CAJ | 학술논문

目的观察莪术醇对人胃癌细胞株BGC823细胞周期的影响,并探讨其作用机制.方法体外培养BGC823细胞,分别加入12.5、25.0、50.0、100.0 mg/L莪术醇,对照组加入10 ml/L无水乙醇,分别孵育24、48 h.采用噻唑蓝(MTT)法、克隆形成实验分析莪术醇对BGC823细胞增殖的影响.流式细胞术测定细胞周期的变化.100 mg/L莪术醇孵育BGC823细胞48 h后,收集细胞,反转录-聚合酶链反应(RT-PCR)测定增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白依赖性激酶4(CDK4)和p16 mRNA的表达,Western blot测定PCNA、Cyclin D1、CDK4和p16蛋白的表达水平.结果 MTT结果显示,莪术醇剂量和时间依赖性抑制BGC823细胞增殖,莪术醇为12.5、25.0、50.0、100.0 mg/L时对BGC823细胞抑制率在24h分别为(13.59±2.74)％、(22.51±4.62)％、(46.09±6.14)％、(73.67±8.24)％,在48 h分别为(15.02±2.26)％、(25.31±4.79)％、(53.62±6.37)％、(82.14±9.65)％;克隆形成实验结果显示,莪术醇具有剂量依赖性抑制BGC823细胞克隆形成率的能力,莪术醇为12.5、25.0、50.0、100.0 mg/L作用14 d时BGC823细胞克隆形成率为(0.88±0.14)％、(0.57±0.09)％、(0.36±0.06)％、(0.11±0.02)％;流式细胞结果显示,莪术醇处理48 h后,G0/G1期的细胞均显著增加,S期细胞明显减少,且随浓度增高变化更为明显(P＜0.05);RT-PCR和Western blot结果显示,100 mg/L莪术醇孵育BGC823细胞48 h后,PCNA和Cyclin D、CDK4mRNA和蛋白表达显著下降,p16 mRNA和蛋白表达显著上升(P＜0.05).PCNA、Cyclin D1、CDK4、p16的mRNA在对照组相对表达量为0.64±0.16、0.57 ±0.12、0.46±0.06、0.26±0.04,在莪术醇组为0.31±0.07、0.19 ±0.05、0.15±0.04、0.59±0.09;各指标蛋白的相对表达量在对照组为0.84 ±0.21、0.62 ±0.17、0.74±0.19、0.35±0.06,在莪术醇组为0.43 ±0.09、0.26±0.07、0.22±0.06、0.64±0.16.结论莪术醇可使BGC823细胞周期阻滞于G0/G1期而抑制其增殖,其主要机制可能与下调PCNA、Cyclin D和CDK4表达、上调p16表达有关. 目的觀察莪術醇對人胃癌細胞株BGC823細胞週期的影響,併探討其作用機製.方法體外培養BGC823細胞,分彆加入12.5、25.0、50.0、100.0 mg/L莪術醇,對照組加入10 ml/L無水乙醇,分彆孵育24、48 h.採用噻唑藍(MTT)法、剋隆形成實驗分析莪術醇對BGC823細胞增殖的影響.流式細胞術測定細胞週期的變化.100 mg/L莪術醇孵育BGC823細胞48 h後,收集細胞,反轉錄-聚閤酶鏈反應(RT-PCR)測定增殖細胞覈抗原(PCNA)、細胞週期蛋白D1(Cyclin D1)、細胞週期蛋白依賴性激酶4(CDK4)和p16 mRNA的錶達,Western blot測定PCNA、Cyclin D1、CDK4和p16蛋白的錶達水平.結果 MTT結果顯示,莪術醇劑量和時間依賴性抑製BGC823細胞增殖,莪術醇為12.5、25.0、50.0、100.0 mg/L時對BGC823細胞抑製率在24h分彆為(13.59±2.74)％、(22.51±4.62)％、(46.09±6.14)％、(73.67±8.24)％,在48 h分彆為(15.02±2.26)％、(25.31±4.79)％、(53.62±6.37)％、(82.14±9.65)％;剋隆形成實驗結果顯示,莪術醇具有劑量依賴性抑製BGC823細胞剋隆形成率的能力,莪術醇為12.5、25.0、50.0、100.0 mg/L作用14 d時BGC823細胞剋隆形成率為(0.88±0.14)％、(0.57±0.09)％、(0.36±0.06)％、(0.11±0.02)％;流式細胞結果顯示,莪術醇處理48 h後,G0/G1期的細胞均顯著增加,S期細胞明顯減少,且隨濃度增高變化更為明顯(P＜0.05);RT-PCR和Western blot結果顯示,100 mg/L莪術醇孵育BGC823細胞48 h後,PCNA和Cyclin D、CDK4mRNA和蛋白錶達顯著下降,p16 mRNA和蛋白錶達顯著上升(P＜0.05).PCNA、Cyclin D1、CDK4、p16的mRNA在對照組相對錶達量為0.64±0.16、0.57 ±0.12、0.46±0.06、0.26±0.04,在莪術醇組為0.31±0.07、0.19 ±0.05、0.15±0.04、0.59±0.09;各指標蛋白的相對錶達量在對照組為0.84 ±0.21、0.62 ±0.17、0.74±0.19、0.35±0.06,在莪術醇組為0.43 ±0.09、0.26±0.07、0.22±0.06、0.64±0.16.結論莪術醇可使BGC823細胞週期阻滯于G0/G1期而抑製其增殖,其主要機製可能與下調PCNA、Cyclin D和CDK4錶達、上調p16錶達有關.
