中华肝胆外科杂志
中華肝膽外科雜誌
중화간담외과잡지
CHINESE JOURNAL OF HEPATOBILIARY SURGERY
2009年
2期
121-125
,共5页
吴灵飞%李国平%苏剑东%蒲泽锦%冯家琳
吳靈飛%李國平%囌劍東%蒲澤錦%馮傢琳
오령비%리국평%소검동%포택금%풍가림
癌,肝细胞%腺苷%细胞凋亡%NF-κB%Caspase-3
癌,肝細胞%腺苷%細胞凋亡%NF-κB%Caspase-3
암,간세포%선감%세포조망%NF-κB%Caspase-3
Carcinoma hepatocellular%Adenosine%Apoptosis%NF-κB%Caspase-3
目的 研究NF-κB、Caspase-3在腺苷(ADO)体外诱导人肝癌HepG2细胞凋亡中的作用.方法 将不同浓度的ADO(0.1～5 mmol/L)作用于HepG2细胞,采用MTT法测定ADO抑制细胞增殖的时间效应和剂量效应.2.0 mmol/L ADO单用或联合NF-κB抑制剂吡咯烷二硫氨基甲酸(PDTC,100 μmol/L)作用HepG2细胞12 h、24 h,采用Hoechst 33342荧光染色法及流式细胞术(FCM)观察细胞凋亡及细胞周期,Western blot技术检测NF-κB蛋白表达;荧光比色法测定Caspase-3酶活性.结果 ADO对HepG2细胞生长具有显著抑制作用,并呈一定的量效和时效关系.药物作用24 h、48 h的IC50分别为2.52 mmol/L和1.89 mmol/L.ADO单独处理HepG2细胞12 h和24 h或联合PDTC处理后,细胞凋亡率分别为8.30%、22.32%;20.18%、30.89%,均显著高于对照组(0.81%,P<0.001);ADO作用HepG2细胞后荧光显微镜观察到细胞凋亡的形态学改变,FCM分析药物处理组显示典型特征性的亚二倍体凋亡峰(sub-G1),细胞生长周期阻滞于G0/G1期;同时伴有Caspase-3活性显著升高(P<0.05).ADO处理后显著增加了NF-κB蛋白表达(P<0.05);PDTC有效抑制了NF-κB表达,同时增加了 Caspase-3活性及ADO的细胞毒作用(P<0.05).结论 ADO诱导了HepG2细胞凋亡并活化Caspase-3.抑制NF-κB活性可通过Caspase-3途径增强ADO的细胞毒作用.
目的 研究NF-κB、Caspase-3在腺苷(ADO)體外誘導人肝癌HepG2細胞凋亡中的作用.方法 將不同濃度的ADO(0.1～5 mmol/L)作用于HepG2細胞,採用MTT法測定ADO抑製細胞增殖的時間效應和劑量效應.2.0 mmol/L ADO單用或聯閤NF-κB抑製劑吡咯烷二硫氨基甲痠(PDTC,100 μmol/L)作用HepG2細胞12 h、24 h,採用Hoechst 33342熒光染色法及流式細胞術(FCM)觀察細胞凋亡及細胞週期,Western blot技術檢測NF-κB蛋白錶達;熒光比色法測定Caspase-3酶活性.結果 ADO對HepG2細胞生長具有顯著抑製作用,併呈一定的量效和時效關繫.藥物作用24 h、48 h的IC50分彆為2.52 mmol/L和1.89 mmol/L.ADO單獨處理HepG2細胞12 h和24 h或聯閤PDTC處理後,細胞凋亡率分彆為8.30%、22.32%;20.18%、30.89%,均顯著高于對照組(0.81%,P<0.001);ADO作用HepG2細胞後熒光顯微鏡觀察到細胞凋亡的形態學改變,FCM分析藥物處理組顯示典型特徵性的亞二倍體凋亡峰(sub-G1),細胞生長週期阻滯于G0/G1期;同時伴有Caspase-3活性顯著升高(P<0.05).ADO處理後顯著增加瞭NF-κB蛋白錶達(P<0.05);PDTC有效抑製瞭NF-κB錶達,同時增加瞭 Caspase-3活性及ADO的細胞毒作用(P<0.05).結論 ADO誘導瞭HepG2細胞凋亡併活化Caspase-3.抑製NF-κB活性可通過Caspase-3途徑增彊ADO的細胞毒作用.
