Δ133p53表达状态对rmhTNF效应的影响及机制研究  

Effect and mechanism of rmh TNF on gastric cancer cell lines with different Δ133p53 status

作　　者：姜琪琪[1] 张红梅[2] 郭爱[1] 张娜[1] 高志星[2] 季万胜[2] 

机构地区：[1]潍坊医学院,山东潍坊261053 [2]潍坊医学院附属医院消化内科,山东潍坊261031

出　　处：《中国癌症杂志》2015年第4期287-293,共7页China Oncology

基　　金：山东省优秀中青年科学家科研奖励基金(BS2010SW034)

摘　　要：背景与目的:p53异构体在胃癌发生中的作用报道较少。该研究旨在探讨p53异构体Δ133p53在重组改构人肿瘤坏死因子(recombinant mutant human tumor necrosis factor,rmh TNF)干预胃癌细胞系生物学效应中的作用,为胃癌诊断和治疗提供新的依据。方法:采用细胞增殖/毒性检测试剂盒(CCK-8)和流式细胞术,检测不同浓度的rmh TNF单独或联合氟尿嘧啶(5-FU)应用于MKN-45(表达Δ133p53)和SGC-7901(不表达Δ133p53)细胞,观察细胞抑制率和细胞凋亡情况。通过巢式逆转录多聚酶链反应(nested reverse transcriptase-polymerase chain reaction,n RT-PCR)和实时荧光定量多聚酶链反应(real-time polymerase chain reaction,RT-PCR)检测Δ133p53、Gadd45α和Cyclin B1 m RNA的表达变化。结果:rmh TNF单独作用于Δ133p53表达阳性的MKN-45细胞有抑制作用,浓度为50、500 IU/m L的rmh TNF作用24 h后,细胞抑制率分别为24.82%、72.33%(t=-9.558,P<0.01),并可提高5-FU的抑制率,且具有显著的量效和时效关系,5-FU(25μg/m L)、rmh TNF(50 IU/m L)+5-FU(25μg/m L)、rmh TNF(500 IU/m L)+5-FU(25μg/m L)作用于MKN-45细胞24 h后,抑制率分别为18.20%、48.66%、59.83%(F=82.742,P<0.01);rmh TNF(50 IU/m L)+5-FU(25μg/m L)作用于MKN-45细胞24、48、72 h后,抑制率分别为48.66%、68.20%、85.23%(F=128.583,P<0.01)。而对于Δ133p53表达阴性的SGC-7901细胞,浓度为50、500 IU/m L的rmh TNF单独抑制率为2.74%、3.25%,抑制作用不明显(t=-0.121,P>0.05)。流式细胞术显示,rmh TNF不仅单独可引起MKN-45细胞凋亡,而且可显著增强5-FU促细胞凋亡作用,rmh TNF(50 IU/m L)、rmh TNF(50 IU/m L)+5-FU(25μg/m L)、rmh TNF(500 IU/m L)+5-FU(25μg/m L)作用于MKN-45细胞24 h后,凋亡率分别为7.21%、10.13%、15.28%(F=123.931,P<0.05)。在MKN-45中,rmh TNF单独或联合5-FU可下调Δ133p53和Cyclin B1基因,上调Gadd45α基因表达水平。n RT-PCR检测对照组及实验组Δ133p53基因相对表达量分别为0.886、0.499、0.330、0.161(F=240.927,P<0.01);ReBackground and purpose： Little about the function of p53 isoforms in gastric cancer was reported. This study was designed to explore the role of Δ133p53 in the effect of recombinant mutant human tumor necrosis factor （rmhTNF） on gastric cancer cells, and provide a new basis for the diagnostics and therapeutics of gastric carcinoma. Methods： MKN45 （with Δ133p53 expression） or SGC7901 （without Δ133p53 expression） cells were treated with rmhTNF of different concentrations only or combined with 5-FU （a traditional gastric cancer cellular killer）, and the growth inhibition rate and apoptosis was detected by CCK-8 and flow cytometry, mRNA expressions of Δ133p53, Gadd45a and CyclinB1 were measured by nested reverse transcription-polymerase chain reaction （nRT-PCR） or real-time polymerase chain reaction（RT-PCR）. Results： On MKN-45 cells with positive Δ133p53 expression, the inhibitory effect of rmhTNF was significant, the inhibition rates of 50 and 500 IU/mL rmhTNF were 24.82%, 72.33% after culturing for 24 h （t=-9.558, P〈0.01）; also, the inhibitory effect of 5-FU was improved by rmhTNF remarkably in time- and dose-dependence, the inhibition rates of 5-FU （25 μg/mL）, rmhTNF （50 IU/mL） combined with 5-FU （25 μg/mL）, rmhTNF （500 IU/mL） combined with 5-FU （25 μg/mL） were 18.20%, 48.66%, 59.83%, separately, after culring for 24 h （F=82.742, P〈0.01）; the inhibition rates of rmhTNF （50 IU/mL） combined with 5-FU （25 μg/mL） were 48.66%, 68.20%, 85.23%, separately, after culturing for 24 h, 48 h and 72 h （F=128.583, P〈0.01）. However, on SGC-7901 cells with negative Δ133p53 expression, no growth inhibition was showed by rmhTNF only, the inhibition rates of 50 and 500 IU/mL were 2.74%, 3.25% after culturing for 24 h （t=-0.121, P〉0.05）. In apoptosis test, the apopto- sis-enhancing effect of rmhTNF was significant on MKN45 cells, and the apoptosis-enhancing effect of 5-FU was fur- ther promoted significantly by rmhTNE the apoptosis of rmhTNF �

关 键 词：重组改构人肿瘤坏死因子 5-FU 胃癌细胞系 Δ133p53 

分 类 号：R73-36[医药卫生—肿瘤]