목적 관찰아술순대인위암세포주BGC823세포주기적영향,병탐토기작용궤제.방법 체외배양BGC823세포,분별가입12.5、25.0、50.0、100.0 mg/L아술순,대조조가입10 ml/L무수을순,분별부육24、48 h.채용새서람(MTT)법、극륭형성실험분석아술순대BGC823세포증식적영향.류식세포술측정세포주기적변화.100 mg/L아술순부육BGC823세포48 h후,수집세포,반전록-취합매련반응(RT-PCR)측정증식세포핵항원(PCNA)、세포주기단백D1(Cyclin D1)、세포주기단백의뢰성격매4(CDK4)화p16 mRNA적표체,Western blot측정PCNA、Cyclin D1、CDK4화p16단백적표체수평.결과 MTT결과현시,아술순제량화시간의뢰성억제BGC823세포증식,아술순위12.5、25.0、50.0、100.0 mg/L시대BGC823세포억제솔재24h분별위(13.59±2.74)％、(22.51±4.62)％、(46.09±6.14)％、(73.67±8.24)％,재48 h분별위(15.02±2.26)％、(25.31±4.79)％、(53.62±6.37)％、(82.14±9.65)％;극륭형성실험결과현시,아술순구유제량의뢰성억제BGC823세포극륭형성솔적능력,아술순위12.5、25.0、50.0、100.0 mg/L작용14 d시BGC823세포극륭형성솔위(0.88±0.14)％、(0.57±0.09)％、(0.36±0.06)％、(0.11±0.02)％;류식세포결과현시,아술순처리48 h후,G0/G1기적세포균현저증가,S기세포명현감소,차수농도증고변화경위명현(P＜0.05);RT-PCR화Western blot결과현시,100 mg/L아술순부육BGC823세포48 h후,PCNA화Cyclin D、CDK4mRNA화단백표체현저하강,p16 mRNA화단백표체현저상승(P＜0.05).PCNA、Cyclin D1、CDK4、p16적mRNA재대조조상대표체량위0.64±0.16、0.57 ±0.12、0.46±0.06、0.26±0.04,재아술순조위0.31±0.07、0.19 ±0.05、0.15±0.04、0.59±0.09;각지표단백적상대표체량재대조조위0.84 ±0.21、0.62 ±0.17、0.74±0.19、0.35±0.06,재아술순조위0.43 ±0.09、0.26±0.07、0.22±0.06、0.64±0.16.결론 아술순가사BGC823세포주기조체우G0/G1기이억제기증식,기주요궤제가능여하조PCNA、Cyclin D화CDK4표체、상조p16표체유관.
Objective To investigate the effecof curcumol on cell cycle of human gastricarcinomcell BGC823 and the mechanism.MethodCultured BGC823 cellwere treated with differenconcentrationof curcumol (12.5, 25.0, 50.0 and 100.0 mg/L) fodifferenduration(24 and 48 h), and the growth inhibition watested by methyl thiazol tetrazolium (MTT) assay and colony formatting assay.Flow cytometry (FCM) waused to measure cell cycle of BGC823 cells.Aftetreatmenwith 100 mg/L curcumol and culture fo48 h, cellwere collected and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting fodetection of the mRNand protein expression of proliferating cell nucleaantigen (PCNA), Cyclin D1, cyclin-dependenkinase 4 (CDK4) and p16.ResultThe MTassay showed thacurcumol could greatly inhibithe growth and proliferation of BGC823 cellin dose-and time-dependenmanne(P ＜ 0.05).When the concentration of curcumol wa12.5, 25.0,50.0 and 100.0 mg/L, the inhibition rate of BGC823 wa(13.59 ±2.74)％, (22.51 ±4.62)％,(46.09 ±6.14)％ and (73.67 ±8.24)％ a24 h, and thawa(15.02 ±2.26)％, (25.31 ±4.79)％,(53.62 ± 6.37) ％ and (82.14 ± 9.65) ％ a48 h, respectively.Clone formation assay revealed thacurcumol could greatly inhibithe colony formation rate of BGC823 cellin dose-dependenmanne(P ＜ 0.05).When the concentration of curcumol wa12.5, 25.0, 50.0 and 100.0 mg/L, the colony formation rate wa(0.88 ± 0.14) ％, (0.57 ± 0.09) ％, (0.36 ± 0.06) ％ and (0.11 ± 0.02) ％ respectively a14 day.FCM showed thathe cell numbeof G0/G1 phase waincreased, while thaof phase wadecreased afteBGC823 cellwere treated with curcumol fo48 h (P ＜ 0.05).Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting analyseindicated thacurcumol decreased PCNA, Cyclin D1 and CDK4 expression awell aincreased p16 expression (P ＜ 0.05).The relative expression of PCNA, Cyclin D1, CDK4, and p16 mRNwa0.64 ±0.16, 0.57 ±0.12, 0.46 ±0.06 and 0.26 ±0.04 in control group, and thain curcumol group wa0.31 ±0.07, 0.19 ±0.05, 0.15 ±0.04 and 0.59 ± 0.09 respectively.The relative expression of PCNA, Cyclin D1, CDK4, and p16 protein wa0.84 ± 0.21,0.62 ± 0.17, 0.74 ± 0.19 and 0.35 ± 0.06 in control group, and thain curcumol group wa0.43 ±0.09, 0.26 ± 0.07, 0.22 ± 0.06 and 0.64 ± 0.16 respectively.Conclusion Curcumol induceBGC823 cell cycle arresin G0/G1 phase, which may be related with the down-regulation of PCNA, Cyclin D1 and CDK4 awell athe up-regulation of p16.