目的 探究新對葉百部堿（NTS）對人胰腺癌細(xì)胞MiaPaCa-2和PANC-1凋亡的影響及作用機(jī)制。方法 以0（對照組）、20、40、60、80、100 μg·mL⁻¹的NTS分別處理MiaPaCa-2、PANC-1細(xì)胞24、48、72 h后，采用MTT法檢測細(xì)胞存活率；分別設(shè)置對照組（0 μg·mL⁻¹）和NTS低、中、高質(zhì)量濃度（20、40、60 μg·mL⁻¹）組，采用平板克隆法檢測NTS對MiaPaCa-2、PANC-1細(xì)胞的增殖能力的影響；采用劃痕愈合實(shí)驗(yàn)檢測細(xì)胞遷移能力；采用Hoechst 33258染色法觀察細(xì)胞的凋亡；采用流式細(xì)胞術(shù)檢測細(xì)胞的凋亡率、線粒體膜電位；采用Western blotting法檢測凋亡相關(guān)蛋白B細(xì)胞淋巴瘤-2（Bcl-2）、Bcl-2相關(guān)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）相關(guān)蛋白表達(dá)水平。結(jié)果 與對照組比較，不同質(zhì)量濃度NTS可顯著降低MiaPaCa-2、PANC-1細(xì)胞存活率（P＜0.01）、克隆形成率（P＜0.05、0.01）、劃痕愈合率（P＜0.05、0.01）、線體膜電位（P＜0.01）；顯著增加MiaPaCa-2和PANC-1細(xì)胞凋亡水平（P＜0.01）；在蛋白水平上，NTS顯著上調(diào)促凋亡相關(guān)因子cleaved-Caspase-3、Bax、Caspase-9蛋白的表達(dá)水平，下調(diào)抑制凋亡因子Bcl-2蛋白表達(dá)水平（P＜0.05、0.01）。結(jié)論 NTS可有效抑制人胰腺癌MiaPaCa-2、PANC-1細(xì)胞增殖和遷移能力，并促進(jìn)其凋亡，其機(jī)制可能與下調(diào)抑制凋亡蛋白Bcl-2并上調(diào)促凋亡蛋白Bax、cleaved-Caspase-3、Caspase-9表達(dá)有關(guān)。

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

廣西壯瑤藥重點(diǎn)實(shí)驗(yàn)室（桂科基字［2014］32號）；壯瑤藥協(xié)同創(chuàng)新中心（桂教科研［2013］20號）；廣西一流學(xué)科中藥學(xué)（民族藥學(xué)）（桂教科研[2018]12號)