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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 the reversal effect of human tumor necrosis factor α (TNFα) combined with bromocriptine (BCT) on the drug resistance of human hepatocarcinoma model in nude mice. Methods Human liver cancer cell line HepG2 and its drug-resistant cell line HepG2ADM induced by adriamycin (ADM) and the drug-resistant cell line HepG2ADMTNFα transfected with TNFα gene were inoculated into the liver of BALB / c nude mice respectively to establish nude mice Orthotopic liver xenograft model. 64 nude mice were divided into 4 groups: HepG2 group (HepG2 cell line xenografted nude mice), ADM group (HepG2ADM cell line xenografted nude mice), TNFα group (HepG2ADMTNFα cell line xenografted nude mice) and BCT group (HepG2ADMTNFα cells Department of nude mice implanted tumor simultaneously oral BCT) were given intraperitoneal injection of 0.15g / kg of fluorouracil + 1.5mg / kg of mitomycin + 10mg / kg ADM intraperitoneally daily for 3 days; BCT group Chemotherapy at the same time another BCT intragastric administration (6.25mg · kg-1 · d-1). B ultrasound observation of tumor size changes, histological observation of histological structure and growth of nude mice and the sensitivity of chemotherapy drugs. Immunohistochemistry and reverse transcriptase polymerase chain reaction (RTPCR) were used to detect the mRNA and protein levels of multidrug resistance-related gene (MDR1) and lung resistance-related protein (LRP) TUNEL-mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis index of tumor tissue after chemotherapy. Results The success rate of in situ implantation in nude mice was 100%. The histological features of the tumor were in line with the characteristics of human hepatocellular carcinoma. The growth of tumor in TNFα and BCT groups was slow, with significant difference compared with HepG2 and ADM (P < 0.05). After 14 days of chemotherapy, the inhibition rate of tumor weight in BCT group was the most significant (67%), which was significantly lower than that in HepG2, TNFα and ADM3 groups (P <0.05). The expression of MDR1 and LRP mRNA in the four groups were statistically significant (P <0.05). Immunohistochemistry showed that the expression of MDR1 protein in tumor tissues of TNFα and BCT groups was lower than that of ADM group (P <0.01) (P> 0.05). The apoptotic index of BCT and TNFα group was higher than that of ADM group (P <0.05), and the difference between TNFα and BCT group was also statistically significant (P < 0.05), but there was no significant difference between HepG2 group and HepG2 group (P> 0.05). Conclusion The TNFα gene can down-regulate the expression of MDR1 and LRP mRNA and protein, and combined with BCT can enhance the sensitivity of chemotherapeutics.