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目的探讨低氧诱导因子1α(HIF-1α)对宫颈癌细胞的生物学行为的影响以及其中可能存在的分子机制。方法通过 CoCl_2化学诱导宫颈癌 HeLa 细胞缺氧;构建靶向 HIF-1α的反义真核表达载体、经脂质体介导转染 HeLa 细胞的方法沉默 HIF-1α的表达。将实验细胞分为常氧未转染对照(NN)组、常氧空质粒转染对照(NI)组、常氧转染 pcDNA3.0/HIF-1α质粒(NT)组、缺氧未转染对照(HN)组、缺氧空质粒转染对照(HT)组、缺氧转染 pcDNA3.0/HIF-1α质粒(HT)组。用四甲基偶氮唑蓝法、Transwell 侵袭小室方法观察各组细胞的增殖、侵袭能力的改变,用流式细胞仪检测各组细胞的凋亡率,用 RT-PCR 技术检测各组细胞目的基因 HIF-1α及其靶基因血管内皮生长因子(VEGF)、葡萄糖转运体1(GLUT1)、多药耐药基因1(MDR1)的表达变化。结果 NT 组细胞在培养12、24、48、72 h 时的活细胞数分别为0.053±0.003、0.074±0.004、0.148±0.015、0.192±0.038,而 HT 组分别为0.069±0.003、0.155±0.022、0.224±0.022、0.308±0.069;NT 和 HT 组的细胞增殖受到抑制,NT组与 NN 及 NI 组、HT 组与 HN 及 HI 组间的活细胞数分别比较,差异均有统计学意义(P<0.01)。各组细胞的凋亡率分别是,NN 组(29.27±0.18)%、NI 组(31.13±0.08)%、NT 组(51.11±0.14)%、HN 组(11.46±0.28)%、HI 组(15.77±0.49)%、HT 组(40.05±0.97)%;HT 组与 HN 及 HI 组、NT组与 NN 及 NI 组间分别比较,差异均有统计学意义(P<0.01)。各组细胞的侵袭能力由高到低依次为 HI、HN、NI、NN、HT、NT 组,分别为(40±9)%、(37±12)%、(28±5)%、(26±7)%、(19±7)%、(10±5)%;NT 组与 NN 及 NI 组、HT 组与 HN 及 HI 组间分别比较,差异均有统计学意义(P<0.05)。NT 组细胞 HIF-1α、VEGF、GLUT1、MDR1 mRNA 的相对表达量分别为0.05±0.12、0.09±0.11、0.08±0.15、0.05±0.15,而 HT 组分别为0.04±0.16、0.16±0.16、0.12±0.20、0.20±0.21;NT 组与 NN及 NI 组、HT 组与 HN 及 HI 组间分别比较,HIF-1α、VEGF、GLUT1、MDR1 mRNA 的相对表达量均有降低,差异均有统计学意义(P<0.01)。结论 HIF-1α可能主要通过其下游的靶基因对宫颈癌细胞的恶性生物学行为产生影响,包括抗凋亡、促增殖、增加血液供应和能量供应、耐药等,且体外抑制 HIF-1α的表达对宫颈癌细胞有抑制作用。
Objective To investigate the effect of hypoxia-inducible factor-1α (HIF-1α) on the biological behavior of cervical cancer cells and the possible molecular mechanisms. Methods The HeLa cells were induced by CoCl 2 to induce hypoxia. Antisense eukaryotic expression vector targeting HIF-1α was constructed and the expression of HIF-1α was silenced by liposome-mediated transfection of HeLa cells. The experimental cells were divided into normoxia untransfected control (NN) group, normoxic empty plasmid transfected control (NI) group, normoxic transfected pcDNA3.0 / HIF-1α plasmid (NT) HN group, HT group, and hypoxia-transfected pcDNA3.0 / HIF-1α plasmid group. The cell proliferation and invasion ability of each group were observed by MTT method and Transwell invasion chamber method. The apoptosis rate of each group was detected by flow cytometry. The purpose of each group of cells was detected by RT-PCR Gene expression of vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1) and multidrug resistance gene 1 (MDR1) in HIF-1αgene. Results The number of viable cells in NT group were 0.053 ± 0.003,0.074 ± 0.004,0.148 ± 0.015,0.192 ± 0.038, respectively, while those in HT group were 0.069 ± 0.003,0.155 ± 0.022, 0.224 ± 0.022,0.308 ± 0.069. The cell proliferation was inhibited in NT and HT groups. There were significant differences in the number of viable cells between NT group and NN group, HT group, HN group and HI group (P < 0.01). The apoptotic rate of each group was 29.27 ± 0.18% in NN group, 31.13 ± 0.08% in NI group, 51.11 ± 0.14% in NT group, 11.46 ± 0.28% in HN group and 15.77 in HI group, respectively ± 0.49)% in HT group and (40.05 ± 0.97)% in HT group. There was significant difference between HT group and HN and HI group, NT group and NN group and NI group (P <0.01). The invasive ability of each group was (40 ± 9)%, (37 ± 12)%, (28 ± 5)%, (26 ± 5)% (7 ± 7)%, (19 ± 7)%, (10 ± 5)% respectively. There were significant differences between NT group and NN group and NI group, HT group and HN group and HI group (P <0.05). The relative expression levels of HIF-1α, VEGF, GLUT1 and MDR1 mRNA in NT group were respectively 0.05 ± 0.12,0.09 ± 0.11,0.08 ± 0.15,0.05 ± 0.15, while those in HT group were 0.04 ± 0.16,0.16 ± 0.16,0.12 ± 0.20, 0.20 ± 0.21 respectively. The relative expression of HIF-1α, VEGF, GLUT1 and MDR1 mRNA in NT group, NN group and NI group were lower than those in HT group and HN group P <0.01). Conclusion HIF-1α may affect the malignant biological behavior of cervical cancer cells mainly through its downstream target genes, including anti-apoptosis, promoting proliferation, increasing blood supply and energy supply, resistance and so on, and inhibiting HIF-1α in vitro The expression of cervical cancer cells have inhibitory effect.