[摘 要] 目的：探讨从小分子化合物库中筛选获得的小分子化合物 WAY-312对胶质母细胞瘤干细胞（GSC）增殖与自我更新 能力的抑制作用及分子机制。方法：通过肿瘤球形成实验与细胞活力检测技术，对小分子化合物库开展系统性化合物筛选；以 筛选得到的小分子抑制剂WAY-302386（按化合物序号将其命名为WAY-312）为研究对象，采用CCK-8 法、WB 法、流式细胞 术及Caspase-Glo 3/7 活性检测法，检测WAY-312 在不同浓度下对GSC 增殖能力、干性标志物表达、细胞周期分布及凋 亡水平的影响，明确WAY-312 对GSC 干性维持、自我更新能力的调控效应，进一步阐明WAY-312对GSC细胞周期进程和细 胞凋亡的作用机制。结果：WAY-312 可有效抑制GSC 存活（P < 0.05）；WAY-312 显著抑制GSC 肿瘤球形成数目和大小 （P < 0.05），WAY-312 处理细胞后降低干性标志物SOX2、OLIG2、BMI1 的表达水平；WAY-312 可有效抑制GSC 的自我更新能力 （P < 0.05）；WAY-312 通过调控细胞周期相关蛋白Cyclin B1、CDK4、CDK6 的表达，诱导GSC 细胞周期阻滞于G2/M 期（P < 0.05）；WAY-312诱导GSC凋亡，增加凋亡相关蛋白cleaved-PARP、BAX的表达，降低BCL2的表达，经WAY-312处理后增强GSC 的Caspase-3/7活性。结论：WAY-312可呈浓度依赖性抑制GSC增殖、干性维持及自我更新能力，诱导GSC周期阻滞于G2/M期 并促进细胞凋亡。

[Abstract] Objective：This study investigated the inhibitory activity and potential mechanism of WAY-312, a small-molecule compound derived through screening of a small-molecule compound library, against the growth and self-renewal capacity of glioblastoma stem cells (GSCs). Methods: Systematic compound screening was conducted on the small molecule compound library through tumor sphere formation assays and cell viability assays; the small molecule inhibitor WAY-302386 (named WAY-312 according to its compound number) obtained through screening was used as the research object. CCK-8 method, Western blot, flow cytometry, and Caspase-Glo 3/7 activity detection method were used to detect the effects of WAY-312 on the proliferation ability, stemness marker expression, cell cycle distribution, and apoptosis level of glioblastoma stem cells at different concentrations. The regulatory effect of WAY-312 on GSC stemness maintenance and self-renewal ability was clarified, and the mechanism of WAY-312 on GSC cell cycle progression and apoptosis was further elucidated. Results: WAY-312 can effectively inhibit the survival of GSCs (P < 0.05); WAY-312 significantly inhibits the number and size of tumor spheroids formed in GSCs (P < 0.05), and treatment with WAY-312 reduces the expression levels of stemness marker proteins SOX2, OLIG2, and BMI1; WAY-312 can effectively inhibit the self-renewal ability of GSCs (P < 0.05); WAY-312 induces GSC cell cycle arrest at the G2/M phase by regulating the expression of cell cycle-related proteins Cyclin B1, CDK4, and CDK6 (P < 0.05); WAY-312 induces apoptosis in GSCs, increases the expression of apoptosis-related proteins cleaved-PARP and BAX, reduces the expression of BCL2, and enhances Caspase-3/7 activity in GSCs after treatment with WAY-312. Conclusion: WAY-312 can inhibit GSC proliferation, stemness maintenance, and self-renewal ability, induce GSC cell cycle arrest at · · 500 车虹宇, 等. 小分子化合物WAY-312通过周期阻滞与凋亡诱导抑制胶质母细胞瘤干细胞增殖及自我更新 G2/M phase, and promote cell apoptosis in a concentration-dependent manner.

[基金项目] 江苏省自然科学基金（BK20251768）