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目的:探讨经RGD修饰的生长抑制因子4(inhibitor of growth 4,ING4)基因与第10染色体缺失与张力蛋白同源的磷酸酶基因(phosphatase and tensin homologue deleted on chromosome ten,PTEN)双基因共表达的腺病毒载体(Ad.RGD-ING4-PTEN)体外对神经胶质瘤U87细胞的增殖、凋亡及侵袭的影响。方法:以Ad.RGD-ING4-PTEN为实验组,Ad.RGD-ING4/-PTEN为单基因对照组,PBS、Ad.RGD/Ad-GFP为空白对照组,分别体外感染U87神经胶质瘤细胞。Western blotting检测目的基因ING4和PTEN在U87细胞中的表达,MTT法检测实验组病毒感染对U87细胞增殖的影响,流式细胞术及Real-time PCR法检测神经胶质瘤细胞凋亡及凋亡相关基因(Bcl-2、Bax、caspase-3、HIF-1α)表达变化,划痕实验及Transwell实验检测实验组病毒感染对U87细胞迁移及侵袭能力的影响,Real-time PCR法检测侵袭相关基因(MMP-2、MMP-9)表达变化。结果:成功检测到ING4和PTEN仅在实验组及相应单基因对照组中表达。实验组第5天细胞抑制率可达(83.1±4.6)%、凋亡率可达(40.7±4.3)%,与单基因组及空白对照组相比差异均有统计学意义(P<0.05);实验组能明显上调U87细胞中Bax、caspase-3和下调HIF-1α、Bcl-2等细胞凋亡相关蛋白的表达(均P<0.05),而且肿瘤侵袭相关分子MMP-2、MMP-9的表达也明显下调(均P<0.05);实验组细胞迁移距离[(70.1±6.2)μm]和穿膜细胞数[(26.6±3.5)个]均明显减少,与单基因组及空白对照组比较差异有统计学意义(均P<0.05)。结论:与单基因腺病毒相比,Ad.RGD-ING4-PTEN双基因具有更显著的抑制U87神经胶质瘤细胞增殖、诱导其凋亡,并抑制其迁移及侵袭能力。
OBJECTIVE: To investigate the coexpression of phosphatase and tensin homolog deleted in chromosome ten (PTEN) genes with inhibitor of growth 4 (ING4) gene and chromosome 10 deletion and tension protein (Ad.RGD-ING4-PTEN) on proliferation, apoptosis and invasion of glioma U87 cells in vitro. Methods: Ad.RGD-ING4-PTEN was used as experimental group, Ad.RGD-ING4 / -PTEN was used as control group, PBS and Ad.RGD / Ad-GFP were used as blank control group, respectively. In vitro infection of U87 glioma cell. The expression of ING4 and PTEN in U87 cells was detected by Western blotting. The effect of viral infection in experimental group on the proliferation of U87 cells was detected by MTT assay. The apoptosis and apoptosis of glioma cells were detected by flow cytometry and Real-time PCR The expression of Bcl-2, Bax, caspase-3 and HIF-1αin the U87 cells were detected by scratch test and Transwell assay. The invasion and metastasis of U87 cells were detected by Real-time PCR. (MMP-2, MMP-9) expression changes. RESULTS: ING4 and PTEN were successfully detected only in the experimental group and the corresponding single-gene control group. The inhibitory rate reached 83.1 ± 4.6% and the apoptosis rate reached 40.7 ± 4.3% on the fifth day in the experimental group, which was significantly different from that in the control group (P <0.05). The experimental group could significantly up-regulate the expression of Bax, caspase-3 and down-regulate the expression of HIF-1α and Bcl-2 in U87 cells (all P <0.05), and the expression of MMP-2 and MMP- (P <0.05). The migration distance [(70.1 ± 6.2) μm] and the number of transmembrane cells in the experimental group [(26.6 ± 3.5)] were significantly decreased compared with those in the single and blank control groups There was statistical significance (all P <0.05). Conclusion: Compared with single-gene adenovirus, the Ad.RGD-ING4-PTEN double gene can significantly inhibit the proliferation of U87 glioma cells, induce its apoptosis, and inhibit its migration and invasion.