大鼠视网膜Müller细胞体外培养特性  

Culture characteristics of rat retinal Müller cells in vitro

作　　者：李春花[1] 郑玉萍[2] 周婷洁[3] 雷晓琴[1] 王润生[1] 宋虎平[1] 马为梅[1] Li Chunhua;Zheng yuping;Zhou Tingjie;Lei Xiaoqing;Wang Yunsheng;Song Huping;Ma Weimei(Department of Ophthalmology,Xi＇an No.4 Hospital,Xi＇an,Shaanxi 710004;Department of Ophthalmology,Second Affiliated Hospital of Xi＇an Jiaotong University,Xi＇an 710002,Shaanxi;Xi＇an Center for Disease Control and Prevention,Xi＇an,Shaanxi 710054,China)

机构地区：[1]西安市第四医院,710004 [2]西安交通大学附属第二医院,710002 [3]西安市疾病预防控制中心,710054

出　　处：《临床眼科杂志》2018年第5期460-464,共5页Journal of Clinical Ophthalmology

基　　金：2017年陕西省中医管理局中医药科学技术研究课题(项目号:JCMS061);陕西省科技厅社会发展科技攻关项目(项目号:2015SF232)

摘　　要：目的探讨视网膜Müller细胞(RMCs)体外培养方法,研究其增殖、凋亡特性。方法将出生7 d的SD大乳鼠处死,分离出视网膜组织并收集于培养皿中,分别加入木瓜蛋白酶(27 U/ml)和0. 25%的胰蛋白酶37℃进行消化30 min,使用20%FBS的DMEM/F12培养基终止消化。用移液枪轻轻吹打,接种于6孔培养板,放入37℃、体积分数为5%的CO2培养箱培养。定时观察细胞的生长情况,2～3 d换液1次。当细胞长满培养板的80%～90%时进行消化终止,按1:3传代,移入放有细胞爬片的6孔培养板,置于5%CO2恒温培养箱中培养。在倒置显微镜下观察细胞形态变化。在原代培养5 d时,将细胞爬片取出,进行谷氨酰胺合成酶(GS)、波型蛋白(Vimentin)免疫荧光染色。结果原代培养的RMCs与神经元细胞共同生长,随接种时间延长,逐渐自接种块周边爬行生长,呈扁平的多边形融合状生长,并有较长的突起,传2代时细胞纯化率高,传3～4代时细胞失去原有秩序,体积约有原代的3～4倍,胞质内见明显束状纤维增生。GS主要在Müller细胞胞核和胞质中表达,胞膜不表达,其阳性表达率达80%以上。Vimentin主要在Müller细胞胞质中表达,其阳性表达率达90%以上。结论本试验通过酶分步消化法以及机械吹打法在体外成功培养并纯化了视网膜Müller细胞,GS和Vimentin均可鉴别视网膜Müller细胞,将二者一起使用可提高Müller细胞鉴别率;随着体外传代培养时间延长,Müller细胞逐渐分化为带有收缩功能的纤维细胞,这可能参与了增生性视网膜病变的发病机制。Objective To investigate the culture methods of retinal Müller cells （RMCs）, and study their proliferation and apoptosis characteristics in vitro. Methods The eyeballs of neonatal （7 days） Sprague-Dawley rats were opened to obtain the retina, then retinal tissues were isolated and collected in petri dishes. After digested with papain （27 U/ ml） and 0.25% trypsin at 37 ° C for 30 min,20% FBS DMEM / F12 medium terminated digestion. Blown by a pipette gently, the mixture was placed in a 6-well plate inoculated at 37 ° C in humidified air containing of 5% carbon dioxide. The medium of the Müller？cell plates was renewed on days 2-3. When the cells were almost confluent （80 90%）, they were digested and passaged by 1： 3, the cell morphology changes were observed under the inverted microscope. After primary culture for 5 days, the cells were taken out for GS （glutathione synthetase） and Vimentin immunofluorescence staining. Results The primary cultured RMCs co-grew with neuron cells. With the prolongation of inoculation time, the RMCs grew gradually from the periphery of the inoculation block, grew into flat polygons and had longer protuberances. The cells were highly purified when subcultured second passage. Third-fourth passage of cells lost their original order, volume increased to 3 times of the original, cytoplasm was full of the bundle-like fiber hyperplasia. In vitro cultured retinal Müller cells expressed glutathione synthetase （GS） and vimentin. GS was expressed in the nuclei and cytoplasm, and positive expression was observed in more than 80% of cells. Vimentin was expressed only in the cytoplasm, and positive expression was observed in more than 90% of cells. Conclusions The present study cultured and purified Müller cells successfully using the enzymatic digestion and mechanical pipetting method. The retinal Müller cells can be identified by both GS and Vimentin, and using them together improves the Müller cell discrimination. With prolonged in vitro culture, Müller ce

关 键 词：视网膜MÜLLER细胞 体外培养 传代培养 增生性玻璃体视网膜病变 

分 类 号：R774.1[医药卫生—眼科]