鼻咽癌肿瘤相关成纤维细胞的实验分析

Isolation, cultivation and identification of cancer associated fibroblasts in nasopharyngeal carcinoma

摘要:

目的 分离、培养和鉴定鼻咽癌肿瘤相关成纤维细胞（CAFs）及正常成纤维细胞（NFs），并鉴定探讨两种成纤维细胞的生物学差异。方法 临床收集新鲜鼻咽癌组织和鼻咽炎组织经胶原酶和胰酶消化后，利用Matrigel胶混合3D成球培养获得成纤维细胞。通过显微镜观察细胞形态，利用细胞免疫荧光染色、Western bolt和CCK8检测CAFs标准物。结果 分离得到的NFs及CAFs形态和生物学形状存在差异，两者都高表达波形蛋白（Vimentin），CAFs高表达血管平滑肌肌动蛋白（a-SMA），且增殖能力增强。结论 利用Matrigel混合3D成球培养的方法能成功分离、培养成纤维细胞；与NFs相比，CAFs高表达a-SMA，且增殖能力增强。

关键词:鼻咽癌;肿瘤相关成纤维细胞;分离;培养;鉴定

Abstract:

Objective To investigate the isolation, cultivation and identification of the cancer associated fibroblasts (CAFs) in nasopharyngeal carcinoma (NPC) and normal fibroblasts (NFs), To explore the biological difference between the two kinds of fibroblasts. Methods Fresh tissues from NPC patients and nasopharyngitis ones were collected and digested by collagenase and tyrisin, and then the cells were mixed with Matrigel. After culturing with the Matrigel 3D pellet-forming method for a week, the CAFs were obtained. The CAFs associated biomarkers were tested by the immunofluorescence staining,Western blot and CCK8. Results The cell morphology and biological shape of the isolated NFs and CAFs were different. Both of them were highly expressed by Vimentin, while CAFs were also highly expressed by a-smooth muscle actin with enhanced proliferation ability. Conclusions The Matrigel 3D pellet-forming culture method can be performed successfully to isolate and culture fibroblasts. Compared with NFs, CAFs has a high expression of a-SMA and enhanced proliferation ability.

Key words:Nasopharyngeal carcinoma;Cancer associated fibroblast;Isolation;Cultivation;Identification