重组腺相关病毒载体表达抗表皮生长因子受体抗体对胰腺癌细胞株抑制作用的研究

Inhibition effect of anti-epidermal growth factor receptor antibody mediated by recombinant adeno-associated virus vector on pancreatic cancer cell lines

目的构建能在体内长期表达抗表皮生长因子受体（EGFR）抗体的重组腺相关病毒（rAAV）载体，观察其对人胰腺癌细胞株的抑制作用。方法将抗人EGFR单链抗体可变区（SCFV）基因插入腺相关病毒载体的Kpn Ⅰ和Bgl Ⅱ位点，构建成含抗EGFR单链抗体基因的重组腺相关病毒载体（rAAV-anti EGFR）。设立对照组（rAAV1-EGFP组）和实验组（rAAV1-anti EGFR组），观察抗EGFR单链抗体蛋白的表达情况。采用Western blot检测抗体蛋白的表达、CCK-8法和流式细胞仪检测6种人胰腺癌细胞株（PCT-3、SWl990、Capan-1、ASPC-1、MiaPaCa-2及PANC-1细胞）的抑制率和凋亡率。两组比较采用t检验。结果抗EGFR单链抗体蛋白能在6种人胰腺癌细胞株中表达，对照组和实验组胰腺癌ASPC-1细胞抑制率分别为1．1％±2．4％和15．1％±3．5％，两组比较，差异有统计学意义（t=6．598，P〈0．05）。对照组和实验组PANC-1细胞凋亡率分别为7．0％±3．0％和11．4％±2．5％，两组比较，差异无统计学意义（t=1．952，P〉0．05）；对照组和实验组SW1990、ASPC-1、Capan-1、PCT-3、MiaPaCa-2细胞凋亡率分别为1．1％±0．8％、1．5％±0．7％、1．7％±1．2％、1．1％±0．7％、2．2％±1．1％和17．6％±2．2％、46．9％±3．9％、20．0％±2．8％、12．1％±1．6％、31．1％±2．5％，两组比较，差异均有统计学意义（t=12．208，19．846，10．405，10．909，18．327，P〈0．05）。结论成功构建了携带抗EGFR单链抗体基因的rAAV载体，其表达的抗EGFR单链抗体蛋白对人胰腺癌细胞株有明显的抑制作用。

Objective To construct a recombinant adeno-associated virus （rAAV） vector containing a human anti-epidermal growth factor receptor （anti-EGFR） single-chain variable fragment antibody gene, and observe its inhibitory effects on pancreatic cancer cell lines. Methods Human anti-EGFR single-chain variable fragment antibody gene was inserted into the Kpn Ⅰ and Bgl Ⅱ sites to construct a rAAV-anti EGFR vector, and then rAAV1-EGFP group and rAAVl-anti EGFR group were established. The expression of anti-EGFR antibody was observed. Antibody expression was detected by Western blot, and the inhibition and apoptosis rates of human pancreatic cancer cell lines （PCT-3, SW1990, Capan-1, ASPC-1, MiaPaCa-2 and PANC-1 cells） were detected by CCK-8 assay and flow cytometry, respectively. All data were analyzed using the t test. Results The results of Western blot assay demonstrated that anti-EGFR antibody was expressed in 6 pancreatic cancer cell lines. The inhibition rates of rAAV1-EGFP and rAAVl-anti EGFR on pancreatic ASPC-1 cells were 1. 1% ± 2. 4% and 15.1%±3.5% , respectively, with a significant difference between the 2 groups （t = 6. 598, P 〈 0.05）. The apoptosis rates of PANC-1 ceils were 7.0% ± 3.0% in the rAAV1-EGFP group and 11.4% ± 2.5% in the rAAV1- anti EGFR group, with no significant difference between the 2 groups （ t = 1. 952, P 〉 O. 05 ）. The apoptosis rates of SW1990, ASPC-1, Capan-1, PCT-3, MiaPaCa-2 cells were 1.1%±0.8% , 1.5%±0.7%, 1.7%± 1.2%, 1.1% ±0.7% and 2.2% ± 1.1% in the rAAV1-EGFP group, and 17.6% ± 2.2%, 46.9% ± 3.9%, 20. 0% ± 2.8%, 12.1% ± 1.6% and 31. 1% ± 2.5% in the rAAVl-anti EGFR group, respectively, with significant differences between the 2 groups （t = 12. 208, 19. 846, 10. 405, 10. 909, 18. 327, P 〈 0.05）. Conclusions A rAAV-anti EGFR vector with human anti-EGFR single-chain variable fragment antibody gene was constructed. Anti-EGFR antibody has obvious inhibition effects on pancreatic cancer cell lines.