目的 探究西紅花苷對(duì)與腫瘤成纖維細(xì)胞（CAFs）共培養(yǎng)的結(jié)直腸癌細(xì)胞放射敏感性的影響，并探究分子機(jī)制。方法 通過實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)（qRT-PCR）和蛋白質(zhì)免疫印記（Western blotting）比較人正常結(jié)腸上皮細(xì)胞NCM460和結(jié)直腸癌細(xì)胞系SW480、SW620、HCT116、LOVO中活化T細(xì)胞核因子c3（NFATc3）表達(dá)差異。建立CAFs與HCT116細(xì)胞共培養(yǎng)體系，西紅花苷處理細(xì)胞后經(jīng)不同劑量（0、2、4、6、8 Gy）X射線照射，細(xì)胞克隆形成實(shí)驗(yàn)分析細(xì)胞存活分?jǐn)?shù)（SF）。再將HCT116細(xì)胞分為對(duì)照組、6 Gy組、CAFs/HCT116＋6 Gy組、CAFs/HCT116＋西紅花苷＋6 Gy組，進(jìn)行對(duì)應(yīng)處理后，MTT法檢測(cè)各處理組HCT116細(xì)胞活性，流式細(xì)胞術(shù)檢測(cè)各處理組HCT116細(xì)胞凋亡率，Hoechst 33258染色觀察各處理組HCT116細(xì)胞凋亡形態(tài)，qRT-PCR和Western blotting測(cè)定各處理組HCT116細(xì)胞中NFATc3表達(dá)水平。結(jié)果 相較于人正常結(jié)腸上皮細(xì)胞NCM460，人結(jié)直腸癌細(xì)胞系SW480、SW620、HCT116、LOVO中的NFATc3 mRNA和蛋白相對(duì)表達(dá)量均顯著上調(diào)（P＜0.05）。與HCT116細(xì)胞比較，與CAFs共培養(yǎng)的HCT116細(xì)胞經(jīng)輻射后的SF顯著增高（P＜0.05）；與CAFs共培養(yǎng)的HCT116細(xì)胞比較，與CAFs共培養(yǎng)的HCT116細(xì)胞經(jīng)西紅花苷處理再進(jìn)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(xì)胞存活率顯著升高（P＜0.05），細(xì)胞凋亡率顯著下降（P＜0.05），細(xì)胞內(nèi)染色也較為均勻，未見致密濃染的凋亡狀細(xì)胞，細(xì)胞中NFATc3 mRNA和蛋白相對(duì)表達(dá)量均顯著上調(diào)（P＜0.05）；而與CAFs/HCT116＋6 Gy組比較，CAFs/HCT116＋西紅花苷＋6 Gy組HCT116細(xì)胞存活率顯著降低（P＜0.05），細(xì)胞凋亡率顯著升高（P＜0.05），細(xì)胞中有較多致密濃染、碎裂熒光塊，同時(shí)，細(xì)胞中NFATc3 mRNA和蛋白相對(duì)表達(dá)量顯著下調(diào)（P＜0.05）。結(jié)論 西紅花苷能夠明顯提高與CAFs共培養(yǎng)的結(jié)直腸癌HCT116細(xì)胞的放射敏感性，從而促進(jìn)細(xì)胞發(fā)生凋亡，該作用可能與抑制NFATc3有關(guān)。

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(xué)基金資助項(xiàng)目(2022D01C302)