目的 探究新对叶百部碱（NTS）对人胰腺癌细胞MiaPaCa-2和PANC-1凋亡的影响及作用机制。方法 以0（对照组）、20、40、60、80、100 μg·mL⁻¹的NTS分别处理MiaPaCa-2、PANC-1细胞24、48、72 h后，采用MTT法检测细胞存活率；分别设置对照组（0 μg·mL⁻¹）和NTS低、中、高质量浓度（20、40、60 μg·mL⁻¹）组，采用平板克隆法检测NTS对MiaPaCa-2、PANC-1细胞的增殖能力的影响；采用划痕愈合实验检测细胞迁移能力；采用Hoechst 33258染色法观察细胞的凋亡；采用流式细胞术检测细胞的凋亡率、线粒体膜电位；采用Western blotting法检测凋亡相关蛋白B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、活化半胱氨酸蛋白酶-3（cleaved-Caspase-3）、半胱天冬酶-9（Caspase-9）相关蛋白表达水平。结果 与对照组比较，不同质量浓度NTS可显著降低MiaPaCa-2、PANC-1细胞存活率（P＜0.01）、克隆形成率（P＜0.05、0.01）、划痕愈合率（P＜0.05、0.01）、线体膜电位（P＜0.01）；显著增加MiaPaCa-2和PANC-1细胞凋亡水平（P＜0.01）；在蛋白水平上，NTS显著上调促凋亡相关因子cleaved-Caspase-3、Bax、Caspase-9蛋白的表达水平，下调抑制凋亡因子Bcl-2蛋白表达水平（P＜0.05、0.01）。结论 NTS可有效抑制人胰腺癌MiaPaCa-2、PANC-1细胞增殖和迁移能力，并促进其凋亡，其机制可能与下调抑制凋亡蛋白Bcl-2并上调促凋亡蛋白Bax、cleaved-Caspase-3、Caspase-9表达有关。

Objective To explore the effects and mechanisms of neotuberostemonine on the apoptosis of human pancreatic cancer cells MiaPaCa-2 and PANC-1. MethodsMiaPaCa-2 and PANC-1 cells were treated with NTS at concentrations of 0, 20, 40, 60, 80, and 100 μg·mL⁻¹ for 24, 48, and 72 h, respectively. Cell viability was detected by the MTT assay. The control group (0 μg·mL⁻¹), the low, medium, high-concentration group of NTS (20, 40, 60 μg·mL⁻¹), were set up. The effect of NTS on the proliferation ability of MiaPaCa-2 and PANC-1 cells was detected by the plate cloning method. The cell migration ability was detected by the scratch healing assay. Apoptosis was observed by Hoechst 33258 staining. The apoptosis rate and mitochondrial membrane potential were detected by flow cytometry. The expression levels of apoptosis-related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), activated Caspase-3 (cleaved-Caspase-3), and Caspase-9 were detected by Western blotting. Results Compared with the control group, different concentrations of NTS significantly reduced the proliferation activity (P < 0.01), colony formation rate (P < 0.05 or 0.01), scratch healing rate (P < 0.05 or 0.01), and mitochondrial membrane potential (P < 0.01) of MiaPaCa-2 and PANC-1 cells. In addition, NTS increased the apoptosis level of MiaPaCa-2 and PANC-1 cells (P < 0.01). At the protein level, NTS significantly upregulated the expression levels of apoptosis-related factors cleaved-Caspase-3, Bax, and Caspase-9 proteins, while the expression level of the apoptosis factor Bcl-2 protein was significantly downregulated (P < 0.05, 0.01). Conclusion NTS can effectively inhibit the proliferation and migration of human pancreatic cancer cells MiaPaCa-2 and PANC-1 and promote their apoptosis. The mechanism may be related to the downregulation of the anti-apoptotic protein Bcl-2 and the upregulation of the pro-apoptotic proteins Bax, cleaved-Caspase-3, and Caspase-9.

R285.5

广西壮瑶药重点实验室（桂科基字［2014］32号）；壮瑶药协同创新中心（桂教科研［2013］20号）；广西一流学科中药学（民族药学）（桂教科研[2018]12号)