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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 a multidrug resistance model of orthotopic liver cancer in nude mice. Methods Human hepatocellular carcinoma cell line HepG2 and multidrug resistance cell line HepG2 / ADM were implanted into the hepatic capsule of nude mice to establish the orthotopic liver cell carcinoma multidrug resistance animal model. With B ultrasound, laparotomy detection of liver tumor growth in both groups. The full-length MDR1 gene of HepG2 / ADM and its corresponding tumor tissue were amplified by PCR and sequenced. The expression of MDR1 mRNA in the two groups were detected by RT-PCR, immunohistochemistry and Western blotting, respectively And P-gp protein expression. Results The tumorigenic rates of orthotopic liver implantation were 100% (30/30) and 95% (19/20) respectively. The long-term PCR was used to detect the drug resistance of HepG2 / ADM and corresponding tumor Tissue MDR1 gene amplification, can amplify a full length of 3.8 Kb gene band, two-way DNA sequencing results are reported in Genebank. The expressions of MDR1 mRNA and P-gp protein in drug-resistant group were significantly higher than those in control group (t1 = 3.72, t2 = 2.98, P <0.01). The expression of P-gp in drug-resistant group was significantly higher than that in control group (42.6 ± 1.7% vs. 2.6 ± 0.1%, t = 6.43, P <0 .01). Conclusion The successful establishment of a multidrug resistance animal model of liver cancer and provide a basis for the study of multidrug resistance reversal of liver cancer.