摘要
目的:通过HBx促进肝癌的发生及DES对其影响的研究,探讨DES干预乙型肝炎进展为肝癌的机理,即是否通过抑制HBxAg诱导的核转录因子NF-kB/p65的高表达。
方法:HepG2.2.15和HepG2细胞株分别以3种不同浓度(1×10~4/ml、3×10~4/ml、10×10~1/ml)接种于3块24孔板中,细胞计数法确定最佳接种浓度和加药时间。DES用DMEM完全培养基溶解,分为3个浓度(100μg/ml、200μg/ml、400μg/ml),以DDP(20μg/ml)为对照,MTT法检测DES对HepG2.2.15和HepG2细胞株是否具有抑制作用,如果有作用,绘制细胞的生长曲线,明确药物工作浓度和时间。在该药物工作浓度和时间下,实验分为6组:HepG2.2.15细胞株DES组、DDP组、正常对照组,HepG2细胞株DES组、DDP组、正常对照组,细胞免疫荧光法检测HepG2.2.15细胞株HBx蛋白的表达强度;收集各组细胞,流式细胞术检测细胞周期、细胞凋亡的表达;抽提各组核蛋白,ELISA法定量检测NF-kB/p65的活性。
结果:HepG2.2.15和HepG2细胞株均以3×10~1/ml为最佳接种浓度,接种后48h进入指数生长期;DES对HepG2.2.15和HepG2细胞株的增殖有抑制作用,与DDP相当(p＞0.05);与对照组相比,DES和DDP均可增加处于S期细胞数并减少G2/M期细胞数,阻碍了肿瘤细胞的分裂生长,两者效果相比,没有显著差异;DES和DDP均能促进细胞凋亡,降低磷酸化NF-kB/p65的表达,并明显干预HBx蛋白的表达(p＜0.05),但二者间差别无统计学意义。HBx蛋白可促进磷酸化NF-kB/p65的表达。
结论:1 DES对HepG2.2.15细胞株的增殖有抑制作用;
2 DES对HepG2.2.15细胞株细胞周期有明确影响;可促进细胞凋亡的表达并抑制HBx蛋白表达;
3 DES可降低HepG2.2.15细胞株磷酸化NF-kB/p65的表达。
Objective:Approach the mechanism which DES prevent HCC from hepatitis B whether or not through inhibit the high express of NF-kB/p65 induced by HBxAg.
Methods:Seed HepG2.2.15 and HepG2 cell line in 24 shadow mask by different concertration(1×10~4/ml、3×10~4/ml,10×10~4/ml ),determined the best culture concertration and time to add drug by cytometry.DES were dissolved with complete DMEM medium;divided into three conch(100μg/ml、200μg/ml、400μg/ml),contrasted by DDP(20μg/ml).And determined the effection of inhibition by MTT method.Every one of cell lines was divided into three group:DES group,DDP group,control group.Determined the expression strength of HBxAg within HepG2.2.15 cell line by immumofluorescent method,the expression of apoptosis within two cell lines by FCM and activity of NF-kB/p65 by ELISA.
Results:The best culture concertration of two cell lines was 3×10~4/ml,and the beginning of exponential growth phase was after 48h culture.DES and DDP all have the effection of inhibition on muitiplication of HepG2.2.15 and HepG2.Contrast with control group,rate of S phase was enhanced as well as of G2/M phase was decreased.There was no significant deviation between DES and DDP.DES and DDP all can decrease the expression strength of HBxAg and the activity of NF-kB/p65,There was no significant deviation between DES and DDP too. HBxAg can stimulate the expression of NF-kB/p65.
Conclusions:
1 DES has the effection of inhibition on multiplication of HepG2.2.15,
2 DES can decrease the expression strength of HBxAg,and has the identify effection on cell cycle and apoptosis of HepG2.2.15,
3 DES can decrease the expression of activited NF-kB/p65 within HepG2.2.15.
引文
[1]Jwo SC,Chiu JH,Chaw GY.Risk:factors linked to tumor recurrence of human hepatocellular carcinoma after hepatic resection.Hepatology,1992,16,1367-1371
[2]Beasley,RP,Hwang LY.Hepatocellular carcinoma and hepatitis-B virus.A prospective study of 22,707 men in Taiwan.Lancet,1981,11,1129-1133
[3]Schrodecr H,and Zentgrahf.Hepatitis-B-virus related hepatocellular carcinoma:chronicity of infection-the opening to different pathways of malignant transformation? Biochim- biophys.Acta,1990,1032,137-156
[4]Slagle,B.L,Butel JS.Hepatitis-B- virus and hepatocellular carcinoma.Aog.med.Virol,1992,39,167-203
[5]Kirk GD,Lesi OA,Mendy M.The Gambia liver cancer study:Infection with hepatitis B and C and the risk of hepatocellular carcinoma in West Africa.Hepatolopy,2004,39;211-219.
