摘要
本文通过体外培养HepG-2细胞探讨了原花青素的抗肿瘤作用,阐述了其诱导HepG-2细胞凋亡的作用及其线粒体途径机制,同时通过体内移植肿瘤实验证明了其体内具有抗肿瘤作用。
体外研究中,原花青素(PC)可抑制人肝癌细胞HepG-2生长。利用MTT法测定原花青素抑制率,对HepG-2细胞的IC50为47.67 pmol·L-1。
原花青素能够诱导HepG-2细胞凋亡且可以通过线粒体途径上诱导HepG-2细胞凋亡。通过形态学鉴定原花青素对HepG-2细胞凋亡作用,用Hoechst33258对HepG-2细胞染色,利用荧光显微镜对其抑制作用进行观察发现肿瘤细胞的核浆比减小,细胞核高度固缩,染色程度加深,染色质聚集,碎裂。产生大小不等的凋亡小体。并且随着原花青素浓度的加大,现象越来越明显,表明细胞凋亡的比例不断增加。采用透射电镜进一步显示给药后,细胞部分核膜向外膨出,胞质有大小不等的空泡,微绒毛数量减少,线粒体、粗面内质网等细胞器减少,细胞发生凋亡。采用流式细胞仪检测原花青素对HepG-2细胞周期的影响,研究发现原花青素可以使HepG-2细胞G0/G1期比例降低,S期细胞的比例增加,且G2/M期比例无明显变化。表明原花青素对HepG-2细胞周期有一定的影响,能够对肿瘤细胞S期有一定的阻滞作用。利用Bcl-2、Bax试剂盒测定原花青素对肿瘤细胞中Bcl-2、Bax蛋白的影响,结果表明,随着原花青素剂量的增加,Bcl-2蛋白表达降低,Bax蛋白表达增加,且呈现一定的剂量依赖关系。利用Rodanmin123进行染色,采用流式细胞仪检测原花青素对HepG-2细胞中线粒体膜电位的影响。研究发现,随着原花青素浓度的不断增大,原花青素能够明显降低HepG-2细胞中线粒体膜电位,且呈现一定的剂量依赖关系。利用Fluo-3/AM探针对HepG-2细胞进行染色,采用激光共聚焦显微镜观察原花青素对HepG-2细胞中钙离子含量的影响。研究发现,原花青素对HepG-2细胞中钙离子的含量有一定的作用,其中高剂量能够显著性的提高肿瘤细胞中钙离子的含量,且呈现一定的剂量依赖关系。利用Caspase-3、9试剂盒测定原花青素对HepG-2细胞中Caspase-3、9含量的影响,研究结果表明,随着原花青素浓度不断增大,肿瘤细胞中Caspase-3、9的含量逐渐增加,且呈现一定的剂量依赖关系。
在体内研究实验中,采用急性毒性实验和剂量筛选方法,测定了原花青素最佳剂量为：120mg/kg-d,60mg/kg-d,30mg/kg-d。采用移植性肿瘤实验对S180进行移植,采用灌胃给药研究发现原花青素高,中,低剂量组均能够抑制S180小鼠肿瘤细胞生长,其中高剂量和中剂量有显著性差异,低剂量组有差异。同时利用Ca2+,Mg2+-ATPnase试剂盒检测原花青素对S180小鼠肿瘤细胞中钙泵含量的影响,研究结果表明：与生理盐水组相比,原花青素能够降低S180小鼠肿瘤细胞中钙泵含量,其中高剂量能够显著性降低S180小鼠肿瘤细胞中钙泵含量。总之,原花青素体内外均具有抗肿瘤作用且原花青素能够通过线粒体途径诱导肿瘤细胞凋亡。
This study explores the actions of the inhibition of tumor cells cultured in vitro by proanthocyanidin(PC) and the apoptosis action and mechanism of mitochondria pathway induced by PC, certificate that PC has anti-tumor action in vivo using the transplantation tumor experiment.
In vitro studies, it has been found that PC can inhibit the growth of human hepatocarcinoma cells HepG-2. Measurements using mononuclear cell direct cytotoxicity assay(the MTT method) shows that its cytotoxic effect on HepG-2 is strong, with IC50 being 47.67μmol·L-1 respectively.
