TRAIL联合阿霉素诱导肝癌耐药株凋亡的实验研究

目的研究肿瘤坏死因子相关凋亡诱导配体(tumornecrosisfactorrelatedapoptosisinducingligand,TRAIL)及TRAIL和阿霉素(ADM)联用对肝癌耐药株诱导凋亡的作用。方法通过浓度递增法用阿霉素处理人肝癌细胞株HepG2获得肝癌耐药株HepG2/ADM。并通过MTT方法鉴定耐药株的耐药性。构建可溶型TRAIL真核表达质粒真核表达质粒pIRES-EGFP-TRAIL。通过脂质体(Lipofectamine)2000介导转染质粒入HepG2/ADM。通过RT-PCR和Westernblot证实转染的HepG2/ADM有sTRAIL的mRNA和蛋白的表达后,用MTT方法和AnnexinVFITCPI染色流式细胞仪检测单纯TRAIL处理和联合阿霉素处理HepG2/ADM的生长抑制率和凋亡率。并用共聚焦显微镜观察处理后细胞凋亡的形态。结果肝癌耐药株筛选成功,sTRAIL质粒构建、转染成功。用MTT测HepG2/ADM抑制率阿霉素处理组40.9%;TRAIL处理组29%;合用组58.4%;联合后抑制率有显著差异。凋亡检测为阿霉素处理组18.37%;TRAIL处理组14.8%;合用组52.71%。但TRAIL处理HepG28.9%和HepG2/ADM8.54%的凋亡率无显著差异。结论TRAIL联合阿霉素能明显增加HepG2/ADM的凋亡从而逆转耐药。同时单纯TRAIL治疗对耐药株作用与亲代细胞作用相仿,说明TRAIL可能有另外的诱发凋亡的途径,受HepG2/ADM耐药性的影响小。

Apoptosis of chemoresistant hepatocellular carcinoma cells synergistically induced by TRAIL and adriamycin

Objective To investigate the effects of TNF-relate d apoptosis-inducing ligand (TRAIL) and combination of TRAIL and adriamycin (ADM) on the induction of apoptosis of chemoresistant hepatocellular carcinoma cells. Methods HepG2/ADM cells were obtained by treatment of human car cinoma hepatocellular cell strain HepG2. Soluble TRAIL (sTRAIL) eukaryotic expre ssion plasmid pIRES-EGFP-TRAIL was constructed and transfected into HepG2/ADM by Lopofectamine 2000. The inhibition and apoptosis rates of HepG2/ADM cells tre ated by TRAIL alone and combination of TRAIL and ADM were detected by MTT method and flow cytometry. The morphology of the apoptotic cells was observed by confo cal laser scanning microscopy. Results MTT assay revealed that the inhibition rate of HepG2/AD M cells was 40.9% in ADM treatment group, 29% in TRAIL treatment group, and 58.4 % in combined treatment group. The inhibition rate was significantly higher afte r combined treatment of TRAIL and ADM. The apoptotic rate was 18.37% in ADM trea tment group, 14.8% in TRAIL treatment group, and 52.71% in combined treatment gr oup. No significant difference in apoptotic rate was found between TRAIL-treate d HepG2 (8.9%) and HepG2/ADM (8.54%). Conclusions Combination o f TRAIL and ADM could reverse the chemoresistance of HepG2/ADM cells by enhancem ent of apoptosis of HepG2/ADM cells. The chemoresistance of HepG2/ADM cells may not be associated with the resistance to TRAIL-mediated apoptosis. The combinat ion of TRAIL and chemotherapy may be a useful therapy for chemoresistant HCC cel ls.