[6]Beasley RP.Hepatitis B virus:the major etiology of hepatocellular carcinoma.Cancer,1988:61:1942-1956.
[7]吴克复.细胞通讯与疾病 北京 科学出版社 2006(1)
[8]陈远能 曹骥.Rel mRNA和NF-kB2 mRNA在人肝细胞癌中的表达及其临床意义 中山大学学报医学科学版 2006,27(6):677-680
[9]张克君 高焱明.核因子-kBp65、IkB-a在肝炎相关性肝细胞癌中的表达及临床意义Journal of PracticalOncology 2004,19(3)
[10]Jang BK,Chung WJ,Park KS.The efficacy of hepatic arterial infusion therapy for advanced hepatocellular carcinoma according to extrahepatic collateral feeding vessels.Kerean J Hepatol,2005,11(4):359-70
[11]李艳芳,符立梧.番荔枝内酯抗肿瘤作用研究进腱.中国药理学通报,2004,20(3):245-247
[12]Kuwabara K,Takada M,Iwata J.Design syntheses and mitochondrial complex I inhibittory activity of novel acetogenin mimics.Eur J Biochem,2000,267(9):2538- 2546
[13]Chiu HF,Chih TT,Hsian YM.Bullatacin,a potent antitumor Annonacesous acetogenin,induces apoptosis through a reduction of intracellular cAMP and cGMP levels in human hepatoma 2.2.15 cells.Biochem Pharmacol 2003,65(3):319-327
[14]任先达,杨政红,周光雄,叶开河,吕艳青.刺果紫玉盘袭J诱导Lovo细胞凋亡的作用.暨南大学学报(医学版),2007,28(2):142-146
[15]陈孝银,周光雄,赵昌林.番荔枝内酯单体Desacetyluvaricin对肝癌细胞株生长和凋亡的影响.中山大学学报(医学版),2007,28(3):254-258
[1]程宝鸾.动物细胞培养技术,广州,华南理工大学出版社,2006(1);
[2]R·I·弗雷谢尼著,章静波,徐存栓译.动物细胞培养-基本技术指南.北京,科学出版社,2004(9);
[3]骆抗先.乙型肝炎基础与临床 北京,人民卫生出版社 2003(2);
[1]张卓然.培养细胞学与细胞培养技术.上海,上海科学技术出版社,2004(1):310-311
[2]方蓉,李芳秋,武建国.MTT比色法的条件探讨.临床检验杂志,2003,21(1):34-35
[3]Jolad DJ,tloffman JJ,Schram KH.Uvaricin,a new antitumor agent from Uvaria accUminata(Annonaceae).J Org Chem,1982;47(16):3135-3153
[4]Hidefumi Makabe,Yuka Kimura,Masaharu Higuchi.Synthesis of (4R,15R,16R,21S)-and(4R,15S,16S,21S)-rollicosin,squamostolide,and their inhibitory action with bovine heart mitochondrial complex Ⅰ.Bioorganic&Medicinal Chemistry 2006,14(9):3119-3130.
