorescence separately. Incomplete co-expression of GRP78 and Myocilin was seen in donor-derived TMCs,and complete co-expression of GRP78 and Myocilin was found in the POAG-derived TMCs in the normal control group
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2017年第4期300-305,共6页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目（81273902、81400424）
陕西省社会发展科技攻关项目（2016SF-100）
陕西省科学技术研究发展计划项目（2014K11-03-07-04）
西安市重点学科优势专科建设项目（[2015]228号-7）
关键词
开角型青光眼/病理
内质网/代谢
糖蛋白/代谢
小梁网/病理
肌纤蛋白
细胞培养
人
Glaucoma, open angle/pathology
Endoplasmic reticulum/metabolism
Glycoproteins/ metabolism
Trabecular meshwork/pathology
Myocilin
Cells, cultured
Human
