熊果酸辅助吉西他滨降低胰腺癌细胞耐药性并增强抗肿瘤作用的实验研究

Ursolic acid as an adjuvant reduces drug resistance of pancreatic cancer cells and enhances its anti-tumor effect in gemcitabine chemotheray

目的:探讨熊果酸(UA)辅助吉西他滨(Gem)通过降低胰腺癌(PC)细胞耐药性增强抗肿瘤效果的药理机制。方法:将体外培养的人胰腺癌细胞株SW1990细胞分别加入磷酸盐缓冲液(PBS)、Gem、UA以及Gem加UA孵育4 h,用噻唑蓝比色法(MTT法)检测细胞增殖,实时定量聚合酶链反应(RT-q PCR)检测细胞中凋亡相关基因的变化;通过皮下注射SW1990细胞构建PC荷瘤小鼠模型,予荷瘤小鼠瘤内注射PBS、Gem5 mg·kg^-1、UA 2 mg·kg^-1及Gem加UA,分别用HE和TUNEL染色观察各组小鼠肿瘤组织病理学变化,并记录所有小鼠存活情况。结果:Gem与UA联合用药对SW1990细胞的杀伤效果大于单独使用Gem和UA杀死细胞之和,且与PBS组比较,Gem联合UA可以显著下调细胞中抗凋亡基因Bcl-2的mRNA表达水平(P<0.05),上调促凋亡基因Bax和Caspase-3的mRNA表达水平(P<0.05);Gem明显提高荷瘤小鼠肿瘤细胞中P-gp蛋白的表达水平(P<0.01);病理学观察结果显示,Gem与UA联合用药后肿瘤组织坏死及细胞凋亡明显,促凋亡作用大于单纯使用Gem与UA之和,最终荷瘤小鼠存活率最高。结论:UA可作为佐剂有效逆转PC对Gem化疗的多药耐药,增强化疗效果,提高荷瘤小鼠存活率。

Objective: To investigate the pharmacological mechanism of gemcitabine(Gem) combined with ursolic acid(UA) for enhancing anti-tumor effect by reducing drug resistance of pancreatic cancer(PC) cells.Methods:SW1990 cells cultured in vitro were incubated with PBS,Gem,UA and Gem + UA for 4 hours,and thereafter the cell proliferation was detected by MTT assay.Real-time quantitative polymerase chain reaction(RT-qPCR) was used to detect the changes of apoptosis-related genes.PC tumor-bearing mouse model was constructed by subcutaneous injection of SW1990 cells,then PBS,Gem 5 mg · kg-1,UA 2 mg·kg-1 and Gem + UA were injected into tumor-bearing mice.The histopathological changes of tumors in each group were detected by HE and TUNEL staining respectively,and the survival rate of all mice were recorded.Results: The lethality of Gem + UA to SW1990 cells was greater than the sum of free Gem and free UA.Compared with PBS group,Gem + UA significantly down-regulated the mRNA expression level of anti-apoptotic gene Bcl-2 in PC cells(P<0.05),and up-regulated the mRNA expression levels of pro-apoptotic genes Bax and Caspase-3(P<0.05).The results showed that Gem increased the protein expression levels of P-gp in tumor cells of the mice significantly(P<0.01).The pathological examination also showed that there were obvious necrosis and apoptosis in tumor tissue after the treatment with Gem + UA,and the pro-apoptotic effect was more potent than the sum of the Gem and UA group used alone.Of all the groups,the survival rate in Gem + UA group was the highest.Conclusion: UA can be used as an adjuvant to effectively reverse the multidrug resistance induced by Gem chemotherapy in PC,which can enhance the effect of chemotherapy and improve the survival rate of tumor-bearing mice.