鼻咽癌变不同时期的体外三维培养模型建立的实验研究

Establishment of Three-dimensional Culture Models Related to Different Stages of Nasopharyngeal Carcinogenesis

背景与目的:三维培养更能模拟体内细胞的微环境,细胞的生物学特性比二维培养更接近于活体组织。本研究建立能反映鼻咽癌变过程中不同时期的鼻咽上皮细胞三维培养模型,为进一步研究鼻咽癌的发病机制提供模型。方法:非癌鼻咽活检标本体外培养用于建立鼻咽正常细胞;早期和晚期传代的正常细胞NPNE2、永生化的鼻咽上皮细胞NP69SV40T、鼻咽癌细胞SUNE-1及其高转移潜能亚株5-8F在Matrigel中培养,建立三维培养模型。结果:从非癌鼻咽活检标本长出具有上皮细胞形态特征的细胞,细胞出现衰老前,在体外能够增殖8～10代,角蛋白免疫组化证实其为上皮起源。除晚期传代的NPNE2外,所有细胞均能在三维体系中增殖,NPNE2及NP69SV40细胞主要形成网状结构,克隆形成数少,边沿清楚而光滑,并且细胞间连接紧密;SUNE-1和5-8F形成大而形态不规则的克隆,细胞间连接松散并且克隆边沿不规则,此外,5-8F尚形成大量的伪足,但不形成网状结构;而晚期传代的NPNE2增殖能力低,无法形成细胞网状结构,也不形成细胞克隆。结论:从非癌鼻咽活检标本成功培养出正常鼻咽上皮细胞,采用正常细胞、永生化鼻咽上皮细胞及肿瘤细胞建立了可能反映体内鼻咽癌发生、发展不同时期的三维培养模型。

BACKGROUND ＆ OBJECTIVE： Since three-dimensional culture simulates in vivo microenvironment better than planar culture, the biological character of cells in three-dimensional culture model are more similar with that of living tissues. This study was to establish three-dimensional culture models that represent different stages of nasopharyngeal caicinogenesis, and provide information to elucidate the related molecular events. METHODS： Non-tumor nasopharyngeal biopsy specimens were used to culture for normal nasopharyngeal epithelial cells. Early and late passages of normal nasopharyngeal epithelial cell line NPNE2 from nasopharyngeal biopsy specimens, immortalized nasopharyngeal epithelial cell line NP69SV40T, nasopharyngeal carcinoma （NPC） cell line SUNE-1 and its high metastatic subclone 5-8F were cultured in Matrigel to establish three- dimensional models. RESULTS： The cells grew out of non-tumor nasopharyngeal biopsy specimens displayed the morphologic characteristics as epithelia, and could proliferate in vitro for 8-10 passages before senescence. Immunocytochemical staining of cytokeratin revealed that the cells were of epithelial origin. All of the cells, except late passages of NPNE2 cells, could proliferate in the three-dimensional culture system. NPNE2 and NP69SV40T cells mainly developed reticular structures in morphology, and formed few clones with clear and smooth edges as well as tight intercellular junctions. SUNE-1 and 5-8F cells formed clones with irregular morphology, unclear edge, and loose intercellular junctions. In addition, the clones formed by 5-8F cells also developed a lot of pseudopodia, but developed no reticular structure. Late passages of NPNE2 cells formed no clone and reticular structure in the three-dimensional culture. CONCLUSIONS. Normal nasopharyngeal epithelial cells can be successfully cultured in vitro from naspharyngeal biopsy specimens. The three-dimensional culture models, established with normal nasopharyngeal epithelial cells, immortalized nasopharyngeal epithelial cells, and NPC cells, may represent the different stages of nasopharyngeal carcinogenesis.