目的 探讨美洲大蠊多肽（PAE₂）逆转人肝癌多药耐药细胞株HepG2/ADM的作用及机制。方法 噻唑蓝（MTT）法检测HepG2/ADM细胞对不同化疗药物的耐药性以及PAE₂联合化疗药物（5-氟尿嘧啶、阿霉素、环磷酰胺、长春新碱、奥沙利铂）对HepG2/ADM细胞增殖的影响；激光共聚焦观察PAE₂对HepG2/ADM细胞中药物累积的影响；Western blotting法检测PAE₂对HepG2/ADM细胞凋亡相关蛋白B细胞淋巴瘤/白血病-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X，Bax）、半胱氨酸的天冬氨酸蛋白水解酶（cysteinyl aspartate specific proteinase 9，Caspase-9）、DNA修复酶（poly ADP-ribose polymerase，PARP）表达的影响；qRT-PCR法检测PAE₂对多药耐药蛋白1（multiple drug resistance 1，MDR1）、乳腺癌耐药相关蛋白（breast cancer resistant protein，BCRP）和酶介导的多药耐药途径中蛋白激酶C（protein kinase C，PKC）、拓扑异构酶Ⅱ（topoisomerase Ⅱ，Top Ⅱ）的mRNA表达。结果 PAE₂抑制HepG2/ADM细胞增殖，呈时间和剂量相关性。PAE₂可逆转HepG2/ADM对不同化疗药物的耐药性；PAE₂可上调HepG2/ADM细胞中阿霉素水平，呈剂量相关性。PAE₂可显著上调耐药细胞中Bax、cleaved Caspase-9 p37蛋白表达水平（P<0.05、0.01），显著下调Bcl-2蛋白表达水平（P<0.01），对cleaved PARP蛋白表达无显著影响。PAE₂均显著下调HepG2/ADM细胞中MDR1、BCRP、PKC、Top Ⅱ mRNA水平（P<0.01）。结论 PAE₂可以通过减少药物外排、促进细胞凋亡、下调耐药蛋白表达等途径逆转HepG2/ADM细胞的多药耐药性。

Objective To investigate the reverse effect of polypeptide from Meizhoudalian (Periplaneta Americana) (PAE₂) on multi-drug resistance of HepG2/ADM cells. Methods The drug resistance of HepG2/ADM cells to different chemotherapeutic drugs and effects of PAE₂ combined with chemotherapeutic drugs on proliferation of HepG2/ADM cells were detected by MTT; The influence of PAE₂ on drug accumulation was observed by Laser Scanning Confocal Microscopy; The expression of Bcl-2 associated X (Bax), B-cell lymphoma-2 (Bcl-2), cysteinyl aspartate specific proteinase 9 (Caspase-9) and poly ADP-ribose polymerase (PARP) were detected by Western blotting; qRT-PCR was used to detect the mRNA expression of multiple drug resistance 1 (MDR1), breast cancer resistant protein (BCRP), protein kinase C (PKC) and topoisomerase Ⅱ (Top Ⅱ). Results Growth of HepG2/ADM cells was inhibited by PAE₂ with a time and dose dependent. PAE₂ reversed the multi-drug resistance of HepG2/ADM cells to different chemotherapeutic drugs; Level of doxorubicin in drug-resistant cells was increased by PAE₂ in a dose-dependent manner. Expressions of Bax, cleaved Caspase-9 p37 were significantly increased (P<0.05, 0.01) and the expression of Bcl-2 was significantly decreased by PAE₂ in HepG2/ADM cells (P<0.01), the expression of cleaved PARP had no changed. Levels of MDR1, BCRP, PKC and Top Ⅱ mRNA in HepG2/ADM cells were significantly reduced by PAE₂. Conclusion PAE₂ reversed the multidrug resistance of HepG2/ADM cells by reducing drug efflux, promoting cell apoptosis and reducing the expressions of drug-resistant protein.

R285.5

国家自然科学基金资助项目（81560600）；云南省应用基础研究重点项目（2017FH001-010）