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目的探讨真核表达质粒pRc/CMV2-FHIT对结肠癌细胞株SW480增殖和侵袭活性的影响。方法将携有FHIT基因的重组真核表达质粒pRc/CMV2-FHIT转化大肠杆菌DH5α并扩增,抽提纯化质粒并进行酶切鉴定,pRc/CMV2-FHIT体外转染SW480细胞(SW480-FHIT),筛选稳定转染的细胞并扩增培养,以转染了空质粒pRc/CMV2的SW480细胞(SW480-pRc/CMV2)和正常SW480细胞为对照,用RT-PCR检测FHIT的表达情况,并用四甲基偶氮唑蓝比色法(MTT法)和细胞侵袭实验分别检测FHIT基因转染前、后SW480细胞增殖和侵袭活性的变化。结果pRc/CMV2-FHIT的酶切鉴定证实含有目的基因FHIT;SW480-FHIT细胞的RT-PCR产物经凝胶电泳有明显的阳性条带,而对照组则无;MTT实验结果显示重组质粒转染组SW480细胞的增殖明显受到抑制;Transwell体外侵袭实验显示转染pRc/CMV2-FHIT能明显抑制SW480细胞的体外侵袭力(P<0.01)。结论应用基因转染技术重表达FHIT基因能有效抑制细胞的生长、增殖及侵袭活性,为以FHIT为靶向的结肠癌基因治疗提供了新的思路和手段。
Objective To investigate the effect of eukaryotic expression plasmid pRc/CMV2-FHIT on proliferation and invasion activity of colon cancer cell line SW480. Methods The recombinant eukaryotic expression plasmid pRc/CMV2-FHIT carrying the FHIT gene was transformed into E.coli DH5α and amplified. The plasmid was extracted and identified by enzyme digestion. SW480 cells were transfected with pRc/CMV2-FHIT in vitro (SW480-FHIT). The stably transfected cells were selected and expanded for culture. SW480 cells (SW480-pRc/CMV2) transfected with empty plasmid pRc/CMV2 and normal SW480 cells were used as controls. The expression of FHIT was detected by RT-PCR and used four times. Methotrexate blue colorimetric assay (MTT assay) and cell invasion assay were used to detect the proliferation and invasion activity of SW480 cells before and after FHIT gene transfection. Results The restriction endonuclease digestion of pRc/CMV2-FHIT confirmed the presence of the target gene FHIT; the RT-PCR product of SW480-FHIT cells showed a positive band by gel electrophoresis, whereas the control group had no positive band; MTT assay results showed that the recombinant plasmid was transfected. The proliferation of SW480 cells was significantly inhibited; Transwell in vitro invasion experiments showed that the transfection of pRc/CMV2-FHIT can significantly inhibit the in vitro invasiveness of SW480 cells (P<0.01). Conclusion Re-expression of FHIT gene by gene transfection technology can effectively inhibit cell growth, proliferation and invasion activity, and provide new ideas and means for gene therapy of colon cancer with FHIT as the target.