神经干细胞标志物nestin在成人Müller细胞中表达特征的初步研究  

Preliminary study on the expression of the neural stem cell marker nestin in human Müller cells

作　　者：张荣[1] 章淑杰[1] 陈君毅[2] 何杰[3] 吴继红[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院实验中心,上海200031 [2]复旦大学附属眼耳鼻喉科医院眼科,上海200031 [3]中国科学院神经科学研究所,上海200031

出　　处：《中国眼耳鼻喉科杂志》2016年第5期305-308,共4页Chinese Journal of Ophthalmology and Otorhinolaryngology

基　　金：国家自然科学基金(81470624);上海市卫生局局级科研项目(20124073);上海市科委医学引导项目(134119a880)

摘　　要：目的研究神经干细胞标志物巢蛋白(nestin)在原位和体外培养成人Müller细胞中的表达特征。方法取正常成人眼球,制备冷冻切片,采用免疫荧光方法对谷氨酸合成酶(GS)和nestin进行双标检测;采用免疫荧光方法检测原代和传代培养的Müller细胞nestin表达;取原代培养的成人Müller细胞给予过氧化氢(H2O2)刺激,采用免疫荧光方法检测胶质纤维酸性蛋白(GFAP)和nestin的表达,并计数分析。结果在成人眼球切片中,原位成人Müller细胞GS免疫荧光染色阳性,但nestin染色阴性。原代培养的Müller细胞中检测到部分nestin阳性表达的细胞,10代以内细胞nestin阳性比例为(15.16%±5.07%)^(22.42%±4.6%),且代次间无显著差异。H2O2刺激原代培养的Müller细胞后,GFAP阳性细胞数增加,是对照组的1.30~2.08倍,但nestin阳性细胞比例未见显著变化。结论正常成人Müller细胞原位时呈静息状态,不表达干细胞标志物nestin;体外培养条件可能作为一种刺激因素使部分Müller细胞离开静息状态呈现干细胞特性;人Müller细胞在体外传代及H2O2刺激后nestin阳性细胞数未见显著变化,提示并非全部Müller细胞均具有干细胞潜能,成人Müller细胞可能存在不同的亚型。Objective To study the stem cell properties of human retinal Mailer cells in situ and in vitro. Methods The expression of glutamate synthase （GS） and nestin in the crynosections of human eyes, the expression of nestin in the primary Muller cell culture, and the expression of glial fibrillary acidic protein （GFAP） and nestin with the treatment of H2 O2 were examined by using immunofluorescence staining. Results In the human eye section, Mailer cells were positive in GS but negative in nestin. In contrast, in the primary culture, nestin was expressed in a small set of Mailer cells, which constituted from 15.16% ±5.07% to 22.42% ± 4.6% of the total Mailer cell population. And such proportion was invariable within ten generations. Upon H2 O2 treatment, the GFAP-positive cells increased while the nestin-expressing cell population remained unchanged. Conclusions In adult human eye, Muller cells were in the resting state and nestin was absent. However, soon after cultured in vitro, a subset of Muller cells started to express nestin, a maker for stem cells, suggesting that the culture condition probably facilitated a portion of the Muller cells to leave the resting state. Upon H2O2 treatment, the number of the nestin-positive cells which potentially have the characteristics of stem cells did not change significantly, suggesting the existence of distinct subtypes of human Mailer cells.

关 键 词：成人Muller细胞 巢蛋白 干细胞潜能 亚型 

分 类 号：R774[医药卫生—眼科]