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该研究通过比较人正常食管鳞状上皮不同的原代培养方法,以期为不同的实验目的提供不同的培养方法。实验用到的正常食管粘膜上皮来源于食管癌患者手术切除的标本,采用组织块法和酶消化法,分别用DMEM/F12混合培养基和K-SFM无血清培养基进行培养。通过直接观察、细胞形态学观察和免疫细胞化学方法观察细胞的生长情况、细胞形态学特征及鉴定所得到的细胞,比较不同方法与不同培养基组合中原代培养细胞的生长状况。用组织块法,在DMEM/F12混合培养基中人正常食管上皮细胞生长较好,细胞融合较快,成纤维细胞污染较少,15～17天上皮细胞铺满瓶底的70%～80%,获得的细胞数量大,但细胞传代后成纤维细胞污染严重。用酶消化法,在K-SFM无血清培养基中人正常食管上皮细胞生长好,细胞融合快,成纤维细胞污染基本消除,细胞纯度高,10～12天细胞便可以铺满瓶底的70%～80%,这种方法培养的细胞可以冻存、复苏和传代。其余各种培养方法所得细胞无论在生长状态、培养周期、成纤维细胞污染和传代方面均较前两种方法差。以上各种方法培养的细胞经免疫细胞化学染色鉴定证实细胞呈广谱细胞角蛋白阳性,确定是食管上皮来源的细胞。酶消化法加K-SFM无血清培养基是本实验获得的原代培养食管上皮细胞的最佳方法,但是费用相对较高。组织块法加DMEM/F12混合培养基价格低廉,但周期较前者稍长且不能用于传代。两者均是适合广泛应用的正常食管上皮细胞培养方法,可以根据不同需要选择不同方法。
This study compares different primary cultures of human normal esophageal squamous epithelium with a view to providing different culture methods for different experimental purposes. The normal esophageal mucosa epithelium derived from the esophageal cancer patients were resected by using the tissue block method and enzyme digestion method, respectively, with DMEM / F12 mixed medium and K-SFM serum-free medium for culture. The growth of primary cultured cells was observed by direct observation, cell morphological observation and immunocytochemistry. The morphological characteristics of cells and the identified cells were compared. The growth of primary cultured cells in different methods and different culture medium was compared. With the tissue block method, human normal esophageal epithelial cells grew well in DMEM / F12 mixed medium with faster cell fusion and less fibroblast contamination. The epithelial cells covered 70% -80% , The number of cells obtained is large, but the cells are heavily contaminated by fibroblasts after passage. Using enzyme digestion, human normal esophageal epithelial cells grow well in K-SFM serum-free medium, cell fusion fast, fibroblast contamination basically eliminated, cell purity is high, 10 to 12 days cells can be covered with 70 % ~ 80%, cells cultured in this way can be frozen, resuscitation and passage. The rest of the culture methods obtained cells in terms of growth status, incubation period, fibroblast contamination and passaging were worse than the first two methods. Cells cultured in the above methods were identified by immunocytochemical staining and confirmed to be broad-spectrum cytokeratin-positive cells, which were identified as esophageal-derived cells. Enzymatic digestion plus K-SFM serum-free medium is the best method for primary culture of esophageal epithelial cells obtained in this experiment, but the cost is relatively high. Tissue block plus DMEM / F12 mixed medium is inexpensive, but the cycle is slightly longer than the former and can not be used for passage. Both are suitable for a wide range of applications of normal esophageal epithelial cell culture methods, according to different needs to choose different methods.