【作者】 陈星； 糜若然； 伊铁忠； 瞿全新； 熊冬生； 许元富； 杨纯正； 邵晓枫； 范冬梅；

【机构】 天津医科大学总医院妇产科；

【摘要】 目的:探讨脂质体介导VEGF—C基因转染人宫颈癌HeLa细胞及对其侵袭转移的分子机制研究。方法:用脂质体介导将真核表达载体pcDNA3．1(+)／VEGF-C转染人宫颈癌HeLa细胞,并加压筛选获得转染成功的细胞株,经半定量RT-PCR检测转染后VEGF-C表达水平,EIASA检测培养上清中VEGF-C的表达。对转染成功的细胞检测NF-κ B、bcl-2、MMP2、MMP9、TIMP-1基因的表达水平及对顺铂敏感性的变化。Transwell小室检测转染细胞侵袭和转移力;黏附实验检测肿瘤细胞黏附力的变化。结果:1、转染组VEGF-C明显高于空载体组和未转染组,ELISA检测转染组(678．73±38．92 mg／ml),也明显高于空载体组(129． 52±50．73 ng／ml),和未转染组(123．05±55．83 mg／ml), 成功构建了高表达VEGF-C的宫颈癌细胞株HeLa／S1;2、在 HeLa／S1组NF-κB的表达(1401．5±58．69 vs 917．5± 10．69 vs 935．0±11．32．P<0．05)、bcl-2的表达(2．02± 0．67 vs 0．41±0．06 vs 0．37±0．06,P<0．05)明显高于空载体组和未转染组;3、在宫颈癌细胞株HeLa／S 1中MMP2 的表达(2．41±0．81vs0．48±0．09vs0．49±0．18,P<0．05)、 MMP9的表达(2．26±0．29 vs 0．93±0．24vs0．79±0．05, P<0．05)明显高于空载体组和未转染组;TIMP-1(0．54± 0．03 vs 1．01±0．06vs1．03±0．06．P<0．05)的表达低于空载体和未转染组,在HeLa／S1细胞中MMP9／TIMP-1比值 4．7;4、在宫颈癌HeLa／S1细胞株中可检测到其对顺铂有弱的耐药性(1．84倍);5、在体外实验侵袭转移和黏附实验中, HeLa／S1细胞较空载体组和未转染组具有较大的侵袭转移力及黏附能力。结论:脂质体介导VEGF—C基因转染人宫颈癌 HeLa可显著增加VEGF-C表达,高表达的VEGF—C可激活 NF-κB,使抗凋亡基因bcl—2高表达;同时引起MMP2、9 高表达,TIMP-1低表达;从而使转染细胞具有较大的侵袭和转移力。更多还原

【Abstract】 Objective: To explore the effect of VEGF-C gene transfection on the expression in the human cervical carcinoma cells HeLa. Methods: The pcDNA3.1(+)/ VEGF-C was transformed into human cervical cancer cells HeLa induced by liposome and was selected by G418.The changes in the expression level of VEGF-C mRNA and protein were determined by semi-quantitive RT-PCR and ELISA. Overexpression VEGF-C HeLa was named as HeLa /Sl.The expression level of NF- κ B, bcl-2, MMP2, MMP9,TIMP-1 mRNA were determined by RT-PCR in transfected cell lines. The sensitivety on cispiatin of the HeLa /S1 was determined by MTT assay. The invasion and adhesion of the cell lines HeLa/S1 were analysis by Transwell filter. Results: 1. After transfection by liposome, the VEGF-C mRNA level and the expression of VEGF-C protein in transfection HeLa cell lines were higher than those of control groups,respectively. Overexpression VEGF-C of HeLa/S1 cell lines is constructed successfully. 2. In HeLa/ S1 cell lines the expression NF- κ B gene ( 1401.5 ± 58.69 vs 917.5 ± 10.69 vs 935.0 ± 11.32), bcl-2 gene mRNA(2.02 ± 0.67 vs 0.41 ± 0.06 vs 0.37 ± 0.06, P < 0.05) level were also higher than those of control groups. 3. In HeLa/S1 cell lines the expression MMP2 gene ( 2.41 ± 0.81 vs 0.48 ± 0.09 vs 0.49 ± 0.18, P < 0.05 ), MMP9 (2.26 ± 0.29 vs 0.93 ± 0.24 vs 0.79± 0.05, P < 0.05 ) gene mRNA level were higher than those of control groups, but TIMP-1 (0.54 ± 0.03 vs 1.01 ± 0.06 vs 1.03 ± 0.06, P < 0.05 ) was lower than those of control groups,the ra- tio of MMP9/TIMP-1 was higher than 1.4. The sensitivety of HeLa/S1 on cisplatin was 1.84.5. The invasion, metastasis and adhesion of HeLa/S1 were enhanced than those of control groups in vitro. Conclusion: After VEGF-C gene transfection by liposome ,the expression level of VEGF-C in human cervical cancer cell lines HeLa/S1 is greatly heightened. The expression of MMP2, MMP9 gene in HeLa/S1 were also increased and TIMP-1 was lower than those of control groups, NF- κ B was also stimulated and induced overexpression bcl-2 gene in HeLa/S1 by VEGF-C.To promote invasion and metastasis and adhesion in the HeLa/S1 phenotype.更多还原