APC不同功能区域对结肠癌细胞株HT-29中β-连环蛋白表达的影响

Effect of recombinant pEGFP-N3-APC vectors carrying various APC functional domains on the expression of β-catenin in HT-29 cells

目的:探讨含有APC蛋白不同功能区域的pEGFP-N3-APC重组质粒对结肠癌细胞株HT-29中β-连环蛋白表达的影响。方法:脂质体介导重组质粒pEGFP-N3-APC1～5转染HT-29,采用绿色荧光及RT-PCR验证重组质粒在细胞中的表达。以Western免疫印迹检测转染后HT-29细胞中β-连环蛋白的表达,以SPSS13.0软件分析电泳条带的灰度值。结果:重组质粒转染HT-29细胞后,绿色荧光及RT-PCR结果显示5个APC蛋白不同功能区域多肽可在细胞中表达。Western免疫印迹结果显示,重组质粒pEGFP-N3-APC1,pEGFP-N3-APC2和pEGFP-N3-APC3对β-连环蛋白表达无影响,而pEGFP-N3-APC4和pEGFP-N3-APC5均可降低结肠癌细胞株HT-29中β-连环蛋白的表达水平,其中以pEGFP-N3-APC5的抑制程度最强。结论:含有APC蛋白中15氨基重复序列+SAMP重复序列的APC5基因片段既可有效降低β-连环蛋白的表达,同时又是相对长度较短的可用于基因治疗的最优片段。

Objective To explore the effect of recombinant pEGFP-N3-APC vectors carrying various APC functional domains on the expression of β-catenin in human colorectal cancer cells HT- 29. Methods The recombinant plasmids were transfected into HT-29 cells mediated by lipofectamineTM 2000, and detected by green fluorescence and RT-PCR. Western blot was applied to detect β- catenin expression level in HT-29 cells after transfection, and gray scales of electrophoresis strips were analyzed by SPSS 13.0. Results Green fluorescence and RT-PCR made clear that all 5 recombinant plasmids were successfully expressed in HT-29 cells. Western blot showed that β-catenin expression level in HT-29 ceils was not affected after being transfected with pEGFP-N3-APC1, pEGFP-N3-APC2 and pEGFP-N3-APC3, and was distinctly affected after being transfected with pEGFP-N3-APC4 and pEGFP-N3-APCS, especially the later one. Conclusion The selected APC5 gene fragment with 15-amino acid repeats and SAMP repeats, whieh is relatively short, ean degrade β-catenin level in HT-29 cells and may be applied in the gene therapy.