抗表皮生长因子受体单抗基因治疗胰腺癌的实验研究

Gene therapy for pancreatic cancer expressing human anti-epidermal growth factor receptor single chain variable fragment antibody

目的EGFR靶向治疗和AAV介导的基因治疗在胰腺癌治疗方面具有广泛前景。本实验将上述两种手段相结合,构建表达人抗EGFR单链可变区抗体的rAAV载体,并筛选出对抗EGFR治疗敏感的胰腺癌细胞株。方法以包含人IX因子基因的rAAV载体中的FIX基因替换成目的基因,转染293细胞,Western Blot法检测目的蛋白:同时将C225作用于六株胰腺癌细胞,通过CCK-8法测定生长曲线,P1染色法及Annexin V-FITC试剂盒检测细胞凋亡。结果Western Blot法检测到目的条带;生长曲线显示C225仅对AsPC-1的生长有明显的抑制作用(P<0.01),Annexin V-FITC试剂盒可检测C225引起AsPC-1细胞的早期凋亡(P<0.05)。结论rAAV载体构建成功,并在293细胞中获得表达;AsPC-1对C225的敏感性明显高于其它五株胰腺癌细胞。

Objective To EGFR-targeted therapy and AAV-mediated gene therapy has been proved to be promising ways to fight pancreatic cancer.This study combined these two methods to challenge pancreatic cancer by constructing a recombinant AAV-2 vector containing a human anti-EG- FR scFv gene.Then a cell line most sensitive to C225 was screened out among 6 pancreatic cancer cell lines in order to provide a basis to in vivo experiments in the future.Methods 1) By overlap extension new cloning sites were introduced into the rAVV backbone vector which contains hFIX gene.Then hFIX cDNA was replaced by target gene.2) The newly constructed vector was transfected into 293 cell line and expression of target gene was determined by Western blot.3) Six pancreatic cancer cell lines were cultured with or without C225.Then inhibition rates of cell growth were investigated by CCK-8 kit,and apoptosis was determined by PI staining flow eytometry or Annexin V-FITC kit.Re- suits Western blot demonstrated that target band did not exist in control.AsPC-1 treated with C225 showed a statistically significant inhibition of growth.Early apoptosis was detected in AspC-1 by An- nexin V/PI double staining flow cytometry.Conclusion 1) Recombinant AAV vector containing tar- get gene has been successfully constructed and expressed in 293 cell line.2) AsPC-1 is more sensitive to C225 than other 5 pancreatic cancer cell lines.