| Lung cance has accounted for the leading cause of cancer related death,and its biological behavior and regulation mechanism are complex and complicated.The establishment of primary lung cancer cell lines can help to explore the molecular mechanism of lung cancer development and provide new targets for the diagnosis and treatment of cancer.CAF are the primary type of stromal cells in the tumor microenvironment and play a pivotal role in tumorigenesis and development.The extreme complexity of CAF's features and their role in tumor development are needed to be further investigated.In this paper we mainly discusses the primary culture of lung cancer cells and cancer-associated fibroblasts,and analyzes the molecular mechanism of cancer-associated fibroblasts in promoting the proliferation and migration of lung cancer cell lines.(1)the primary culture of lung cancer cells and CAFIsolation of lung cancer primary cells and CAF from fresh lung cancer tissue samples by tissue block adherence method and enzymatic digestion method.We used different media and added cytokines to improve cell adhesion and survival,and ultimately failed to isolate and culture stablelung cancer cells.Among the 10 samples we obtained,the success rate of lung cancer-associated fibroblasts was >50%,suggesting that it may be related to the proliferative properties of cancer-associated fibroblasts and the persistence of viability.(2)Identification of CAFThe cell morphology of cancer-associated fibroblasts was "long spindle shape" under phase contrast microscopy,which was significantly different from the polygonal shape of lung cancer cell line A549.Immunofluorescence assay showed high specific expression of fibronectin and vimintin,and no expression of epithelial keratin 8/18(CK8/18).the results shows that we successfully isolated cancer-associated fibroblasts.(3)Analysis of proliferation characteristics of lung adenocarcinoma CAFThe growth characteristics of CAF and A549 were analyzed by CCK-8 reagent and flow cytometry.The proliferation curve of CAF and A549 were S-shaped curves.Both of them had approximate growth characteristics,indicating that CAFs are cell populations with high proliferative properties in lung adenocarcinoma tissues.The percentage of cells in the S phases measured by flow cytometry was significantly higher in CAFs,indicating that the synthesis of DNA as CAFs was more vigorous.while The percentage of cells in G2/M measured by flow cytometry was significantly higher in A549,indicating that the proliferation of A549 wasmore fast.(4)CAF promote proliferation and migration of lung cancer cellsThe effect of CAF conditioned medium on the proliferation of A549 was analyzed by CCK-8 reagent.Compared with serum-free DMEM medium,CAF conditioned culture can significantly promote the proliferation of A549,suggesting that the secretion of CAF during growth can promote the proliferation of lung cancer cell line A549.We analyzed the effect of CAF on the migration ability of A549 by non-contact co-culture through wound healing experiments and Transwell migration experiments.It was evident that CAF promoted the movement and migration of A549 when A549 was co-cultured with CAF.This part of the results suggests that lung cancer-associated fibroblasts can obviously promote the proliferation and migration of lung cancer cells.(5)Effect of CAF on gene expression of lung cancer cellsWe used qRT-PCR to detect the changes of key genes involved in tumor proliferation and migration in A549 co-cultured with CAFs.qRT-PCR results showed that the expression levels of PRR11,c-Myc,PIM1,SOCS3 and CCND1 were significantly increased after 72 hours of serum-free non-contact co-culture,and the expression level of IRF1 gene was significantly decreased.After contact co-culture,the levels of STAT3,IL-6,SOCS3,CCND1,Snail,and Zeb1 genes were significantly increased.This part of the results suggests that CAFs are likely to promote theproliferation and migration of lung cancer cells by activating the STAT3 signaling pathway. |
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