【摘要】 目的：应用组织块培养大鼠视网膜干细胞的有效性。方法:机械分离出生10d大鼠睫状体组织，剪成细小组织块，置入20μg/L 碱性成纤维细胞生长因子、20μg/L表皮生长因子及1×B27添加剂的无血清DMEM/F12培养液中培养，同时取大鼠胚胎脑组织，应用常规机械酶消化法进行神经干细胞的分离培养作为阳性对照。应用免疫荧光染色检测神经干细胞特异性抗原神经巢蛋白(nestin)的表达，应用含100mL/L的胎牛血清的培养基促进其分化以确定其神经干细胞特性。结果:原代培养48h后在组织块边缘开始出现细胞团，折光性强，呈球形或者桑葚状悬浮生长。培养4～5d后，悬浮生长的细胞团数量增多，出现较大的细胞团，原代培养7d后传代，传代后细胞能重新形成细胞团。应用免疫荧光方法可检测到细胞内nestin阳性表达，且视网膜干细胞可在以血清为诱导条件下变为贴壁生长，并分化出具有神经细胞形态的细胞。其形态、增殖及可分化特性与作为阳性对照的神经干细胞相似。结论：组织块培养法对细胞损伤小，操作简便，可成功对大鼠视网膜干细胞进行分离培养。
【关键词】 组织块培养法;视网膜干细胞;分离;培养;鉴定
Isolation, cultivation and identification of retinal stem cells by tissue cultivation
ShouNan Qi, GuanFang Su, ChenGuang Wang, ShuRong Wang, Yu Mu
Foundation item:Youth Fund of Science and Technology Projects of Jilin Province, China(No.20080136)
Department of Ophthalmology,the Second Hospital of Jilin University, Changchun 130041, Jilin Province,China
Correspondence to:GuanFang Su.Department of Ophthalmology,the Second Hospital of Jilin University, Changchun 130041, Jilin Province,[email protected]
Received:20100603 Accepted:20100806
Abstract
AIM: To observe the efficiency of cultivation of retinal stem cells (RSC) in vitro by tissue cultivation method.
METHODS: The tissue of ciliary body of postnatal 10 days rat was isolated and divided into small bits, then cultivated with nonserum DMEM/F12 medium, 20μg/L bFGF, 20μg/L EGF and 1×B27 supplement at the same time, the embryonic brain derived neural stem cells (NSC) were isolated and cultivated with routine method as positive control. These two kinds of cells were identified by detection of nestin with immunofluorescence method, which was known as a marker of neural stem cells. The characteristic of differentiation of RSC was identified in the induced condition of 100mL/L FBS medium.
RESULTS: In the primary cultivation, cell spheres with high refraction were observed 48 hours after isolation and cultivation. The cell spheres enlarged and the amount increased gradually in the fourth or fifth day of cultivation. The passage of the cells occurred in the seventh day of cultivation, and the cells of the next passage can also formed the sphere shape. The marker nestin was detected both in RSC and NSC by immunofluorescence method. The RSC became adherent with the bottom of the culture dish in the induced condition with serum, and some of the cells differentiated into cells with neural cell shapes. The characteristics of the shape, proliferation and differentiation of RSC were similar with NSC.
CONCLUSION: Tissue cultivation has the characteristics of less damage and convenience, which can be used in the isolation and cultivation of RSC successfully.