PC can induce tumor cells apoptosis and can induce tumor cells apoptosis via mitochondria pathway(HepG-2).Measurement of action of apoptosis using morphology with Hoechst 33258 stain show that the karyoplasmic ratio of tumor cells diminish, cell nucleus pyconsis,the staining extent become deeper,chromatin prosperous aggregation, fragmentation. The cells breaks up into several apoptotic bodies of different sizes. As PC concentration is increased, these morphological changes under the microscope become more and more clear, indicating that the proportion of cells undergoing apoptosis is gradually increasing. Measurement the tumor cell cycle dealed with PC using flow cytometry shows that PC leads to a decrease in the proportion of cells in the G0/G1 phase, and increase in the proportion of cells in the S phase and G2/M phase, but have no obvious change in the G2/M phase. This shows that PC has the influence in the tumor cell cycle and can block the tumor cells in the S phase. The Kit of Bcl-2 and Bax was used in the HepG-2 cells of Bcl-2 and Bax protein detection.When increasing the doses of PC, Bcl-2 protein expression was decreased and Bax protein expression was increased. Measurement the mitochondrial membrane electric potentiall(Δψm) treated with PC using the flow cytometry with Rodanmin 123 stain shows that PC can obviously cut down theΔψm of the tumor cells with the increasement of the dose. This effect shows dose-dependent. Measurement the content of Ca2+ using confocal laser scanning microscope with Fluo-3/AM stain shows that the PC has influence in the content of Ca2+, the high dose can increase the content of Ca2+ in tumor cells.This effect shows dose-dependent. Measurement the activities of Caspase-3 and Caspase-9 using the kit of Caspase-3,9 shows that the activities of Caspase-3 and Caspase-9 in tumor cells have gradually increase with the dose of PC. This effect shows dose-dependent too.
In vivo study, Measurement the best dose of PC using acute toxicity test and dosage bloting method shows that the best dose of PC is 120mg/kg·d,60mg/kg·d,30mg/kg·d.Using the transplantation tumor test in S180 and intragastric administration method shows that all of high,middle and low dose group can inhibit the tumor cells growth. The dose of high and middle group have significance difference,the low dose group has disparity.At the same time,measurement the content of calcium pump using the kit of Ca2+,Mg2+-ATPnase shows that PC can cut down the content of calcium pump in tumor cells comparing with the isotonic Na chloride group. The high group of PC can obviously cut down the content of calcium pump in tumor cells. In a word, PC has the anti-tumor effect in vitro or vivo and PC can induce the tumor cell apoptosis via mitochondria pathways.
引文
[1]董志勇.新世纪肿瘤防治的目标[J],中华肿瘤杂志,2002,24：311-312
[2]Thornton DE,Jones KH,Jiang Zch,et al.Antioxidant and cytotoxic tocophonyl quinons in normal and cancer cells[J].Free Radic Biol Med,1995,18(6):963-967
[3]Wells ww,Rocque PA,Xu DP,et al.Acsorbic acid and cell survival of adriamyin resistant and sensitive MCF-7 breast cells[J].Free Radic Biol Med,1995,18(4):699-708
