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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的表达载体ｐＣＭＶ－ＡＳ－ＥＧＦＲ，转染已经反义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、原位凋亡检测细胞凋亡。结果双重转染细胞系有外源ＴＧＦα基因的整合及表达，内源性ＥＧＦＲ及ｃｙｃｌｉｎＤ１ｍＲＮＡ表达下调、细胞表面ＥＧＦＲ受体表达下降，与反义ＴＧＦα单独作用比较，反义ＥＧＦＲ与反义ＴＧＦα的联合作用更强。３Ｈ－ＴｄＲ掺入率由２５％降至１４．５％。生长抑制率由７８．８％提高到８６．０％、软琼脂集落形成能力完全丧失。双重抑制后，细胞更容易发生凋亡。结论对胰腺癌中异常信号传导途径的阻断，能明显地抑制肿瘤恶性生物学行为，使肿瘤细胞恶性表型部分逆转。
Objective To study the effect of blocking TGFα-EGFR autocrine loop on the growth of pancreatic cancer cells. Methods The antisense EGFR expression vector pCMV-AS-EGFR was constructed and transfected into PC-7 cells which had been transformed with antisense TGFα. The cells were stained with G418 to obtain a stable double-stranded transformed cell line PC-7 / AS-TGFα / AS -EGFR. The integration and expression of double-transfected cell lines were analyzed by Southern blot, Northern blot and 125I-EGF binding assay. DNA gel electrophoresis, flow cytometry, apoptosis in situ detection of apoptosis. Results The double transfected cell lines had the integration and expression of exogenous TGFα gene, the expression of endogenous EGFR and cyclinD1 mRNA was down-regulated and the expression of EGFR receptor on the cell surface was decreased. Compared with the antisense TGFα alone, the combination of antisense EGFR and antisense TGFα More effective. 3H-TdR incorporation decreased from 25% to 14.5%. Growth inhibition rate increased from 78.8% to 86.0%, soft agar colony formation ability completely lost. After double inhibition, cells are more prone to apoptosis. Conclusion The blockade of abnormal signal transduction pathways in pancreatic cancer can significantly inhibit the malignant biological behavior of tumor and partially reverse the malignant phenotype of tumor cells.