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...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。展开更多
As CD40 transduces activation signals involved in inflammatory and immune disorders,we explored the expression and response to CD40 engagement in human glioma cell lines in this study.The CD40 expression in BT-325 and...As CD40 transduces activation signals involved in inflammatory and immune disorders,we explored the expression and response to CD40 engagement in human glioma cell lines in this study.The CD40 expression in BT-325 and U251 cells was flow cytometrically detected.The cells were incubated with srhCD40L for 72 h to assess its effects on cell growth in vitro.TNF-α expression was quantified by real-time PCR,and protein expression was analyzed by ELISA.The I-κb mRNA was detected by RT-PCR.I-κB expression decreased after stimulation with 1 μg/mL srhCD40L,but it was upregulated after the cells were pretreated with CD40 antibody.srhCD40L significantly inhibited the proliferation of the CD40+ human glioma cells.The stimulation of CD40+ glioma cells with soluble CD40L (CD154) up-regulated the expression of TNF-α at both mRNA and protein levels.We are led to conclude that CD40L/CD40 could inhibit human glioma cells through I-κb signaling pathway.Interferon-γ can augment CD40 expression and the inhibitory effect of CD40 ligand on cell growth in vitro.These results suggest that srhCD40L may benefit the therapy strategy of glioma.展开更多
文摘目的: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。
基金supported by a grant from the Ministry ofEducation for New Teachers,China (No.20070487132)
文摘As CD40 transduces activation signals involved in inflammatory and immune disorders,we explored the expression and response to CD40 engagement in human glioma cell lines in this study.The CD40 expression in BT-325 and U251 cells was flow cytometrically detected.The cells were incubated with srhCD40L for 72 h to assess its effects on cell growth in vitro.TNF-α expression was quantified by real-time PCR,and protein expression was analyzed by ELISA.The I-κb mRNA was detected by RT-PCR.I-κB expression decreased after stimulation with 1 μg/mL srhCD40L,but it was upregulated after the cells were pretreated with CD40 antibody.srhCD40L significantly inhibited the proliferation of the CD40+ human glioma cells.The stimulation of CD40+ glioma cells with soluble CD40L (CD154) up-regulated the expression of TNF-α at both mRNA and protein levels.We are led to conclude that CD40L/CD40 could inhibit human glioma cells through I-κb signaling pathway.Interferon-γ can augment CD40 expression and the inhibitory effect of CD40 ligand on cell growth in vitro.These results suggest that srhCD40L may benefit the therapy strategy of glioma.
