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目的研究外源FHIT基因对不同FHIT基因背景肺癌细胞系恶性增殖的影响并探讨抑癌机理。方法用Lipofectamine介导PEGFP-FHIT真核表达质粒转染受体细胞系并建立稳定转染细胞系。RT-PCR、免疫组织化学及Western blot方法鉴定转染细胞中外源FHIT基因和蛋白的表达。细胞集落形成实验研究外源FHIT基因对肺癌细胞恶性增殖的影响,流式细胞仪分析细胞周期和凋亡。Western blot检测母系及转染细胞系中p21Waf/cip蛋白的表达。结果3种受体细胞转染外源FHIT基因后均检测到FHIT mRNA和蛋白的表达。集落形成实验801D-FHIT(46.3%)、A2-FHIT(43.7%)、A549-FHIT(32.7%)细胞集落形成率比801D(58.2%)、A2(60.3%)、A549(52.8%)的低,差异有显著性(P<0.01)。FACS分析细胞周期显示:801D-FHIT、A2-FHIT和A549-FHIT细胞分别出现了G1、S和G2/M期阻滞。转染FHIT基因的801D-FHIT、A2-FHIT和A549-FHIT细胞p21Waf/cip蛋白表达增加。结论外源FHIT基因能够抑制不同FHIT基因背景的肺癌细胞的恶性增殖,并引起细胞周期改变,不同FHIT基因背景的细胞产生细胞周期阻滞的时间不同。FHIT基因通过促进p21Waf/cip蛋白的表达来调节细胞周期。
Objective To investigate the effect of exogenous FHIT gene on the malignant proliferation of lung cancer cell lines with different FHIT gene backgrounds and to explore the mechanism of tumor suppressor. Methods Lipofectamine-mediated PEGFP-FHIT eukaryotic expression plasmid was transfected into recipient cell lines and stable transfected cell lines were established. RT-PCR, immunohistochemistry and Western blot were used to identify the expression of exogenous FHIT gene and protein in transfected cells. The effects of exogenous FHIT gene on malignant proliferation of lung cancer cells were studied by cell colony formation assay. Cell cycle and apoptosis were analyzed by flow cytometry. Western blot was used to detect p21Waf / cip protein expression in maternal and transfected cell lines. Results FHIT mRNA and protein expression were detected in all three recipient cells transfected with exogenous FHIT gene. The colonization rate of colony-forming experiments 801D-FHIT (46.3%), A2-FHIT (43.7%) and A549-FHIT (32.7%) were lower than that of 801D (58.2%), A2 (60.3%) and A549 , The difference was significant (P <0.01). FACS analysis showed that G1, S and G2 / M arrests were observed in 801D-FHIT, A2-FHIT and A549-FHIT cells, respectively. The expression of p21Waf / cip protein in 801D-FHIT, A2-FHIT and A549-FHIT transfected with FHIT gene increased. Conclusion Exogenous FHIT gene can inhibit the proliferation of lung cancer cells with different FHIT gene backgrounds and cause cell cycle changes. The cell cycle arrest in different FHIT gene backgrounds is different. The FHIT gene regulates the cell cycle by promoting the expression of p21Waf / cip protein.