口腔癌细胞/成纤维细胞三维共培养模型的建立和评价

Establishment and evaluation of three-dimensional co-culture model of oral cancer cells/fibroblasts

目的:建立口腔癌细胞/成纤维细胞三维共培养模型。方法:体外分离培养口腔黏膜正常成纤维细胞(NFs),与口腔癌细胞Cal27共培养构建三维模型,通过HE及免疫荧光染色观察三维共培养模型中Cal27和NFs的生长情况及之间的相互关系。结果:通过原代培养成功获取NFs。三维共培养悬浮培养4 d,胶原凝胶收缩,Cal27细胞呈单层生长;胶原凝胶转移到支架上,气液相界面培养条件下,Cal27细胞呈多层生长,培养3 d时癌细胞基底部出现类基底膜样结构,培养9 d时基底膜样结构趋于完整。结论:三维气液相界面培养模型不同于二维细胞培养,其构建了类似于人体口腔组织的上皮层及其下面的间质细胞层,使口腔癌细胞在生长模式上更接近人体组织,从而为研究口腔癌的侵袭转移提供新的方法和手段。

Objective: To establish a three-dimensional co-culture model of oral cancer cells/fibroblasts and provide a new research method for the follow-up study on the invasion and metastasis of oral cancer. Methods: The method of tissue block was used to isolate and cultivate primary oral mucosa of normal fibroblasts( NFs). NFs and oral cancer cells( Cal27) were used to construct the three-dimensional co-culture model. HE and immunofluorescence staining were used to observe the growth and relationship between Cal27 and NFs of the co-culture model. Results: NFs were successfully harvested by primary culture. Using a floating culture for 4 days,the collagen gel contracted and Cal27 cells showed monolayer growth. When the collagen gel was transferred to the air-liquid interface culture conditions,Cal27 cells showed multilayer growth after 3 days,and the tumor cells exhibited a basement membrane like structure. After9 days,the basement membrane like structure grew integrated. Conclusions: The three-dimensional air-liquid interface culture model is different from two-dimensional cell culture,which is similar to human tissue structure to form the oral epithelium and the underlying stromal cell layer,so that the oral cancer cells more resemble the human tissue in the growth pattern. Thus it provides a new method and approach for the study of oral cancer.