摘要
目的改良SpragueDawley(SD)新生大鼠视网膜Müller细胞培养方法。方法取新生1~2d的SD,分2d完成视网膜Müller细胞的提取,第1天将眼球浸泡于含体积分数1%双抗的无血清高糖达尔伯克改良伊戈尔培养基(DMEM)中,置于37℃、含体积分数5%CO2的培养箱内过夜;第2天,去培养基,以2.5g·L^-1胰蛋白酶消化眼球1h,分离视网膜组织,加入含体积分数1%双抗和体积分数10%胎牛血清的高糖DMEM培养基,制备细胞悬液接种培养;24h后换液,6~8d后传代,传至第4代,细胞免疫荧光染色法鉴定Müller细胞纯度。结果Müller细胞贴壁生长良好,细胞体宽大,细胞质丰富,细胞核呈圆形或椭圆形,无神经元共生长,Müller细胞纯度>95%。结论改良的SD新生大鼠视网膜Müller细胞培养方法操作简单,细胞纯度高。
Objective To improve the primary culture method of Müller cells in the retina of newborn Sprague Dawley(SD) rats. Methods Retinal tissues from newborn(1-2 days) SD rats were selected,and the Müller cells in the retina were completed in two days. On the first day,the eyeballs were soaked in high-sugar Dulbecco’s modified Eagle medium(DMEM)without serum which contained volume fraction 1% double antibody. The eyeball was incubated overnight at 37 ℃ with 5% of the volume fraction of CO2. On the second day,retinal tissues were isolated after digestion with 2. 5 g·L^-1 trypsin solution for one hour and cultured in the DMEM which contained volume fraction 10% fetal bovine serum and volume fraction 1% double antibody. After 24 hours,the medium was replaced,and after 6-8 days,the cells could be passaged until to the fourth generation. Then the purity of Müller cells was determined by immunofluorescence staining. Results Müller cells attached well,with great somas,abundant cytoplasm,round or oval nucleus,no neuron co-growth,and the purity was > 95%.Conclusion This improved method of Müller cells culture in the retina of SD newborn rat is simple and convenient,while has a high cells purity.
作者
杨梅
范围
黄瑶
袁容娣
YANG Mei;FAN Wei;HUANG Yao;YUAN Rong-di(Department of Ophthalmology,the Second Affiliated Hospital of PLA Army Medical University,Chongqing 400037,China)
出处
《新乡医学院学报》
CAS
2019年第9期815-818,共4页
Journal of Xinxiang Medical University
基金
国家自然科学基金资助项目(编号:81470630)
