目的：研究Notch4 信号通路对口腔鳞癌PJ15 和PJ41 细胞的肿瘤干细胞（cancer stem cell，CSC）自我更新能力的影响及其作用机制。方法：利用TCGA数据库分析Notch4 基因在头颈部肿瘤中的表达。2 μmol/L 顺铂处理口腔鳞癌PJ15 和PJ41 细胞48 h，再加入Notch 通路抑制剂DAPT作用24 h，采用流式细胞术检测CSC比例，Western blotting 法和qPCR法检测CSC标志物（Sox2、Bmi-1、Oct4、Nanog）和Notch4 信号通路中的下游基因（JAG1、HEY1、HEY2、HES1、HES2、DLL4 等）的表达。采用siRNA技术敲降Notch4，Western blotting 法和qPCR 法检测si-Notch4 和顺铂对CSC标志物表达水平的影响，干细胞成球实验检测CSC的自我更新能力。结果：头颈部肿瘤组织中Notch4 基因呈高表达（P=0.046）。顺铂处理后，PJ15 和PJ41 细胞中NICD4 和CSC标志物（Sox2、Bmi-1、Oct4、Nanog ）和Notch4 信号通路的下游基因（JAG1、HEY1、HEY2、HES1、HES2、DLL4）的表达水平较对照组均显著增加（均P<0.05），ALDH1 阳性细胞群体比例显著增加（P<0.05）；再加入Notch 通路抑制剂DAPT 作用24 h 后上述基因表达量均显著下降（P<0.05 或P<0.01）。敲降Notch4 后，与对照组相比，si-Notch4 组PJ15 细胞[ （1.33±0.47）vs（8.00±0.82）个，P<0.01]和PJ41 细胞[ （1.00±0.82）vs（7.67±1.25）个，P<0.01]球体的数量和大小均显著减少；再加入2 μmol/L DDP作用24 h，si-Notch4 组Nanog 和Bmi-1 基因表达量与对照组无显著差异。结论：激活Notch4 信号通路可增加口腔鳞癌PJ15 和PJ41 细胞的干细胞自我更新能力。

Objective: To study the effect of Notch4 signaling pathway on the self-renewal capacity of cancer stem cells (CSCs) of oral squamous cell carcinoma PJ15 and PJ41 cells and its mechanism. Methods: The expression of Notch4 gene in head and neck tumors was analyzed using TCGA database. 2 μmol/L cisplatin was used to treat oral squamous cell carcinoma PJ15 and PJ41 cells for 48 h,following with the treatment of Notch pathway inhibitor DAPT for 24 h. Flow cytometry was used to detect the ratio of CSCs, Western blotting and qPCR were used to detect the expressions of CSCs markers (Sox2, Bmi-1, Oct4, Nanog) and downstream genes in Notch4 signaling pathway (JAG1, HEY1, HEY2, HES1, HES2, DLL4, etc.). Notch4 was knocked down by siRNA technology. Notch4 was silenced with siRNA technology. Western blotting and qPCR were used to detect the effect of si-Notch4 and cisplatin on the expression level of CSCs markers. The stem cell pelleting test was used to detect the self-renewal ability of CSCs. Results: Notch4 gene was highly expressed in head and neck tumor tissues (P=0.046). After cisplatin treatment, compared with the control group, expressions of NICD4 and CSCs markers (Sox2, Bmi-1, Oct4, Nanog) as well as downstream genes of Notch4 signaling pathway (JAG1, HEY1, HEY2,HES1, HES2, DLL4, etc.) increased significantly (all P<0.05), and the proportion of ALDH1 positive cells increased significantly (P<0.05); with the further addition of DAPT, the expressions of the above genes decreased significantly (P<0.05 or P<0.01). After knocking down Notch4, compared with the control group, the size and number of spheres of PJ15 cells [1.33±0.47] vs [8.00±0.82],P<0.01) and PJ41 cells ([1.00±0.82] vs [7.67±1.25], P<0.01) were significantly lower than that of the control group; after the addition of 2 μmol/L DDP for 24 h, there was no significant difference in the expressions of Nanog and Bmi-1 gene between si-Notch4 group and control group. Conclusion: Activation of Notch4 signaling pathway can enhance the self-renewal ability of CSCs in oral squamous cell carcinoma PJ15 and PJ41 cells.

上海市自然科学基金资助项目（No. 16ZR1416100）