树鼩角膜内皮细胞体外原代培养方法的建立  

Establishment of in Vitro Primary Culture Method for Tree Shrews Corneal Endothelial Cells

作　　者：苗雨润[1] 宋庆凯 陈玲霞[1] 尹博文[1] 李晓飞[1] 代解杰[1] 

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室树鼩种子资源中心,昆明650118

出　　处：《实验动物科学》2016年第5期1-8,共8页Laboratory Animal Science

基　　金：国家科技支撑计划项目(No.2014BAI01B01);联合支持国家计划项目(No.2015GA009)

摘　　要：目的建立树鼩角膜内皮细胞(corneal endothelial cells,CECs)体外分离、培养及纯化的稳定技术,为人类眼科角膜疾病提供新的实验材料。方法通过比较揭膜法、双酶消化法及揭膜-消化两步法,确定每种树鼩原代CECs取材方法的优劣性。分别使用DMEM/F12,M199,Ham’s F12/M199三种培养液以确定树鼩CECs原代细胞最适培养液。使用TGF-β抑制剂、低血清培养法及梯度消化法来探讨树鼩CECs纯化的可行性。苏木精-伊红染色观察细胞形态,根据树鼩神经元烯醇化酶(NES)设计并筛选特异性引物,使用PCR方法检测细胞NES表达情况并用NES抗体进行细胞免疫荧光染色鉴定。结果揭膜法可以快速、有效得到高纯度的树鼩CECs细胞。Ham’s F12/M199培养液是树鼩CECs的最适培养液。使用TGF-β抑制剂可以达到CECs的纯化目的。结论优化的树鼩CECs细胞培养纯化方法对树鼩CECs的体外培养更适宜,优化条件下分离培养的细胞符合CECs的一般特征。Objective To establish an efficient and stable technique for primary isolation,culture and purification of tree shrew corneal endothelial cells（ CECs）,and provide a new experimental material for human ophthalmic corneal diseases. Method For the purpose of seeking the most advantageous way of acquiring tree shrew CECs,in our study,we compared the distrip method,double digests method and method Combining distrip and double digest. We used DMEM/F12,M199,Ham’s F12/M199 cell culture mediums respectively to determine which is the most suitable medium for CECs cultivation. TGF-β inhibitor,low-serum culture method and gradient digestion were also employed to explore the feasibility of tree shrews CECs’ purification. Using hematoxylin-eosin staining to observe the morphology of CECs. We designed and filtrated specific-primers according to tree shrews neuronspecific enolase（ NES） forecast sequences,utlised the PCR method to detect the expression of NES and NES antibody was also used to identify cell immunofluorescence. Result Cornea peeled off method is the most convenient and efficient way to obtain tree shrews CECs with high purificationin comparison with other methods.Ham’s F12/M199 medium is the most suitable medium for tree shrews CECs cultivation. Applying TGF-β inhibitor can help to purify CECs. Conclusion Optimized culture and purification method are more appropriate for in vitro tree shrew CECs culture,and the cells isolated by this manner more conform to CECs characteristics.

关 键 词：树鼩 角膜内皮细胞 体外 原代培养 优化 

分 类 号：Q2-33[生物学—细胞生物学]