[4]Hahn SM, Sullivan FJ, Deluca AM, et al.Evaluation of tempol radio protectionin a murine tumor model[J]. Free Radic Biol Med,1997,22(7):1211-1216
[5]Fadeel B,Orrenius S and Zhivotovsky B. Apoptosis in human disease:a new skin for the old ceremony [J].Biochem Biophys Res Commun,1999,266:699-717
[6]贾林涛,于翠娟,许彦鸣,等.Caspase-3融合蛋白基因的构建及其对Hela细胞凋亡的诱导作用[J].第四军医大学学报,2001,22(12)：1057-1058
[7]Pandey S, Smith BP,Walker,et al.Caspase-dependent and independent cell death in rat hepatoma 5123tc cells[J].Apoptosis,2005,5:265-275
[8]Desagher S Martinou JC. Mitochondria as the central control point of apoptosis [J]. trends in Cell Biol 2000,10:369-377
[9]Schendel SL,Azmov R, Pawlowskiet K, et al.Ion channel activity of the BH3 only Bcl-2 family member, BID[J]. Biol Chem 1999,274:21932-21936
[10]Shimizu S Narita M, Tsujimoto Y.Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondria channel VDAC[J].Nature 1999,3999:483-487
[11]Zamzami N, Marchetti P, Castedo M. Sequential reduction of mitochondrial transmennbrane potential and generation of reactive oxygen species in early programmed cell death[J]. Exp Med,1995,182(2):367-377
[12]Ma YX, Ogino T, Kawabata T, et al. Cupric nitrilotriacetate-induced apoptosis in HL-60 cells association with lipid peroxdation, release of cytochrome c from mitochondria, and activation of caspase-3[J]. Free Radic Biol Med,1999,27(1-2):227-233
[13]Hengartner MO. Apoptosis:Death cycle and swiss army knives[J]. Nature,1998,391:441-442
[14]Michael R.Duchen. Mitochondria and calcium:from cell signalling to cell death[J]. Physiology,2000,529:57-68
[15]Michael T.Crow,Kartik Mani,Young-Jae,et al. The Mitochondrial Death Pathway and Cardiac Myocyte Apoptosis[J].Circulation Research,2004,95:957-970
[16]Skulachev VP. Cytochromec in the apoptotic and antioxidant cascades[J]. FEBSLett, 1998,423(2):275-280
[17]Valeria Petronilli,Daniele Penzo,Luca Scrrano, et al. The mitochondrial permeability transition release of cytochrome c and cell death[J]. Biological Chemistry,2001,275(15):12080-12084
[18]Dmitri.Krysko,Frank Roels,Luc Leybaert,etal.Mitochondrial transmembrane potential potential changes support the concept of mitochondrial heterogeneity during appotosis[J]. Histochemistry,2001,49(10):1277-1284
[19]Korsmeyer SJ. Bcl-2 gene family and the regulation of programmed cell death[J].Cancer Res 1999,59(7):1693-1700
[20]Joslyn K,Brunelle,Anthony Letai. Control of mitochondrial apoptosis by the Bcl-2 family[J].Cell Science,2009,122:237-441
[21]Haldar S, Basu A, Croce CM. Bcl-2 is the guardian of microtubule integrity[J]. Cancer Res 1997, 57(2):229-233
[22]Asa B,Gustafsson,Roberta A.Gottlieb. Bcl-2 family members and apoptosis, taken to heart[J]. Cell Physiol,2007,292:45-51
[23]施勤,刘继明,张学光.细胞凋亡的线粒体调控与Bcl-2基因家族[J].上海免疫学
[24]Enari M, Sakahira H, Yokoyama H, et al. A caspase-activated DNase that degrades DNA during apoptois, and inhibitor ICAD[J]. Nature,1998,391:43-50
[25]Yang J, Liu XS, Bhalla K, et al. Prevention of apoptosis by Bcl-2:release of cytochrome c from mitochondria blocked[J]. Sicence,1997,275(5303):1129-1132
[26]Henartner MO. Death cycle and Swiss army knives[J]. Nature,1998,391 (6666):441-442
