裸鼠原位肝细胞癌多药耐药模型的建立

The establishment of multidrug resistant model in nude mice via orthotopic unplantation of human multidrug resistant hepatocellular carcinoma cells

目的建立裸鼠原位肝癌多药耐药模型。方法分别在开腹直视下将人肝癌细胞系 HepG2和多药耐药细胞系HepG2／ADM两种细胞种植于裸鼠的肝包膜下建立原位肝细胞癌多药耐药动物模型。用B超、剖腹探查检测两组肝脏肿瘤生长情况。利用长链PCR技术对耐药细胞系 HepG2／ADM和相应种植瘤组织MDR1基因全长进行扩增并测序,再分别用RT—PCR、免疫组化、 Western blotting方法检测两组肿瘤耐药基因MDR1mRNA和P—gp蛋白的表达。结果原位肝脏种植肿瘤成瘤率均为100％(30／30),耐药诱导成功率为95％(19／20);用长链PCR技术对耐药细胞系 HepG2／ADM和相应种植瘤组织进行MDR1基因扩增,均能扩增出全长为3．8 Kb的基因条带,双向 DNA测序结果均与Genebank报道一致。耐药组MDR1mRNA和P-gp蛋白的表达均明显高于对照组 (t1=3．72,t2=2．98,P<0．01)。耐药组P-gp蛋白表达为(42．6±1．7)％远高于对照组(2．6± 0．1)％,差异有统计学意义(t=6．43,P<0．01)。结论成功建立肝癌多药耐药动物模型并且为研究肝癌多药耐药逆转提供基础。

Objective To establish muhidrug resistant （mdr） model in nude mice via orthotopic implantation of human muhidrug resistant hepatocellular carcinoma cells. Methods Orthotopic mdrl hepatoma were obtained by injecting human hepatocellular carcinoma cells HepG2 and muhidrug resistant human hepatocellular carcinoma cells HepG2/ADM subserosally into the mouse liver. Ultrasonography and laparotomy were used to detect tumor growth, and integrity of MDR1 gene from muhidrug resistant human hepatocellular carcinoma cells HepGJADM. Implantated tumor tissues were amplificated by long PCR technique and sequenced. Furthermore, the MDR1 mRNA and P-gp protein expression were evaluated by reverse transcription and polymerase chain reaction （RT-PCR）, Western blotting, immunohistochemistry （IHC）. Results The successful rate of tumor implantation was 100% and multidrug resistant induced rate was 95% （19/20） respectively. 3.8 kb mdrl integrity gene band was identified from both HepG2/ADM cells line and corresponding implantated tumor tissues by long RT-PCR technique. MDR1 gene sequence was in coincidence to that reported in genebank. The expression of MDRlmRNA and P-gp protein in multidrug resistant groups were significantly higher than that in control groups. P-gp protein expression in multidrug resistant groups （42.6%± 1.7%） was higher than controls （2.6% ± 0.1%）, P 〈 0.01. Conclusions Multidrug resistant model in nude mice via orthotopic implantation of multidrug resistant human hepatocellular carcinoma cells was successfully established.