初断乳大鼠视网膜微血管周细胞的分离培养  

Isolation and Cultivation of Retinal Microvascular Pericytes from Weanling Rats

作　　者：杨密清 刘光辉[2] 王航[1] 黄陈稳 郑永征[2] 任秉仪[2] 周子鑫[2] 杨巍[1] 纪斌峰 孟春[1] 

机构地区：[1]福州大学生物科学与工程学院,福州350108 [2]福建中医药大学附属人民医院,福州350004 [3]同济大学生命科学与技术学院,上海200092

出　　处：《中国细胞生物学学报》2014年第7期963-969,共7页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81102619);福建省自然科学基金(批准号:2011J01197)资助的课题~~

摘　　要：探讨、优化初断乳大鼠视网膜微血管周细胞（retinal microvascular pericytes,RMPs）的分离、培养方案。分别从15只初断乳大鼠及15只成年大鼠中剜取眼球,采用眼科显微手术器械分离获取视网膜,经碎化、消化、过滤处理,收集视网膜微血管片段,予接种培养。MTT法测绘RMPs生长曲线,通过倒置显微镜观察RMPs形态,免疫荧光法鉴定周细胞标记物。比较2组之间视网膜分离操作时间、完整性、原代细胞数量、周细胞形态和表面标记物表达。结果显示,初断乳大鼠视网膜均成功分离,其中24眼视网膜呈整片分出,6眼视网膜破裂呈碎片状,单个眼球视网膜分离时间为14.3~45.5 s。视网膜分离操作时间及完整性与成年大鼠差异无统计学意义（P〉0.05）,初断乳大鼠原代RMPs细胞产量较成年大鼠高,细胞增殖能力强,细胞形态及表面标记物与成年大鼠一致。研究结果表明,初断乳大鼠视网膜是一种可用于RMPs培养的良好组织原料,该实验成功建立了初断乳大鼠RMPs分离培养体系。The aim of this work was to culture retinal microvascular pericytes （RMPs） from weanling rat and optimize the protocol for isolation and cultivation of rat RMPs. The retinas were obtained separately from weanling rats （n= 15） and adult rats （n= 15） by ophthalmic microsurgery instruments. RMPs were isolated from the retinas by mechanical morcel, enzymatic digestion and filtration, and were cultivated. Cell growth was assessed by MTT. Morphological examination of RMPs was performed by inverted microscopy, and further characterization was analyzed by immunofluorescence. The differences in isolating-time, integrity-ratio, total-numbers, morphol-ogy, and marker-expression were observed. All retinas of weanling rats were successfully isolated. 24 of them were intact and 6 of them were broken fragmentarily. The time for isolating retina from a single eye was about 14.3~45.5 seconds. The isolating-time, integrity-ratio of 2 groups showed no significant difference （P〉0.05）. There was no significant difference in morphology and marker expression between the 2 groups. But the total numbers of primary RMPs and the proliferative ability of the cells from weanling rats were higher than those from adult rats. These resuits suggested that the retinas of weanling rats were favorable tissue source for RMPs culture. Here we established a protocol for the isolation and cultivation of RMPs from weanling rats.

关 键 词：初断乳大鼠 视网膜微血管 周细胞 分离 培养 

分 类 号：R774.1[医药卫生—眼科]