目的 探究西紅花苷對與腫瘤成纖維細胞（CAFs）共培養(yǎng)的結直腸癌細胞放射敏感性的影響，并探究分子機制。方法 通過實時熒光定量聚合酶鏈式反應（qRT-PCR）和蛋白質免疫印記（Western blotting）比較人正常結腸上皮細胞NCM460和結直腸癌細胞系SW480、SW620、HCT116、LOVO中活化T細胞核因子c3（NFATc3）表達差異。建立CAFs與HCT116細胞共培養(yǎng)體系，西紅花苷處理細胞后經不同劑量（0、2、4、6、8 Gy）X射線照射，細胞克隆形成實驗分析細胞存活分數（SF）。再將HCT116細胞分為對照組、6 Gy組、CAFs/HCT116＋6 Gy組、CAFs/HCT116＋西紅花苷＋6 Gy組，進行對應處理后，MTT法檢測各處理組HCT116細胞活性，流式細胞術檢測各處理組HCT116細胞凋亡率，Hoechst 33258染色觀察各處理組HCT116細胞凋亡形態(tài)，qRT-PCR和Western blotting測定各處理組HCT116細胞中NFATc3表達水平。結果 相較于人正常結腸上皮細胞NCM460，人結直腸癌細胞系SW480、SW620、HCT116、LOVO中的NFATc3 mRNA和蛋白相對表達量均顯著上調（P＜0.05）。與HCT116細胞比較，與CAFs共培養(yǎng)的HCT116細胞經輻射后的SF顯著增高（P＜0.05）；與CAFs共培養(yǎng)的HCT116細胞比較，與CAFs共培養(yǎng)的HCT116細胞經西紅花苷處理再進行輻射的SF顯著降低（P＜0.05）。與6 Gy組比較，CAFs/HCT116＋6 Gy組HCT116細胞存活率顯著升高（P＜0.05），細胞凋亡率顯著下降（P＜0.05），細胞內染色也較為均勻，未見致密濃染的凋亡狀細胞，細胞中NFATc3 mRNA和蛋白相對表達量均顯著上調（P＜0.05）；而與CAFs/HCT116＋6 Gy組比較，CAFs/HCT116＋西紅花苷＋6 Gy組HCT116細胞存活率顯著降低（P＜0.05），細胞凋亡率顯著升高（P＜0.05），細胞中有較多致密濃染、碎裂熒光塊，同時，細胞中NFATc3 mRNA和蛋白相對表達量顯著下調（P＜0.05）。結論 西紅花苷能夠明顯提高與CAFs共培養(yǎng)的結直腸癌HCT116細胞的放射敏感性，從而促進細胞發(fā)生凋亡，該作用可能與抑制NFATc3有關。

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

新疆維吾爾自治區(qū)自然科學基金資助項目(2022D01C302)