二氢青蒿素联合吉西他宾治疗胰腺癌的实验研究  

Experimental study of the function and mechanism combining dihydroartemisinin and gemcitabine in treating pancreatic cancer

作　　者：王双佳[1] 孙备[1] 潘尚哈[1] 陈华[1] 孔瑞[1] 李军[1] 薛东波[1] 白雪巍[1] 姜洪池[1] 

机构地区：[1]哈尔滨医科大学附属第一医院肝胆胰外科,150001

出　　处：《中华外科杂志》2010年第7期530-534,共5页Chinese Journal of Surgery

基　　金：国家自然科学基金资助项目（30901437,30972907）;教育部新世纪优秀人才支持计划资助项目（NCET-07-0248）;黑龙江省杰出青年科学基金资助项目（JC200717）

摘　　要：目的探讨二氢青蒿素联合吉西他宾治疗胰腺癌的作用及其机制。方法培养胰腺癌细胞株BxPC-3和Panc-1，通过MTY法检测二氢青蒿素联合吉西他宾对胰腺癌细胞生长的抑制作用；通过AnnexinV—FITC／PI染色流式细胞术和激光共聚焦显微镜检测细胞凋亡情况；通过EMSA检测NF—kB与DNA结合活性的改变；通过Western blot法检测细胞中NF-kB／P65和NF-kB下游增殖、凋亡相关蛋白的表达情况。裸鼠皮下注射BxPC-3细胞建立胰腺癌裸鼠移植瘤，监测给药后肿瘤体积的变化，TUNEL染色检测肿瘤细胞凋亡情况。结果二氢青蒿素联合吉西他宾对BxPC-3和Panc-1两株细胞的增殖抑制率可达（81．1±3．9）％和（76．5±3．3）％；凋亡率可达（53．6±3．8）％和（48．3±4．3）％，与吉西他宾组[（24．8±2．9）％和（21．8±3．5）％]相比，差异有统计学意义（P〈0．01）。在裸鼠体内，各治疗组均可抑制胰腺癌裸鼠移植瘤的生长；联合组肿瘤的体积和增殖凋亡指数分别为（262±37）mm。和（50±4）％，与吉西他宾组[（384±56）mm^2和（25±3）％]相比，差异有统计学意义（P〈0．05）。EMSA和Western blot结果显示，二氢青蒿素能抑制胰腺癌细胞NF—kB与DNA结合活性，联合组较吉西他宾组NF—kB活性有显著的降低；二氢青蒿素下调BxPC-3和Panc-1细胞核P65的表达，联合组较对照组显著下调NF—KB靶基因蛋白CyclinD1、Bcl—xL、Bcl-2的表达，上调Bax的表达，降低Bcl-2／Bax的比例，进一步增加Caspase-3的活化。结论二氢青蒿素抑制吉西他宾所诱导激活的NF—kB的活性，下调NF—kB下游靶基因蛋白的表达可能是其增敏吉西他宾抗胰腺癌作用的分子机制。Objective To investigate the anti-tumor activity of combined gemcitabine with dihydroartemisinin, and the mechanism of the anti-tumor effect of gemcitabine enhanced by dihydmartemisinin on pancreatic cancer. Methods For cultured cells, cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis and confocal laser scanning microscope stained with Annexin V-FITC/PI. The nuclear extract for determining NF-kB DNA-binding activity was analyzed by EMSA,while nuclear P65 and its downstream gene expression was determined by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to agents. TUNEL assay was used to assess tumor cell apoptosis in tumor tissue. Results After combination of gemcitabine and dihydroartemisinin treatment, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and Pane-1 reached up to （81. 1 ± 3.9 ）% and （76.5±3.3）%,and the apoptosis rates were up to （53.6 ±3.8）% and （48.3 ±4.3）%,the differences were significantly （ P 〈 0. 01 ） compared with gemcitabine [ （ 24. 8 ± 2. 9 ） % and （ 21.8 ± 3.5） % ]. All the treatment groups inhibited the growth of pancreatic xenograft tumors in nude mice. The tumor volume and apoptosis index were （262 ± 37 ）mm^3 和 （50 ± 4 ）% respectively in the combined treatment,compared to those of [ （ 384 ± 56） mm3 and （ 25 ± 3 ） % ] in gemcitabine, the differences were significantly（P 〈 0. 05 ）. EMSA showed that gemcitabine alone obviously enhanced its DNA-binding activity compared to control. However, dihydroartemisinin significantly reduced its DNA-binding activity, so that abrogated the inducing effect of gemcitabine on NF-KB activation. Western blot assay indicated that dihydroartemisinin downregulated expression of nuclear P65, and combined treatment not only downregnlated the expression of Cyclin D1, Bcl-xL and Bcl-2 while upregulated Bax, thus red

关 键 词：胰腺肿瘤 抗药性 肿瘤 二氢青蒿素 吉西他宾 

分 类 号：R735.9[医药卫生—肿瘤]