目的 探究西红花苷对与肿瘤成纤维细胞（CAFs）共培养的结直肠癌细胞放射敏感性的影响，并探究分子机制。方法 通过实时荧光定量聚合酶链式反应（qRT-PCR）和蛋白质免疫印记（Western blotting）比较人正常结肠上皮细胞NCM460和结直肠癌细胞系SW480、SW620、HCT116、LOVO中活化T细胞核因子c3（NFATc3）表达差异。建立CAFs与HCT116细胞共培养体系，西红花苷处理细胞后经不同剂量（0、2、4、6、8 Gy）X射线照射，细胞克隆形成实验分析细胞存活分数（SF）。再将HCT116细胞分为对照组、6 Gy组、CAFs/HCT116＋6 Gy组、CAFs/HCT116＋西红花苷＋6 Gy组，进行对应处理后，MTT法检测各处理组HCT116细胞活性，流式细胞术检测各处理组HCT116细胞凋亡率，Hoechst 33258染色观察各处理组HCT116细胞凋亡形态，qRT-PCR和Western blotting测定各处理组HCT116细胞中NFATc3表达水平。结果 相较于人正常结肠上皮细胞NCM460，人结直肠癌细胞系SW480、SW620、HCT116、LOVO中的NFATc3 mRNA和蛋白相对表达量均显著上调（P＜0.05）。与HCT116细胞比较，与CAFs共培养的HCT116细胞经辐射后的SF显著增高（P＜0.05）；与CAFs共培养的HCT116细胞比较，与CAFs共培养的HCT116细胞经西红花苷处理再进行辐射的SF显著降低（P＜0.05）。与6 Gy组比较，CAFs/HCT116＋6 Gy组HCT116细胞存活率显著升高（P＜0.05），细胞凋亡率显著下降（P＜0.05），细胞内染色也较为均匀，未见致密浓染的凋亡状细胞，细胞中NFATc3 mRNA和蛋白相对表达量均显著上调（P＜0.05）；而与CAFs/HCT116＋6 Gy组比较，CAFs/HCT116＋西红花苷＋6 Gy组HCT116细胞存活率显著降低（P＜0.05），细胞凋亡率显著升高（P＜0.05），细胞中有较多致密浓染、碎裂荧光块，同时，细胞中NFATc3 mRNA和蛋白相对表达量显著下调（P＜0.05）。结论 西红花苷能够明显提高与CAFs共培养的结直肠癌HCT116细胞的放射敏感性，从而促进细胞发生凋亡，该作用可能与抑制NFATc3有关。

Objective To investigate the effect of crocin on the radiosensitivity of colorectal cancer cells co-cultured with tumor fibroblasts (CAFs), and to explore the molecular mechanism. Method The expressions of activated T nuclear factor c3 (NFATc3) in human normal colon epithelial cells and colorectal cancer cell lines SW480, SW620, HCT116 and LOVO were compared by real-time quantitative PCR and Western blotting. The co-culture system of CAFs and HCT116 cells was established. Cells treated with crocin were irradiated with different doses (0, 2, 4, 6, 8 Gy), cell survival fraction (SF) was analyzed by cell cloning and formation experiment. Then HCT116 cells were divided into control group, 6 Gy group, CAFs/HCT116 + 6 Gy group, CAFs/HCT116 + crocin + 6 Gy group, after corresponding treatment, the activity of HCT116 cells in each treatment group was detected by MTT assay, the apoptosis rate of HCT116 cells in each treatment group was detected by flow cytometry, the apoptotic morphology of HCT116 cells in each treatment group was observed by Hoechst 33258 staining, the expression level of NFATc3 in HCT116 cells of each treatment group was determined by qRT-PCR and Western blotting. Results Compared with human normal colon epithelial cells NCM460, the relative expression levels of NFATc3 mRNA and NFATc3 protein in human colorectal cancer cell lines SW480, SW620, HCT116, and LOVO were significantly up-regulated (P < 0.05). Compared with HCT116 cells, the SF of HCT116 cells co-cultured with CAFs was significantly increased after irradiation (P < 0.05), compared with HCT116 cells co-cultured with CAFs, the SF of HCT116 cells co-cultured with CAFs and treated with crocin after radiation treatment was significantly decreased (P < 0.05). Compared with the 6 Gy group, the survival rate of HCT116 cells in CAFs/HCT116 + 6 Gy group was significantly increased (P< 0.05), while the apoptosis rate was significantly decreased (P < 0.05), the intracellular staining was more uniform, and no dense and densely stained apoptotic cells were observed, the relative expression levels of NFATc3 mRNA and NFATc3 protein were significantly up-regulated (P < 0.05). Compared with CAFs/HCT116 + 6 Gy group, the survival rate of HCT116 cells in CAFs/HCT116 + crocin + 6 Gy group was significantly decreased (P < 0.05), and the apoptosis rate was significantly increased (P < 0.05), there were more dense dense staining and fragmentation fluorescence blocks in the cells, the relative expression levels of NFATc3 mRNA and NFATc3 protein in cells were significantly down-regulated (P < 0.05). Conclusions Crocin can significantly improve the radiosensitivity of colorectal cancer HCT116 cells co-cultured with CAFs, thus promoting cell apoptosis, and the effect may be related to the inhibition of NFATc3.

R285

新疆维吾尔自治区自然科学基金资助项目(2022D01C302)