人正常食管鳞状上皮不同原代培养方法的比较  

Comparison of Different Methods in Primary Culture of Human Normal Esophageal Squamous Epithelium

作　　者：何晓琳[1] 李月红[1] 张祥宏[1] 严霞[2] 王俊灵[2] 

机构地区：[1]河北医科大学第二医院病理科,石家庄050000 [2]河北医科大学病理研究室,石家庄050017

出　　处：《中国细胞生物学学报》2011年第6期681-687,共7页Chinese Journal of Cell Biology

基　　金：河北省自然科学基金(No.C2005000763)资助项目~~

摘　　要：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。The purpose of this study was to compare the different methods in primary culture of human normal esophageal epithelial cells （HNEECs） and look for appropriate methods to acquire abundant and well growth esophageal epithelial cells for next experiment. The human normal esophageal epithelial cells were isolated from surgically excisional normal esophagus of patients with esophageal cancer. Tissue explant and enzyme digestion methods were used to clutrue HNEECs with DMEM/F 12 mixed medium and with serum-free medium K-SFM. Gross examination, inverted microscopy observation and immunocytochemistry had been used to observe and identify growth, morphological characteristics of the regenerate esophageal epithelial cells. In DMEM/F12 mixture medium using tissue explant method, the regenerated esophageal epithelial cells, shaping slabstone appearance, grew fast with little fibroblasts pollution, and covered 70%-80% area of culture flask after 15-17 days, but the passage cells grew with more fibroblasts pollution. In the serum-free medium K-SFM using enzyme digestion method, the cells grew as well as the former, covered 70%-80% area of culture flask after only 10-12 days. Moreover, the cells can be used for cryopreservation, recovery and passage. The cells cultured with the other methods were not as good as the cells cultured with these two methods. About 90% of the regenerated cells, cultured with above methods, were cytokeratin positive staining with immunohistochemistry, which indicated that they were all epithelial cells. For primary culture of human esophageal epithelial cells, enzyme digestion method with the serum-free medium K-SFM is the best, but expensive. Tissue explant method with DMEM/F12 mixture medium is cheap with a long culture time, but not for passage. The two methods both can be choosen for different experiments.

关 键 词：食管 上皮细胞 原代培养 

分 类 号：R735.1[医药卫生—肿瘤]