[摘要] 目的：探讨桦木酸（BEA）提高胰腺癌Panc-1、Miapaca-2 细胞对吉非替尼的敏感性及其潜在的作用机制。方法：细胞培养完成后，将对吉非替尼不敏感的Panc-1、Miapaca-2 细胞随机分为4 组：对照组、BEA组、吉非替尼组及BEA联合吉非替尼组，分别予以不处理、BEA、吉非替尼及BEA联合吉非替尼处理。MTS法检测BEA对2 种细胞的增敏效果，集落形成实验检测BEA协同吉非替尼的治疗效果，WB实验检测BEA对Panc-1 细胞凋亡相关蛋白的影响，流式细胞术检测BEA对Panc-1 细胞凋亡的影响，表面等离子体共振（SPR）实验验证信号转导子和转录激活子3（STAT3）和BEA的直接结合，分子对接和分子动力学模拟实验预测STAT3 和BEA的结合模式。结果：BEA协同增强Panc-1、Miapaca-2 细胞对吉非替尼的敏感性（P<0.05），使其对两种细胞的IC50值均降低至原值的50%以下。吉非替尼联合BEA较单用吉非替尼或BEA促进Panc-1 细胞的凋亡以及凋亡相关蛋白cleaved-PARP和Bax 的表达，减少对凋亡抑制蛋白Bcl-2 的表达（均P<0.05 或P<0.01）。BEA对Panc-1 细胞中STAT3 的活化有剂量依赖性抑制作用（P<0.01）。BEA通过与STAT3 的Lys-591、Ser-613 形成氢键而稳定BEA与STAT3 的结合作用，同时BEA稳定在STAT3 的蛋白结合位点内，以此阻断STAT3 二聚发挥增敏作用。结论：联用BEA和吉非替尼显著抑制胰腺癌Panc-1、Miapaca-2 细胞的增殖并促进其凋亡，这种增敏作用可能是由BEA对STAT3 抑制作用所介导。

[Abstract] Objective: To investigate the effects and the underlying mechanisms of betulinic acid (BEA) on sensitizing pancreatic cancer cell lines Panc-1 and Miapaca-2 to gefitinib. Methods: After the cell culture was completed, Panc-1 and Miapaca-2 cells were randomly divided into 4 groups: control group (without treatment), BEA group, gefitinib group and BEA combined with gefitinib group, respectively.The sensitization effect of BEA on gefitinib-insensitive pancreatic cancer cells was detected by MTS assay. The treatment effects of combined treatment of gefitinib and BEA against Panc-1 and Miapaca-2 cells were evaluated by colony formation assay. Flow cytometry was used to examine the effect of BEA on apoptosis of Panc-1 cells while WB was applied to determine the effect of BEA onapoptosis-related proteins. Surface plasmon resonance (SPR) experiment was used to detect the direct combination between signal transducer and activator of transcription 3(STAT3) and BEA; Molecular docking and molecular dynamics simulation experiments were adopted topredict the combining mode between STAT3 and BEA. Results: BEA synergistically enhanced the gefitinib-sensitivity of pancreatic cancer Panc-1 and Miapaca-2 cells (P<0.05), and IC50 of gefitinib on two cells were reduced by over 50%. Compared with single treatment, the combined treatment of BEA and gefitinib promoted the apoptosis and up-regulated the expressions of apoptosis-relatedproteins (cleaved-PARP and Bax), but reduced the apoptosis-inhibitory protein Bcl-2 (all P<0.05 or P<0.01). Moreover, the inhibitory effect of BEA on STAT3 activation in Panc-1 cells was in a dose-dependent mannar (P<0.01). BEA stabilizes its binding to STAT3 by forming hydrogen bonds with Lys-591 and Ser-613 of STAT3; in the meanwhile, BEA stabilized inthebinding site of STAT3,there by blocking STAT3 dimerization to enhance the drug sensitivity. Conclusion: Combined use of BEA and gefitinib could significantly inhibit the proliferation and induce apoptosis of Panc-1 and Miapaca-2 cells, which might be mediated by the inhibition of BEA on STST3.