树鼩角膜内皮细胞体外原代培养方法的建立

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。

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

Abstract: 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．