[27]Koseki T, Inohara N, Chen s, et al. ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectively with caspase[J].Pro Natl Acad Sci USA,1998,95(9):5156-5160
[28]Raff M. Cell suicide for beginners[J]. Nature 1998,396 (6707):119-122
[29]Deveraux QL Takahashi R, Salvesen GS, et al. X-liked IAP is a direct inhibitor of cell-death proteases[J]. Nature 1997,388(6639):300-304
[30]徐璐,郑建仙.松树皮提取物的研究进展[J].食品工业,2005,4：36
[31]高羽,董志.原花青素的药理学研究现状[J].中国中药杂志,2009,3(34)：651
[32]Simonetti P, Ciappellano S, Gardana C, et al. Procyanidins from vitis vinifera seeds:in vivo effects on oxidative stress[J]. J Agric Food Chem,2002,50(21):6217
[33]林春兰,蒋建伟,吴美玉,等.松树皮原花青素的离体抗氧化作用[J].中药材,2001,24(12)：882
[34]Puiggros F, Llopiz N, Ardevol A, et al. Grape seed procyanidins prevent oxidative injury by modulating the expression of antioxidantenzymesystems [J]. J Agric Food Chem,2005, 53(15):6080
[35]Louren C F, Gago B, Barbosa R M, et al. LDL isolated from plasma-loaded red wine procyanidins resist lipid oxidation and tocopherol depletion [J]. J Agric Food Chem,2008,56 (10):3798
[36]Al-Makdessi S, Sweidan H, Jacob R. Effect of oligomer procyanidins on reperfusion arrhythmias and lactate dehydrogenase release in the isolated rat heart[J]. Arzneimittelforschung,2006,56 (5):317
[37]Zhai W, Chang J, Lin K, et al. Crosslinking of decellularized porcine heart valve matrix by procyanidins [J]. Biomaterials,2006,27 (19):3684
[38]黄晓瑾,毛峻琴.葡萄籽原花青素抗大鼠脑缺血再灌注损伤的研究[J].中国药师,2005,8(7)541
[39]贾玉洁,闵连秋,季占胜,等.原花青素对大鼠局灶性脑缺血的保护作用[J].中国脑血管病杂志,2005,2(12)：558
[40]贾玉洁,闵连秋,季占胜,等.原花青素对脑缺血后抑凋亡基因蛋白Bcl-2和促凋亡基因蛋白Bax的影响[J].中国临床康复,2006,10(23)：65,72
[41]吴秀香,卢晓梅,杜莉莉,等.原花青素对脑缺血再灌注小鼠脑组织细胞因子、一氧化氮合酶和血脑屏障的影响[J].中国动脉硬化杂志,2006,14(8)：665
[42]Kenny T P, Keen C L, Jones P, et al. Cocoa p rocyanidins inhibit proliferation and angiogenic signals in human dermalmicrovascular endothelial cells following stimulation by low-level H2O2[J]. Exp BiolMed (Maywood),2004,229 (8):765
[43]Fitzpatrick D F, Bing B, Maggi D A, et al. Vasodilating procyanidins derived from grape seeds[J]. Ann N Y Acad Sci,2002,957:78
[44]蒋辛,赵良中,张海龙,等.原花青素对大鼠实验性血栓形成的影响[J].中国实验血液学杂志,2007,15(3)：617
[45]Del Bas J M, Fernandez-Larrea J, Blay M, et al. Grape seed procyanidins improve atherosclerotic risk index and induce liver CYP7A1 and SHP expression in healthy rats [J]. FASEB J,2005,19 (3):479
[46]朱慧娟,孟繁岳,汪志国,等.葡多酚对大鼠脂质代谢紊乱的干预作用[J].南京医科大学学报,2006,26(12)：1179
[47]闫少芳,李勇,吴娟,等.葡萄籽提取物原花青素调节血脂作用及机理研究[J].中国食品卫生杂志,2003,15(4)302
[48]Pinent M, Blay M, Blade M C, et al. Grape seed-derived procyanidins have an antihyperglycemic effect in strep to zotocin-induced diabetic rats and insulinom imetic activity in insulin-sensitive cell lines[J]. Endocrinology,2004,145 (11):4985
[49]Terra X, Valls J, Vitrac X, et al. Grape2seed p rocyanidins act as antiinflamma-tory agents in endotoxin-stimulated RAW 264.7 macrophages by inhibiting NFkB signaling pathway[J]. J Agric Food Chem,2007,55 (11):4357
[50]徐晓云,潘思轶,谢笔钧,等.沙棘原花青素对大鼠应激性胃溃疡的预防作用[J].营养学报,2007,29(1)：58
[51]徐晓云,潘思轶,谢笔钧,等.沙棘原花青素对应激性胃溃疡大鼠上皮细胞凋亡与增殖的影响[J].中国药理学通报,2006,22(11)：1329
[52]刘辉,钟进义,于萍,等.葡多酚对小鼠肝细胞蛋白激酶C和增殖细胞核抗原表达的影响[J].卫生研究,2007,36(1)：31
[53]张波,沈新南,张亚东,等.原花青素对小鼠乙醇性肝损伤的保护作用机制[J].卫生研究,2007,36(3)：295
[54]钟进义,李杰,刘辉,等.葡多酚对肝细胞内Ca2+浓度和增殖活性的影响[J].卫生研究,2006,35(5)：567
