人肺腺癌中癌相关成纤维细胞的分离培养与初步分析

Isolation and identification of cancer-associated fibroblasts from human lung adenocarcinoma

摘要:

目的：分离鉴定人肺腺癌组织中的癌相关成纤维细胞（cancer-associataed fibroblasts，CAFs），分析CAFs对肺癌细胞增殖和迁移能力的影响，为进一步探索CAFs在肺癌发生发展中的作用及机制奠定基础。方法：获取人肺腺癌患者手术样本，分离并培养癌组织中的成纤维细胞。采用相差显微镜观察细胞形态，采用间接免疫荧光实验分析关键分子标志物的表达。采用CCK-8法检测细胞增殖，采用流式细胞仪分析细胞周期；采用Transwell实验分析细胞迁移能力。采用共培养和制备条件培养基方法分析CAFs对肺腺癌细胞A549增殖和迁移能力的影响。采用qRT-PCR检测关键基因表达的变化。结果：从人肺腺癌新鲜组织中分离获得CAFs，其形态为长梭形，表达纤粘蛋白和波形蛋白等成纤维细胞标志物，不表达上皮细胞角蛋白8/18（CK8/18）等上皮细胞标志物。肺腺癌CAFs增殖活跃。肺腺癌CAFs能明显促进A549的增殖和迁移能力，并明显上调A549细胞中c-Myc、PIM1、SOCS3和CCND1基因的表达。结论：成功分离了人肺腺癌CAFs，其能明显促进肺癌细胞的增殖和迁移能力。

Abstract:

Objective：To isolate and identify cancer-associated fibroblasts（CAFs） from human lung adenocarcinoma tissue，to investi－gate the effect of CAFs on the proliferation and migration of lung cancer cells，and to lay a foundation for further research on the role and mechanism of CAFs in the development and progression of lung cancer. Methods：Surgical specimens were obtained from patients with lung adenocarcinoma，and fibroblasts were isolated and cultured. A phase-contrast optical microscope was used to observe cell morphology，and indirect immunofluorescence assay was used to measure the expression of key molecular markers. CCK-8 assay was used to evaluate cell proliferation，flow cytometry was used to analyze cell cycle，and Transwell assay was used to assess cell migration ability. The co-culture method and a conditioned medium were used to analyze the effect of CAFs on the proliferation and migration of lung adenocarcinoma A549 cells，and qRT-PCR was used to measure the change in the expression of key genes. Results：CAFs were isolated from fresh human lung adenocarcinoma tissue，with a spindle-like shape. Immunofluorescence assay showed the expression of the fibroblast markers fibronectin and Vimintin in CAFs，while epithelial keratin 8/18 was not found in CAFs. The CAFs isolated from lung adenocarcinoma tissue showed active proliferation and significantly promoted the proliferation and migration of A549 cells and remarkably upregulated the mRNA expression of c-Myc，PIM1，SOCS3，and CCND1 in A549 cells. Conclusion：CAFs are successfully isolated from human lung adenocarcinoma tissue and such CAFs can significantly promote the proliferation and migration of lung cancer cells.