中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2008年
8期
583-587
,共5页
熊娟%李一荣%汤兆明%窦丽芳%王琳%胡丽华
熊娟%李一榮%湯兆明%竇麗芳%王琳%鬍麗華
웅연%리일영%탕조명%두려방%왕림%호려화
p21%survivin%肝肿瘤%基因表达调控
p21%survivin%肝腫瘤%基因錶達調控
p21%survivin%간종류%기인표체조공
p21%survivin%Hepatocellular neoplasms%Gene expression regulation
目的 研究细胞周期依赖性激酶抑制蛋白p21对人肝癌细胞survivin转录抑制的调控作用,并探讨其相应机制.方法 使用多柔比星处理人肝癌细胞株HepG2,采用脂质体法将质粒pEGFP-C2-p21导入HepG2细胞,用G418筛选转染后的HepG2-p21和HepG2-pEGFP细胞;采用实时定量聚合酶链反应(RQ-PCR)检测p21、p53和survivin的mRNA表达;采用流式细胞仪分析转染后细胞周期的变化;采用逆转录聚合酶链反应(RT-PCR)检测转染后E2F-1和p300的mRNA表达.结果 经多柔比星处理后,HepG2细胞中p21和p53 mRNA的表达先增高,然后逐渐降低;survivin mRNA的表达则相反.转染后,HepG2-p21细胞中p21 mRNA的表达明显增高,分别为HepG2和HepG2-pEGFP细胞的2100.1倍和980.9倍;survivin mRNA的表达却明显降低,分别为HepG2和HepG2-pEGFP细胞的0.5%和0.6%;p53 mRNA的表达差异无统计学意义.流式细胞仪检测显示,HepG2-p21细胞的细胞周期明显阻滞在G0/G1期.HepG2-p21细胞中E2F-1和p300 mRNA的表达水平分别为0.75±0.04和0.18 ±0.06,与HepG2和HepG2-pEGFP细胞比较,含量均明显减低(P＜0.05).结论 p21可能通过抑制survivin的转录调控因子E2F-1和p300 mRNA的表达,从而抑制细胞中survivin的表达,并导致细胞出现G0/G1期阻滞.
目的 研究細胞週期依賴性激酶抑製蛋白p21對人肝癌細胞survivin轉錄抑製的調控作用,併探討其相應機製.方法 使用多柔比星處理人肝癌細胞株HepG2,採用脂質體法將質粒pEGFP-C2-p21導入HepG2細胞,用G418篩選轉染後的HepG2-p21和HepG2-pEGFP細胞;採用實時定量聚閤酶鏈反應(RQ-PCR)檢測p21、p53和survivin的mRNA錶達;採用流式細胞儀分析轉染後細胞週期的變化;採用逆轉錄聚閤酶鏈反應(RT-PCR)檢測轉染後E2F-1和p300的mRNA錶達.結果 經多柔比星處理後,HepG2細胞中p21和p53 mRNA的錶達先增高,然後逐漸降低;survivin mRNA的錶達則相反.轉染後,HepG2-p21細胞中p21 mRNA的錶達明顯增高,分彆為HepG2和HepG2-pEGFP細胞的2100.1倍和980.9倍;survivin mRNA的錶達卻明顯降低,分彆為HepG2和HepG2-pEGFP細胞的0.5%和0.6%;p53 mRNA的錶達差異無統計學意義.流式細胞儀檢測顯示,HepG2-p21細胞的細胞週期明顯阻滯在G0/G1期.HepG2-p21細胞中E2F-1和p300 mRNA的錶達水平分彆為0.75±0.04和0.18 ±0.06,與HepG2和HepG2-pEGFP細胞比較,含量均明顯減低(P＜0.05).結論 p21可能通過抑製survivin的轉錄調控因子E2F-1和p300 mRNA的錶達,從而抑製細胞中survivin的錶達,併導緻細胞齣現G0/G1期阻滯.
목적 연구세포주기의뢰성격매억제단백p21대인간암세포survivin전록억제적조공작용,병탐토기상응궤제.방법 사용다유비성처리인간암세포주HepG2,채용지질체법장질립pEGFP-C2-p21도입HepG2세포,용G418사선전염후적HepG2-p21화HepG2-pEGFP세포;채용실시정량취합매련반응(RQ-PCR)검측p21、p53화survivin적mRNA표체;채용류식세포의분석전염후세포주기적변화;채용역전록취합매련반응(RT-PCR)검측전염후E2F-1화p300적mRNA표체.결과 경다유비성처리후,HepG2세포중p21화p53 mRNA적표체선증고,연후축점강저;survivin mRNA적표체칙상반.전염후,HepG2-p21세포중p21 mRNA적표체명현증고,분별위HepG2화HepG2-pEGFP세포적2100.1배화980.9배;survivin mRNA적표체각명현강저,분별위HepG2화HepG2-pEGFP세포적0.5%화0.6%;p53 mRNA적표체차이무통계학의의.류식세포의검측현시,HepG2-p21세포적세포주기명현조체재G0/G1기.HepG2-p21세포중E2F-1화p300 mRNA적표체수평분별위0.75±0.04화0.18 ±0.06,여HepG2화HepG2-pEGFP세포비교,함량균명현감저(P＜0.05).결론 p21가능통과억제survivin적전록조공인자E2F-1화p300 mRNA적표체,종이억제세포중survivin적표체,병도치세포출현G0/G1기조체.
Objective To observe the inhibitory effect of cyclin-dependent kinase inhibitor p21 on regulation of survivin transcription in human liver caner HepG cells.and explore the related mechanisms.Methods Doxorubicin(DOX)was used to treat HepG ceHs.Eukaryotic vector pEGFP-C2-p21 was transfected into HepG cells by lipofectamine and positive clones were screened out by G418.The mRNA expression of p21,p53 and survivin were detected by real-time fluorescent quantitative polymerase chain reaction(RQ-PCR).Flow cytometry was used to determine the cell cycle phases,and reverse transcription polymerase chain reaction(RT-PCR)was used to measure the levels of E2F-1 or p300.Results After treatment with DOX.the expression of D53 and D21 was increased.wheleas that of survivin was reduced during 24 hours of the treatment.Mter transfection the p21 level was 2100.1-fold or 980.9-fold enhanced in comparison with that in HepG2 cells or HepG2-pEGFP cells.Survivin level was markedly down-regulated to 0.5%or 0.6%relative to that in the other two groups,nevertheless,significant r63 changes were not observed.Overexpression of p21 resuhed in G1/G0 phase arrest(F=31.59,P＜0.01),meanwhile,E2F-1 mRNA or p300 mRNA were less expressed compared with that in the other controls(FE2F-1=125.28,P＜0.05;Fp300=46.01,P＜0.01).Conclusion p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cells,which misht be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.
