人肺癌相关成纤维细胞的原代培养及其放射激活初探

Activation of Human Lung Cancer-associated Fibroblasts Induced by Irradiation

[目的]探讨在体外培养条件下,不同剂量的X线照射对人肺癌相关成纤维细胞(cancer-associated fibroblasts,CAFs)表型的影响。[方法]用经组织块贴壁法从手术标本中提取人肺癌CAFs并原代培养。使用0Gy、4Gy、8Gy、12Gy、16Gy的不同剂量X线照射CAFs,通过Western blot、qRT-PCR检测其平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、波形蛋白(Vimentin)、成纤维细胞活化蛋白(fibroblast activation protein,FAP)、平足蛋白(podoplanin,PDPN)的蛋白和RNA水平;使用CCK8、划痕实验及流式细胞术检测细胞增殖和迁移及凋亡情况。[结果]与其他组比较,8Gy照射组的人肺CAFs中α-SMA、Vimentin、FAP、PDPN在蛋白和RNA水平表达更高。划痕实验提示8Gy照射组在48h首先愈合。CCK8研究提示该组细胞增殖活性明显高于其他照射组;流式细胞术显示照射后该组细胞凋亡率为37%,与0Gy组(36%)接近,明显低于其他照射剂量组。[结论]经过一定剂量的X线照射后,人肺CAFs可表达更高水平的标志蛋白、同时表现出更强的生长和迁移能力,而该表型变化在8Gy照射组最为显著,为将来深入探讨CAFs的放射激活机制奠定基础。

[Objective] To investigate the effect of irradiation on the activation of human lung cancer-associated fibroblasts(CAFs) in vitro. [Methods] The primarily cultured lung cancer-associated fibroblasts were irradiated with different dosage of X-ray (0,4,8,12 and 16 Gy). The protein and mRNA expressions of α-SMA,vimentin,FAP and PDPN were detected by Weston blot and qRT-PCR,respectively. The proliferation and the migration ability of CAFs were determined by CCK8,wound healing and flow cytometry. [Results] Compared with other groups,the expression levels of α-SMA,vimentin,FAP,PDPN proteins and mRNA were significantly higher,and the migration ability was also significantly higher in the 8 Gy. The cell proliferation was higher and apoptosis rate was lower in 8 Gy group than those of other groups. [Conclusion] The irradiation of X-ray at dosage of 8 Gy can up-regulate the expression of fibroblast molecular markers and enhance cell proliferation and migration,as well as inhibit cell apoptosis of human lung cancer-associated fibroblasts in vitro,indicating that the irradiation at appropriate dosage can induce the activation of CAFs.