干扰素-γ消除肿瘤相关成纤维细胞介导非小细胞肺癌细胞辐射抵抗的实验研究

Interferon-γeliminates radiation resistance in non-small cell lung cancer cells mediated by cancer-associated fibroblasts

目的探讨干扰素-γ消除肿瘤相关成纤维细胞(CAFs)介导的非小细胞肺癌细胞辐射抵抗的效果。方法从2020年12月至2021年6月温州市中心医院胸外科行根治性切除的15例肺癌患者临床标本中提取CAFs,通过流式细胞术、HE染色、免疫组化对分离出的CAFs进行鉴定。将肺腺癌细胞A549和肺鳞癌细胞H460置于直线加速器中辐射。采用Transwell和CAFs细胞上清液培养两种方法对CAFs与辐射后的A549和H460共培养,流式细胞术检测肿瘤细胞凋亡占比,CCK-8法检测肿瘤细胞活性;高效液相色谱法检测CAFs细胞上清液中谷胱甘肽(GSH)、半胱氨酸、胱氨酸水平;Western blot检测CAFs细胞内胱氨酸转运蛋白xCT、肿瘤细胞内DNA损伤修复蛋白γH2AX、信号传导及转录激活蛋白1(STAT1)表达情况。结果CAFs与辐射后肿瘤细胞共培养后,辐射后肿瘤细胞凋亡占比下降,细胞活性增加;CAFs提高了肿瘤细胞内GSH水平,而干扰素-γ降低CAFs细胞上清液中GSH、半胱氨酸水平,提高胱氨酸水平,进而减少肿瘤细胞活性;干扰素-γ下调xCT在CAFs中表达,干扰素-γ作用后CAFs组的肿瘤细胞中γH2AX表达相应增加,CAFs细胞内STAT1获得激活并使其成为磷酸化STAT1。结论CAFs通过释放GSH来诱导非小细胞肺癌细胞对辐射抵抗,而干扰素-γ则下调xCT在CAFs中表达,进而消除其介导的辐射抵抗。

Objective To explore the effect of interferon-γon radiation resistance of non-small cell lung cancer(NSCLC)cells mediated by cancer-associated fibroblasts(CAFs).Methods CAFs were isolated from resected NSCLC samples and identified by the flow cytometry,HE staining and immunohistochemistry,human lung adenocarcinoma A549 cells and squamous lung cancer H460 cells were irradiated in linear accelerator and co-cultured with CAFs by Transwell and CAFs supernatant.The cell apoptosis rate was detected by flow cytometry,and the cell viability was detected by CCK-8.The concentrations of glutathione,cysteine and cystine in CAFs supernatant were detected by HPLC.The protein levels ofγH2AX and xCT were detected by Western blot.Results The protein expression group the proportion of cancer cell apoptosis was decreased,and the cancer cell activity was increased,which was significantly different from that of the control group(P<0.01).CAFs increased the expression level of GSH in cancer cells.Interferon-γdecreased the levels of glutathione and cysteine and increased cystine level in the supernatant of CAFs,while the viability of tumor cells was also decreased(P<0.01).Interferon-γdown-regulated the expression of xCT in CAFs,and up-regulated theγH2AX expression in tumor cells.Conclusion CAFs induce resistance to radiation of NSCLC cells by releasing GSH,while interferon-γcan downregulate xCT expression in CAFs,eliminating CAFs-mediated radioresistance of NSCLC cells.