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（〖STBX〗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 explore effect of the adenovirus vector (Ad.RGD-ING4-PTEN) modified with RGD and co-expressing with double genes coding inhibitor of growth 4 (ING4) and phosphatase and tensin homologue deleted on chromosome ten (PTEN) on proliferation, apoptosis and invasion of human neuroglioma cells in vitro.Methods: Neuroglioma U87 cells were in vitro infected with Ad.RGD-ING4-PTEN as experimental group, Ad.RGD-ING4/-PTEN as monogenge control group and PBS, Ad.RGD/RGD-GPF as blank control group. Expressions of the target genes, ING4 and PTEN in the U87 cells were detected by Western blotting. Effect of infection with the Ad.RGD-ING4-PTEN of experimental group on proliferation of the U87 cell was detected by MTT assay. Changes in apoptosis of the neuroglioma cells and expressions of apoptosis-related genes, Bcl-2, Bax, caspase-3 and HIF-1α, were respectively examined with flow cytometry and Real-time PCR assays. Effects of infection with the Ad.RGD-ING4-PTEN of experimental group on migration and invasion abilities were detected with scratch and Transwell assays respectively. Expression of invasion-associated genes, MMP-2 and MMP-9, was detected by Real-time PCR. Results: Expressions of ING4 and PTEN were successfully detected only in the experimental group and the corresponding monogene control groups. Inhibitory and apoptosis rate of the U87 cell on 5th day in experimental group were (83.1±4.6)% and (40.7±4.3)%, respectively, compared to the blank control and monogene control groups, the differences were statistically significant (P<0.05). In the experimental group, expressions of apoptosis-related proteins, Bax and caspase-3, were remarkably up-regulated, while expressions of HIF-1α and Bcl-2 proteins down-regulated in the U87 cells (all P<0.05), and expressions of invasion-associated molecules MMP-2 and MMP-9 also down-regulated significantly (all P<0.05 ). Migration distances(\[70.1±6.2\] μm) of the cells and number of the penetrated cells (\[266±3.5\] cell) in the experiment group were significantly lesser than those in the monogene control and blank control groups(all P<0.05). Conclusion: The adenovirus with double-gene ING4 and PTEN (Ad.RGD-ING4-PTEN) could more significantly inhibit proliferation and induce apoptosis of the neuroglioma U87 cells, compared to those in the adenovirus with monogene, and also could inhibit migration and invasion abilities of the U87 cells.

国家自然科学基金青年基金资助项目（No.81001016)