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目的研究外源性脆性组胺酸三联体(FHIT)基因在体内外对人肺腺癌A549细胞恶性表型的影响。方法用脂质体介导外源FHIT基因转染A549细胞,建立单克隆细胞系FHITA-549和PEGFP-A549。逆转录聚合酶链反应(RTPCR)、免疫组化方法检测外源FHIT基因在A549细胞的表达状况。采用细胞生长曲线、集落形成试验、流式细胞仪及裸鼠移植瘤试验研究外源FHIT基因对A549细胞体外增殖、凋亡、细胞周期和体内成瘤性的影响。结果RTPCR试验证实FHIT-A549中有FHITmRNA表达,免疫组织化学染色显示FHIT-A549细胞FHIT蛋白表达强阳性,而A549细胞和转空载体的PEGFPA549细胞FHIT基因和蛋白表达均阴性。FHIT-A549细胞的集落形成率为2.6%,显著低于A549细胞的50.1%和转染空载体PEGFPA549细胞的53.6%,三者相比差异有统计学意义(P<0.01)。流式细胞仪分析显示FHIT-A549细胞95.8%阻滞在G2期。FHITA549细胞移植瘤的瘤重为(0.04±0.03)g,显著低于A549细胞的(0.24±0.11)g和转染空载体PEGFPA549细胞的(0.25±0.07)g,三者差异有统计学意义(P<0.01)。结论A549细胞内外源FHIT基因的转导并表达能显著抑制其恶性增殖和分裂,诱导其凋亡,调节其细胞周期、抑制成瘤性。
Objective To investigate the effect of exogenous FHIT gene on the malignant phenotype of human lung adenocarcinoma A549 cells in vitro and in vivo. Methods A549 cells were transfected with exogenous FHIT gene by liposome, and the monoclonal cell lines FHITA-549 and PEGFP-A549 were established. Reverse transcriptase polymerase chain reaction (RTPCR) and immunohistochemistry were used to detect the expression of exogenous FHIT gene in A549 cells. The effects of exogenous FHIT gene on the proliferation, apoptosis, cell cycle and in vivo tumorigenicity of A549 cells were studied by cell growth curve, colony formation assay, flow cytometry and nude mice xenografts. Results FHIT-A549 FHIT mRNA expression was confirmed by RTPCR. FHIT protein expression in FHIT-A549 cells was strongly positive by immunohistochemical staining. FHIT gene and protein expression in A549 cells and empty vector transfected PEGFPA549 cells were all negative. The colony formation rate of FHIT-A549 cells was 2.6%, which was significantly lower than that of A549 cells and 53.6% of the transfected empty vector PEGFPA549 cells. The difference was statistically significant (P <0.01). Flow cytometry analysis showed 95.8% of FHIT-A549 cells arrested in G2 phase. The tumor weight of FHITA549 cells was (0.04 ± 0.03) g, significantly lower than that of A549 cells (0.24 ± 0.11) g and (0.25 ± 0.07) g transfected with empty vector PEGFPA549 cells P <0.01). Conclusion The transduction and expression of both FHIT gene and exogenous FHIT gene in A549 cells can significantly inhibit the proliferation and division of malignant cells, induce apoptosis, regulate cell cycle and inhibit tumorigenicity.