人肿瘤坏死因子α联合溴隐亭对人肝癌裸鼠耐药模型耐药性逆转的研究

Synergistic effect of bromocriptine combining tumor necrosis factor-α on reversing multidrug resistance in a nude mouse model of liver neoplasm

目的探讨人肿瘤坏死因子α(TNFα)联合溴隐亭(BCT)对人肝癌裸鼠耐药模型耐药性的逆转作用。方法将人肝癌细胞系HepG2及其经阿霉素(ADM)诱导建立的耐药细胞系HepG2ADM和转染TNFα基因后的耐药细胞系HepG2ADMTNFα分别原位种植BALB/C裸鼠肝脏,建立裸鼠原位肝移植瘤模型。64只裸鼠分为4组:HepG2组(HepG2细胞系种植瘤裸鼠),ADM组(HepG2ADM细胞系种植瘤裸鼠),TNFα组(HepG2ADMTNFα细胞系种植瘤裸鼠)和BCT组(HepG2ADMTNFα细胞系种植瘤裸鼠同时口服BCT),成瘤后均给以每天腹腔内注射0.15g/kg的氟脲嘧啶+1.5mg/kg的丝裂霉素+10mg/kg的ADM,连续3d;BCT组化疗的同时另行BCT灌胃治疗(6.25mg·kg-1·d-1)。B超观察种植瘤的大小变化,病理观察组织学结构及裸鼠生长状况和对化疗药物的敏感性。采用免疫组织化学和逆转录聚合酶链反应(RTPCR)检测各组种植瘤的多药耐药相关基因(MDR1)和肺耐药相关蛋白(LRP)在mRNA水平、蛋白水平的变化,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL法)检测化疗后肿瘤组织凋亡指数情况。结果细胞系原位种植裸鼠肝脏成功率100%,种植瘤组织学特点符合人肝癌特征;TNFα组和BCT组种植瘤生长速度慢,与HepG2和ADM两组比较差异有统计学意义(P<0.05)。化疗14d后,BCT组重量抑瘤率(67%)最明显,与HepG2、TNFα和ADM3组比较差异有统计学意义(P<0.05)。4组均有MDR1和LRPmRNA表达,组间差异具有统计学意义(P<0.05);免疫组化显示TNFα和BCT组肿瘤组织MDR1蛋白表达比ADM组低,差异具有统计学意义(P<0.01),与HepG2组比较差异无统计学意义(P>0.05);BCT、TNFα组凋亡指数比ADM组高(P<0.05),且TNFα和BCT两组之间差异亦有统计学意义(P<0.05),但与HepG2组之间比较差异均无统计学意义(P>0.05)。结论TNFα基因能下调MDR1和LRPmRNA及蛋白表达,联合BCT能加强对化疗药物的敏感性。

Objective To investigate synergistic effect of bromocriptine （BCT） combining tumor necrosis factor-α （TNF-α） on reversing muhidrug resistance in a nude mouse model of liver neoplasm. Methods Human hepatocarcinoma cell line HepG2 （HepG2 group ）, drug resistant hepatocarcinoma cell line HepG2/adriamycin （HepG2/ADM group ） and hepatocarcinoma cell line transfected with TNF-α gene HepG2/ADM/TNF （TNF group and BCT group）were injeceted into the liver of nude mice via orthotopic implantation to establish muhidrug resistance model of liver neoplasm in vivo. All the mice were injected with 5-fluouracil ＋ adriamycin ＋ mitomycin in abdominal cavity for 7 d. The mice in BCT group was simultaneously given bromocriptine through gastric canal. Size and weight of the tumor were measured. Furthermore tumor histological character and growth of the nude mice was observed and its chemosensitivity was tested. MDR associated genes and proteins （ MRP, LRP ） of implantated tumors were detected by imunohistomchemical staining and reverse transcriptive polymerase chain reaction （RT-PCR）, and apoptosis rate of hepatocarcinoma cells was detected by TUNEL assay. Results The nude mouse model of each cell line was all inoculated successfully. The tumor growth rate and weight were significantly different among groups（P 〈 0. 05 ）. After chemotherapy tumor growth inhibition rate was higher in BCT group （67%） compared to ADM and TNF groups （ P 〈 0. 01 ）, and similar to HepG2 group（54% ）. MDR1 and LRP mRNA could be detected in all groups, but TNF-α was detected only in TNF-α and BCT groups. Furthermore, MDR1 and LRP protein expression of tumors in TNF-α and BCT groups was low similar to HepG2 group. The apoptosis rate of hepatocarcinoma cells was much higher in BCT group than in other groups（ P 〈 0. 05 ） with TUNEL assay. Conclusions TNF-α gene can down-regulate the MDR associated genes and proteins expression for example MDR1 ,LRP, and lower its tumorgenesis. Moreover, bromocriptine can enhance the susceptibility of HepG2/ADM cells to cytotoxic drugs.