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目的:改良和补充兔Tenon’s囊组织原代成纤维细胞的分离培养、鉴定方法,为开展抗青光眼术后滤过泡瘢痕化的药物研究提供成熟的原代成纤维细胞。方法:应用组织块培养法,无菌条件下摘取组织,机械剪碎,于含10%胎牛血清的DMEM中培养,显微镜下观察细胞形态,免疫荧光法鉴定,实时荧光定量PCR法检测细胞波形蛋白、角蛋白的mRNA水平。结果:分离培养的细胞具有成纤维细胞形态,7-10d从组织块周围大量爬出,15d铺满培养皿;免疫荧光法鉴定显示,波形蛋白表达阳性,角蛋白阴性表达;实时荧光定量PCR检测出细胞中波形蛋白mRNA水平远远高于角蛋白。结论:改良和补充了兔Tenons囊组织成纤维细胞原代培养与鉴定方法,为后续青光眼术后滤过泡抗瘢痕化的药物研究奠定基础。
OBJECTIVE: To improve and supplement the isolation, cultivation and identification of primary fibroblasts from rabbit Tenon’s sacs and to provide mature primary fibroblasts for the study of anti-glaucomatous scarring after filtration surgery. Methods: Tissue explant culture was used. The tissues were removed under sterile conditions, mechanically excised, cultured in DMEM containing 10% fetal bovine serum. Cell morphology was observed under a microscope. Immunofluorescence assay and real - time quantitative PCR were used to detect the cells Vimentin, keratin mRNA levels. Results: The isolated and cultured cells had fibroblast morphology. Large numbers of outgrowths were found around the tissue mass in 7-10 days and petri dishes were covered 15 days later. Immunofluorescence showed that the vimentin was positive and keratin was negative. Real-time fluorescence quantitative PCR The level of vimentin mRNA in cells is much higher than keratin. Conclusion: The methods of primary culture and identification of rabbit Tenons capsule fibroblasts were improved and supplemented, which lays the foundation for the study of anti-scarring drugs after filtration surgery of glaucoma.