KEYWORDS: tissue cultivation; retinal stem cell; isolation; cultivation; identification
Qi SN, Su GF, Wang CG, et al. Isolation, cultivation and identification of retinal stem cells by tissue cultivation. Int J Ophthalmol
(Guoji Yanke Zazhi) 2010;10(9):16651667
视网膜干细胞(retinal stem cell, RSC)是近年来发现的成体干细胞之一。RSC在体外培养中可自我更新和增殖，亦具有多向分化的潜能，可在一定条件下诱导分化成为神经元细胞及神经胶质细胞。RSC在体外培养、分化与移植方面的研究进展为我们最终应用这些研究成果来有效的治疗致盲性眼病带来了希望，因而，成功对其进行分离，体外培养及鉴定则为进一步研究其分化机制及细胞治疗、基因治疗奠定基础。图1原代干细胞(IM×100) A:视网膜干细胞;B:神经干细胞。图2Nestin免疫荧光染色阳性(IF×100) A:第2代视网膜干细胞;B:第2代神经干细胞。
1材料和方法
1.1材料
孕18d Wistar大鼠及出生10d大乳鼠，由吉林大学动物中心提供。DMEM/F12培养基、胎牛血清(Gibco公司)，B27添加剂(Invitrogen公司)，胰蛋白酶、EDTA(Sigma公司)，神经巢蛋白(nestin)兔抗大鼠多克隆抗体，FITC羊抗兔抗体(武汉博士德公司)， CO2培养箱(日本SANYO公司)，倒置相差显微镜( Olympus)，超净工作台(苏州净化设备厂)等。
1.2方法
将出生10d的Wistar大乳鼠断颈处死后置于750mL/L乙醇中消毒5min，取出眼球，浸泡于DMEM/F12培养液的培养皿中。更换新的无菌器械，在解剖显微镜下，首先剔除眼球外组织及视神经，在角巩膜缘后3～4mm处环形剪开眼球，仔细去除晶状体、玻璃体及视网膜。取出睫状体组织放入另一无菌皿内，应用微观剪刀尽量将组织剪成碎块，加入DMEM/F12培养液吹打均匀，移入24孔板中培养，并于孔板中加入20μg/L大鼠表皮生长因子(EGF)，20μg/L大鼠碱性成纤维生长因子(bFGF)及1×B27添加剂，于37℃,50mL/L CO2、饱和湿度条件下培养。每天以倒置显微镜观察细胞形态变化和生长状况。当细胞形成较大团体时予以传代，首先离心收集细胞，用2.5g/L含有EDTA的胰蛋白酶500μL消化细胞约2min，以含100mL/L胎牛血清的培养液中止消化，用移液枪吹打使细胞分散，离心去上清后，应用DMEM/F12培养液洗涤1遍，按1∶2比例传代，每3d半量换液，约7d传代1次。应用免疫荧光法对RSC及对照组神经干细胞进行鉴定。分别应用多聚赖氨酸处理的玻片进行细胞爬片，取出玻片，以0.1mol/L PBS冲洗1次，应用40g/L多聚甲醛固定20min，以PBS冲洗5min×3次，2g/L Triton 100作用10min，0.1mol/L PBS洗4min×3次，加入血清室温封闭20min，倾去血清，加入一抗 (1∶100)孵育过夜(4℃)， PBS洗3次,每次5min。空白对照用PBS代替一抗。加入FITC标记的羊抗兔抗体，避光室温放置30min，0.1mol/L PBS洗3次,每次5min。中性树脂封片，荧光显微镜下观察。另取孕18d大鼠，脱颈处死后浸泡于750mL/L乙醇5min。用碘酊及750mL/L乙醇进一步消毒小鼠腹部，无菌器械剪开孕鼠腹部皮肤，取出受孕的子宫，移入呈有PBS液的培养皿中。PBS液冲洗两遍，剪开离体的子宫，取出胚胎，应用眼科剪剪开头骨，小心将脑组织移入另一培养皿中，应用DMEM/F12培养液冲洗，去除脑膜及血管，并应用微观剪刀将组织剪碎，移入加有1mL 2.5g/L含有EDTA的胰蛋白酶的离心管内，置于37℃下消化15min，每5min振荡1次。加入含有100mL/L胎牛血清的DMEM/F12培养液终止消化，离心弃上清，应用无血清DMEM/F12培养液洗涤1遍，200目筛网过滤，制成单细胞悬液，以密度为109 /L将细胞接种于6孔板中，培养条件同RSC。收集第2代RSC及神经干细胞，应用含100mL/L胎牛血清的DMEM/F12培养液诱导分化，37℃,50mL/L CO2、饱和湿度条件下培养，倒置显微镜下观察其分化情况。
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