A 150 bp epidermal growth factor (EGF) cDNA fragment and a 1024 bp epidermal growth factor receptor (EGFR) cDNA fragment were inserted into 5.05 kb pBabe-puro retroviral vectors between BamH I and EcoR I sites in 3...A 150 bp epidermal growth factor (EGF) cDNA fragment and a 1024 bp epidermal growth factor receptor (EGFR) cDNA fragment were inserted into 5.05 kb pBabe-puro retroviral vectors between BamH I and EcoR I sites in 3'-5' and / or 5'-3' orientation. The vectors were ligated with EGF and EGFR fragments by T-4 Ligase. The recombinant retroviral vectors were then packaged with packaging cell line PA317 through calcium phosphate mediated transfection. The viral supernatant of transfected PA317 cell lines were used to infect the human pancreatic carcinoma cell line PC-7. The resultant transformant cell lines: PC-7 / AS-EGF, PC-7 / S-EGFR, PC-7 / AS-EGFR and PC-7 / pBabe were tested for their endogenous EGF and EGFR mRNA expressions, cell growth rate, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice. The results showed that there were noticeable inhibitions of cell growth, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice in PC-7 / AS-EGF and PC-7 / AS-EGFR transformant cell lines. The endogenous EGF mRNA expression was blocked in PC-7 / AS-EGF cell line and the endogenous EGFR mRNA was significantly down-regulated in PC-7 / AS-EGFR cell line.展开更多
文摘A 150 bp epidermal growth factor (EGF) cDNA fragment and a 1024 bp epidermal growth factor receptor (EGFR) cDNA fragment were inserted into 5.05 kb pBabe-puro retroviral vectors between BamH I and EcoR I sites in 3'-5' and / or 5'-3' orientation. The vectors were ligated with EGF and EGFR fragments by T-4 Ligase. The recombinant retroviral vectors were then packaged with packaging cell line PA317 through calcium phosphate mediated transfection. The viral supernatant of transfected PA317 cell lines were used to infect the human pancreatic carcinoma cell line PC-7. The resultant transformant cell lines: PC-7 / AS-EGF, PC-7 / S-EGFR, PC-7 / AS-EGFR and PC-7 / pBabe were tested for their endogenous EGF and EGFR mRNA expressions, cell growth rate, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice. The results showed that there were noticeable inhibitions of cell growth, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice in PC-7 / AS-EGF and PC-7 / AS-EGFR transformant cell lines. The endogenous EGF mRNA expression was blocked in PC-7 / AS-EGF cell line and the endogenous EGFR mRNA was significantly down-regulated in PC-7 / AS-EGFR cell line.
