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目的探讨Smad4基因对人胰腺癌细胞BxPC3 E-cadherin、β-catenin表达及细胞增殖的影响,为试图运用Smad4对胰腺癌进行基因治疗提供实验依据。方法经脂质体介导将含有Smad4重组表达质粒转染人胰腺癌细胞Bx-PC3,用G418筛选及RT-PCR、Western blot鉴定;分别采用RT-PCR和Western blot检测转染阳性细胞克隆E-cadherin、β-catenin表达改变;又分别采用噻唑盐(MTT)比色法及5-溴-2-脱氧尿苷(BrdU)掺入法检测阳性细胞克隆的增殖活性。结果成功建立表达Smad4的阳性BxPC3克隆(S4-8,S4-23),并证实其E-cadherin、β-catenin mRNA及蛋白表达均明显升高。MTT法和BrdU掺入法显示,S4-8与S4-23细胞克隆增殖显著降低。结论 Smad4可增加胰腺癌细胞E-cadherin及β-catenin的表达并抑制其增殖,Smad4可能对胰腺癌具有基因治疗的效果。
Objective To investigate the effect of Smad4 gene on the expression of BxPC3 E-cadherin, β-catenin and cell proliferation in human pancreatic cancer cells. In order to provide an experimental basis for the use of Smad4 in gene therapy of pancreatic cancer. Methods The recombinant plasmid containing Smad4 was transfected into human pancreatic cancer cell line Bx-PC3 by Lipofectamine 2000. The cells were screened by G418 and identified by RT-PCR and Western blot. The expression of clone E -cadherin, and β-catenin. The proliferation activity of positive cell clones was detected by MTT assay and BrdU incorporation assay respectively. Results Positive BxPC3 clones expressing Smad4 were successfully established (S4-8, S4-23), and the mRNA and protein expression of E-cadherin and β-catenin were significantly increased. MTT assay and BrdU incorporation assay showed that the proliferation of S4-8 and S4-23 cells was significantly reduced. Conclusion Smad4 can increase the expression of E-cadherin and β-catenin in pancreatic cancer cells and inhibit its proliferation. Smad4 may have a therapeutic effect on pancreatic cancer.