목적 연구NF-κB、Caspase-3재선감(ADO)체외유도인간암HepG2세포조망중적작용.방법 장불동농도적ADO(0.1～5 mmol/L)작용우HepG2세포,채용MTT법측정ADO억제세포증식적시간효응화제량효응.2.0 mmol/L ADO단용혹연합NF-κB억제제필각완이류안기갑산(PDTC,100 μmol/L)작용HepG2세포12 h、24 h,채용Hoechst 33342형광염색법급류식세포술(FCM)관찰세포조망급세포주기,Western blot기술검측NF-κB단백표체;형광비색법측정Caspase-3매활성.결과 ADO대HepG2세포생장구유현저억제작용,병정일정적량효화시효관계.약물작용24 h、48 h적IC50분별위2.52 mmol/L화1.89 mmol/L.ADO단독처리HepG2세포12 h화24 h혹연합PDTC처리후,세포조망솔분별위8.30%、22.32%;20.18%、30.89%,균현저고우대조조(0.81%,P<0.001);ADO작용HepG2세포후형광현미경관찰도세포조망적형태학개변,FCM분석약물처리조현시전형특정성적아이배체조망봉(sub-G1),세포생장주기조체우G0/G1기;동시반유Caspase-3활성현저승고(P<0.05).ADO처리후현저증가료NF-κB단백표체(P<0.05);PDTC유효억제료NF-κB표체,동시증가료 Caspase-3활성급ADO적세포독작용(P<0.05).결론 ADO유도료HepG2세포조망병활화Caspase-3.억제NF-κB활성가통과Caspase-3도경증강ADO적세포독작용.
Objective To investigate the role of NF-κB and caspase-3 in adenosine (ADO)-in-duced apoptosis in human HepG2 hepatocellular carcinoma cells. Methods HepG2 cells were treated with different concentrations of ADO for 12-48 h, and the effect of adenosine on cell proliferation was evaluated by MTT assay. HepG2 cells were stimulated with ADO (2.0 mmol/L) alone or co-treated with PDTC (pyrrolidine dithiocarbamate, 100 μmol/L), an inhibitor of NF-κB, for 12 h and 24 h. The effects of ADO or ADO+PDTC on cell apoptosis and cell cycle were evaluated by Hoechst 33342 fluorescent staining and flow cytometric analysis. The protein expression of NF-κB was assayed by Western blot and caspase-3 activity measured by Fluorometric assay. Results The viability of HepG2 cells decreased significantly in a dose-and time-dependent manner. IC50 (24 h and 48h) of ADO was 2.52 mmol/L and 1.89 mmol/L, respectively. The apoptostic index of HepG2 cells in ADO treatment alone for 12h and 24h or ADO with PDTC treatment were 8.30%, 22.32% and 20.18%, 30.89%, respectively. All of them were higher than that in the control group (0.81%, P<0.001). Character-istic morphological changes of cell apoptosis were observed under fluorescent microscopy and sub-G1 peak, G0/G1 cell cycle arrest appeared on the FCM diagram after ADO treatment. ADO induced apop-tosis and significantly increased NF-κB protein expression (P<0.05). PDTC diminished NF-κB ex-pression and increased caspase-3 activation and cytotoxicity in HepG2 cells (P<0.05). Conclusion ADO induces HepG2 cell apoptosis and activates NF-κB. Inhibition of NF-κB could potentiate apopto-sis through caspase-3 pathway in HepG2 cells.
