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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: Three-dimensional culture can mimic the microenvironment of cells in vivo. The biological characteristics of cells are more similar to that of two-dimensional culture. In this study, a three-dimensional culture model of nasopharyngeal epithelial cells that can reflect different stages of nasopharyngeal carcinogenesis was established to provide a model for further study on the pathogenesis of nasopharyngeal carcinoma. Methods: Non-tumor nasopharyngeal biopsy specimens were used to establish normal nasopharyngeal cells in vitro. NPNE2, NP69SV40T, nasopharyngeal epithelial cells SUNE-1 and their metastatic potential subtypes 5-8F in Matrigel culture, the establishment of three-dimensional culture model. Results: The cells with morphological characteristics of epithelial cells were obtained from non-cancerous nasopharyngeal biopsy specimens. The cells proliferated for 8 to 10 generations in vitro before senescence. Keratin immunohistochemistry confirmed the origin of epithelial cells. In addition to the late passage of NPNE2, all cells can proliferate in the three-dimensional system, NPNE2 and NP69SV40 cells mainly formed a network structure, a small number of cloned, clear and smooth edges, and tight junctions; SUNE-1 and 5-8F In addition, 5-8F still formed a large number of pseudopodia, but did not form a network structure; and late passage of NPNE2 proliferation of low, unable to form cells Reticular structure, nor the formation of cell clones. CONCLUSION: Normal nasopharyngeal epithelial cells are successfully cultured from non-cancerous nasopharyngeal biopsy specimens. Three-dimensional culture models that may reflect the occurrence and development of nasopharyngeal carcinoma in vivo are established using normal cells, immortalized nasopharyngeal epithelial cells and tumor cells.