阻断TGF α—EGFR自泌环对胰腺癌细胞体外生长的影响

目的 研究阻断ＴＧＦα－ＥＧＦＲ自泌环对胰腺癌细胞生长的影响。方法 构建了反义ＥＧＦＲ 表达载体ｐＣＭＶ－ＡＳ－ＥＧＦＲ。转染反应反义ＴＧＦα转化的胰腺癌ＰＣ－７细胞,经Ｇ４１８筛选,获得稳定的双重转化细胞系ＰＣ－７／ＡＳ－ＴＧＦα／ＡＳ－ＥＧＦＲ。经Ｓｏｕｔｈｅｒｎｂｌｏｔ,Ｎｏｒｔｈｅｒｎｂｌｏｔ,＾１２５Ｉ－ＥＧＦ结合试验分析双重转染细胞系基因的整合及表达。ＤＮＡ凝胶电泳,流式细胞术,。原位

Effects of blocking the TGF alpha-EGFR autocrine loop on the growth of human pancreatic carcinoma cells in vitro

OBJECTIVE: Investigate the effects of blocking the TGF alpha-EGFR autocrine loop on the growth of human pancreatic carcinoma. METHODS: The pCMV-AS-EGFR, a recombinant vector expressing antisense EGFR under the control of human CMV promotor was constructed and transfected the transformant PC-7/AS-TGF alpha cell line cells, which had been transfected by a recombinant retroviral vector expressing antisense TGF alpha. G418 resistant colonies were isolated and identified as PC-7/AS-TGF alpha/AS-EGFR. The integration and expression of exogenous and endogenous genes were detected by Southern blot and Northern blot analysis. Apoptosis was detected by DNA fragmentation, flow cytometry and in situ cell death detection. RESULTS: The cells which were double-transfected by the recombinant vectors showed the integration and expression of exogenous genes, and the downregulation of endogenous EGFR and cyclin D1 mRNA. 125I-EGF ligand binding test showed the binding affinity of the EGFR on the cell surface also reduced. The inhibition effect of cotransfecting of antisense TGF alpha and antisense EGFR was remarkable as compared with that of antisense TGF alpha alone. The incorporation rate of 3H-TdR reduced from 25% to 14.5%, and the growth inhibition rate increased from 78.6% to 86.0%. The ability of soft agar colony-formation was completely suppressed. CONCLUSIONS: These observations strongly support that the blockage of the expression of autocrine growth factor TGF alpha and its receptor EGFR was a potent way in circumventing the malignant properties of the pancreatic carcinoma cells.