摘要
背景视网膜Muller细胞是视网膜重要的胶质细胞，也是视网膜干细胞的来源之一，研究Muller细胞的生物学行为对研究视网膜的各种生理病理过程和视网膜干细胞治疗研究具有重要意义，而建立稳定的视网膜Muller细胞培养体系是相关研究的基础。目的优化分离培养和纯化人视网膜Muller细胞的方法，为相关的实验研究提供良好优质的Muller细胞来源。方法分离人供体眼视网膜组织，然后将剪碎的视网膜组织置入质量分数0．25％胰蛋白酶＋100U透明质酸酶混合溶液中进行消化，添加含体积分数10％胎牛血清的DMEM／F12培养液1～2ml终止消化，然后加入含体积分数20％胎牛血清的RPMI1640培养液培养72h，行半量换液，再以含10％胎牛血清的培养液进行传代。光学显微镜下根据细胞的形态特征进行细胞鉴定，并分别采用免疫荧光技术和免疫组织化学法检测胶质细胞标志物胶质纤维酸性蛋白（GFAP）和Muller细胞特异性标志物谷氨酰胺合成酶（GS）的表达，对培养的细胞进行鉴定。结果应用0．25％胰蛋白酶＋100U（商品单位）透明质酸酶的混合酶消化法可成功获取人视网膜Muller细胞，原代培养后24h细胞贴壁，培养后9—10d细胞融合。培养的细胞胞体呈长圆形，细胞质丰富，部分细胞体两端锥状长突起，末端膨隆，细胞核大。传代后的细胞胞体大，呈扁平多角形，符合Muller细胞的形态。细胞免疫组织化学和免疫荧光技术检测显示95％以上的细胞中GFAP呈阳性表达，90％以上的细胞中GS呈强阳性表达。结论应用透明质酸酶和胰蛋白酶混合液消化法可以成功分离人视网膜Muller细胞，分别用含20％胎牛血清和含10％胎牛血清的RPM11640培养液培养和传代的人视网膜Muller细胞产量高且纯度好。
Background Retinal Mailer cells are important gliocytes and the source of retinal stem cells. Researching the biological behavior of Mailer cells is of important significance to the study on retinal physiopathological process and stem cell therapy of retinal diseases. To establish a stable culture method of Muller cells is a solid basis of relative basic research. Objective This study was to establish a simple and stable method of isolation and culture of human retinal Mailer cells and provide sufficient and high-quality Muller cell source. Methods Human retinal Muller cells were isolated from healthy human donor eyes. The mixture solution of hyaluronidase （ 100 U） and 0. 25% trypsin were used to digest chopped retinal tissue. The DMEM/F12 medium with 20% fetal bovine serum （FBS） was added to stop the digestion process. RPMI1640 medium with 20% FBS was used to cuhure the cell for 72 hours and then replaced the half medium. The cells were passaged by the RPMI1640 medium with 20% FBS. The morphology of the cells were examied under the optical microscope, and the expressions of glial fibrillary acidic protein （GFAP） , a marker of gliocytes, and glutamine synthetase （GS） , a special marker of retinal Mtiller cells, were detected by immunochemistry and immunofluorescence technology. Results Human retinal Mailer cells were successfully isolated by enzyme mixture solution of hyaluronidase （100 U） and 0. 25% trypsin. The cells were adherent to wails 24 hours after primary culture and completely merged 9-10 days after culture. The ceils showed oval in shape with abundant cytoplasm,and a part of cells presented with cone-shaped bulge bilaterally and ectasia in the posterior containing large nuclei. After cells passage,the cells were enlarged and grew toward polygonal shape. The positive expression of GFAP was observed in more than 95% cells and strongly positive expression of GS was observed in more than 90% cells by immunohistochemstry and immunofluorescent staining. Conclusions Human retinal MUller ce
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2017年第1期22-25,共4页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目（81570876）
广东省医学科研基金项目（A2013214）