[5]符立梧,潘启超.番荔枝内酯抗肿瘤作用研究进展.癌症,1998,17(16):471-473
[6]周光雄.番荔枝科植物刺果紫玉盘抗肿瘤化学成分研究.博士研究生毕业论文.中国医学科学院-中国协和医科大学,1996
[7]Raynaud S,Fourneau C,Hocquemil]er R,Sevenet T.Acetogenins from the bark of Uvaria pauciovulata.Phytochemistry 1997;46(2):321-326
[8]符立梧,潘启超.阿蒂莫耶番荔枝总内酯体外抗肿瘤作用.癌症,1997,16(6):409-410
[9]Miyoshi tt,Ohshima M,Shimada tt.Essential structural factors of annonaceous ace togenins as potent inhibitors of mitochondrial complex Ⅰ.Biochimicaet Biophysica Acta-Bioenerzetics,1998,1356(3):443-452
[10]赵昌林.琼玉膏和DES干预HBxAg致HCC的机理研究.暨南大学硕士学位论文.2007,5
[1]张卓然.培养细胞学与细胞培养技术.上海,上海科学技术出版社,2004(1):176-177
[1]王建中.临床流式细胞分析.上海,上海科学技术出版社,2005,559
[2]程斌,林松挺,杨玉珍,黎培员.转基因细胞模型L02/HBx的构建及HBx对细胞周期的影响.世界华人消化杂志,2007;15(13):1488-1493
[3]Toshihiko Okazaki,Daitoku Sakamuro.lnduction of Fas(CD95/APO-1) ligand is essential for p53-dependent apoptosis in an in vitro renal carcinoma model system.J Cancer Res and Clinical Oncol,2007,133:581-588
[4]Diao J,Khine AA,Sarangi F,Hsu E,Iorio C,Tibbles LA,Woodgett JR.X Protein of Hepatitis B Virus Inhibits Fas-mediated Apoptosis and Is Associated with Up-regulation of the SAPK/JNK Pathway.J Biol Chem,2001,276(11):8328-8340
[5]Zhaorui Lian,Jie Liu,Jingbo Pan.A cellular gene up-regulated by hepatitis B virus- encoded X antigen promotes hepatocellular growth and survival.Hepatology,2001,34(1):146-157
[6]赵昌林.琼玉膏和DES干预HBxAg致HCC的机理研究.暨南大学硕士学位论文.2007,5
[7]Miyoshi H,Ohshima M,Shimada H.Essential structural factors of annonaceous acetogenins as potent inhibitors of mitochondrial complex I.Biochimica et Biophysica Acta-Bioenergetics,1998,1356(3):443-452
[8]朱孝峰,谢冰芬,李志铭.番荔枝内酯诱导白血病细胞凋亡的机理.药学学报,2001,36(7):498-501
[9]陈孝银,周光雄,赵昌林.番荔枝内酯单体Desacetyluvaricin对肝癌细胞株生长和凋亡的影响.中山大学学报(医学版),2007,28(3):254-258
[1]吴克复.细胞通讯与疾病.北京,科学出版社,2006(1)
[2]Suyun Huang,Ariel DeGuzman,Corazon D Bucana and Isaiah J Fidler.Level of interleukin-8 expression by metastatic human melanoma cells directly correlates with constitutive NF-kB activity Cytokines,Cellular&Molecular Therapy 2000,6:9-17
[3]朱波,罗成基.核转录因子.kB与细胞凋亡.国外医学临床生物化学与检验学分册.2001,22:18-19
[4]Wang CY,Guttridge DC,Mayo MW.NF-kappa B induces expression of the Bcl-2homologue A1 / Bfl-1 to preferentially suppress chemotherapy-induced apoptosis.Mol Cell Biol 1999;19:5923-5929
[5]Wang CY,MayO MW,KomelUk RG.NF-kappa B antiapoptosis:induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation.Science 1999;281:1680-2483
[6]袁淑芳,张跃新.肿瘤坏死因子a、caspase、核因子kBmRNA在病毒性肝炎中的表达与肝细胞凋亡.中华肝脏病杂志.2006;14:129-131
[7]Atsushi Kato,Michael J.Edward.Gene deletion of NF-kB p50 does not alter the hepatic inflammatory response to ischemia/reperfusion Journal of Hepatology 2002,37:48-55
[8]陈远能 曹骥.Rel mRNA和NF-kB2 mRNA在人肝细胞癌中的表达及其临床意义 中山大学学报医学科学版.2006,27(6):677-680
[9]张克君 高焱明.核因子-kBp65、IkB-a在肝炎相关性肝细胞癌中的表达及临床意义.Journal of PracticalOncology 2004,19(3)216-218
[10]Doria M,Klei N,Lucito R.The hepatitis B virus HBx proteins a dual specificity cytoplasmic activitor of Ras and nuclear activitor of transcription factors.EM BO J,1995,14(2):4747-4757.
[11 ]Su FS, Chneider KJ. Hepatitis B virus HBX protein activates NF-kB by acting on multiple cytoplasmic inhibitors of rel-related proteins . J Virol, 1996, 70(8): 4558- 4560.
[12]Matsumoto M , Heieh TY, Zhu N. Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor. J Virol, 1997, 71(11) :1301 - 1309.
[13]Zhu N , Khoshnan R. Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor receptor and enhances TNF-induced apoptosis. J Virol, 1998, 72 (9): 3691-3697.