摘要
目的 探讨腺病毒介导的人生长抑制因子4（ING4）基因对人膀胱移行细胞癌T24细胞体外抑癌效应及其分子机制.方法 将含ING4的重组腺病毒（Ad-ING4）转染T24细胞,以空载体组、未转染组细胞作对照.应用半定量反转录-聚合酶链反应（RT-PCR）和Western印迹法检测各组ING4基因及蛋白质在T24细胞中的表达 四甲基偶氮唑盐（MTT）法检测各组T24细胞生长情况 流式细胞术（FCM）和Hochest 33258染色法检测各组T24细胞凋亡变化 半定量RT-PCR法检测各组细胞中凋亡相关基因B细胞淋巴瘤/白血病基因-2（Bcl-2）、B细胞淋巴瘤/白血病基因伴随蛋白x（Bax）、p53、缺氧诱导因子（HIF）1α和半胱氨酸蛋白酶3（caspase-3）的表达 Western印迹法检测各组细胞中caspase-3蛋白的表达.结果 腺病毒介导的ING4基因在T24细胞中成功表达,能明显诱导T24细胞凋亡,其凋亡率可达17.2%±4.1%,高于空载体组（4.7%±1.2%）和未转染组（4.8%±1.2%,P〈0.05） 外源性ING4基因可引起T24细胞中p53、Bax基因表达上凋（1.40±0.11比0.27±0.04,1.50±0.12比0.60±0.05）和Bcl-2、HIF-1α基因表达下凋（0.19±0.02比1.20±0.08,0.33±0.03比0.98±0.06,均P〈0.05）,并促进caspase-3活化.结论 腺病毒介导的ING4基因在体外可通过上凋p53、Bax/Bcl-2基因和下凋HIF-1α基因表达,导致caspase-3的活化而抑制人膀胱移行细胞癌T24细胞的生长,并诱导其凋亡.
Objective To explore the inhibitory effect and anti-cancer mechanisms of adenovirusmediated ING4 gene on the human bladder cancer T24 cells in vitro.Methods The methods of reverse transcription-polymerase chain reaction （RT-PCR） and Western blot were used to detect the expression of ING4 in human bladder urothelial carcinoma T24 line.The influence of Ad-ING4 transfection on cell proliferation was evaluated by MTT assay and cell apoptosis by Hochest33258 staining and flow cytometry.RT-PCR and Western blot were performed to detect the transcriptional level of such apoptosis-related genes as Bcl-2, Bax, p53, HIF-1α and caspase-3.Results Human ING4 was successfully transcribed in T24 cell.Apoptosis rate of T24 cell in Ad-ING4 group was significant higher than that in control groups（17.2% ±4.1% vs 4.7% ± 1.2% and 4.8% ± 1.2% ,P 〈0.05）.Ad-ING4 not only up-regulated the expression of p53 and Bax （1.40 ± 0.11 vs 0.27 ± 0.04,1.50 ± 0.12 vs 0.60 ± 0.05） and down-regulated the expression of Bcl-2 and HIF-1α（0.19 ±0.02 vs 1.20 ±0.08,0.33 ±0.03 vs 0.98 ±0.06,all P 〈0.05）, but also enhanced the caspase-3 activation and eventually led to apoptosis.Conclusion Adenovirus-mediated ING4 gene exhibits anti-tumor capacity in T24 human bladder cancer cell and induces in vitro apoptosis.It may be related to the up-regulation of P53 and Bax/Bcl-2, and the down-regulation of HIF-lα.Thus the caspase-3 activation is enhanced so as to lead to apoptosis.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2010年第42期3008-3013,共6页
National Medical Journal of China
基金
基金项目：国家自然科学基金（K112214710）
江苏省医学重点人才项目（RC2007080）
江苏省卫生厅科研课题（H200610）