[55]段玉清,张海晖,唐瑛,等.莲房原花青素对60 Go-y射线致亚急性辐射损伤防护的研究[J].营养学报,2005,27(6)：491
[56]袁小英,刘玮,谢艳飞,等.葡萄籽提取物原花青素对皮肤急性光损伤Fas蛋白及Bcl-2蛋白表达的影响[J].中国美容医学,2008,17(3)：387
[57]Sugiyama H, Akazome Y, Shoji T, et al. Oligomeric procyanidins in appleolyphenol are main active components for inhibition of pancreatic lipase and triglyceride absorp tion [J]. J Agric Food Chem,2007,55 (11):4604
[58][58] Le Bourvellec C, Le QuereJ M, Sanoner P, et al. Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products[J]. J Agric Food Chem,2004,52 (1): 122
[59]Actis-Goretta L, Ottaviani J I, Keen C L, et al. Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins[J]. FEBS Lett,2003,555 (3):597
[60]谭毓治,万晓霞,赖娟娟,等.葡萄籽原花青素对学习记忆的影响[J].中国药理学通报,2004,20(7)：804
[61]龚玉石,唐瑛,肖俊松,等.莲房原花青素改善小鼠学习记忆障碍的研究[J].营养学报,2006,28(4)：318
[62]Miura T, Chiba M, Kasai K, et al. App le p rocyanidins induce tumor cell apoptosis through mitochondrial pathway activation of caspase-3 [J]. Carcinogenesis,2008,29 (3):585
[63]Gali H U, Perchellet E M, Gao X M, et al. Comparison of the inhibitory effects of monomeric, dimeric, and trimeric procyanidins on the biochemicalmarkers of skin tumor p romotion inmouse epidermis in vivo[J]. PlantaMed,1994,60 (3):235
[64]Li K, Li Q, Li J, et al. Antitumor activity of the procyanidins from pinus koraiensis bark on mice bearing U14 cervical cancer[J]. Yakugaku Zasshi,2007,127 (7):1145
[65]吴自勤,黄治,丁雪梅,等.原花青素对前列腺癌LNCaP细胞增殖和凋亡的影响[J].南方医科大学学报,2007,27(4)：499
[66]GosseF, Guyot S, Roussi S, et al. Chemop reventive p roperties of apple procyanidins on human colon cancer-derived metastatic SW620 cells and in a ratmodel of colon carcinogenesis[J]. Carcinogenesis,2005,26 (7):1291
[67]Bagchi D,Garg A,Krohn R L.Protective effects of grape seed proanthocyanidins and selected antioxidants against TPA-Induced hepatic and brain lipid peroxidation and DNA fragmentation, and peritoneal macrophage activation in mice[J].Pharmac,1998,30(5):771-776
[68]Rao J,Hiroshi Y,Hiroshi O,et al.Occurrence of antioxidant and radical scavenging proanthocyanidins from the Indianminor spicenagkesa[J]. Bioorganic & Medicinal Chemistry, 2004,12:31-36
[69]Shacter E,Weitzman S A.Chronic inflammation and cancer[J].Oncology,2002,16:217-226.
[70]Coussens L M,Werb Z. Inflammation and cancer[J].Nature Rev2002,420:19-26
[71]Sharma S D,Katiyar S K. Ditary grape-seed proanthocyanidin inhibition of ultravioletB-induced immune suppression isassociated withinduction of IL-12[J]. Carcinogenesis,2006 (1):95-102
[72]Manetti R, Parronchi P, Giudzi M G. Natural killer cell stimulatoryfactor (interleukin-12[IL-12]) induces Thelper type1(Th1)-specific immune responses and inhibits the development of IL-4-produc-ing Th cells[J]. Exp Med,1993,177:1199-1204
[73]Hsieh C, Macatonia S E, Tripp C S. Development of Thl CD4+ T cells through IL-12 produced by Listeria-induced macrophages[J]. Science,1993,260:547-549
[74]Aggarwal B B, Shishodia S. Molecular targets of dietary agents for prevention and therapy of cancer[J]. Biochem.Pharmacol,2006,14:1397-1421
[75]Meeran S M. Proanthocyanidins inhibit mitogenic and survival-signaling invitro and tumor growth in vivo[J]. FrontBiosci,2008,13:887-897
[76]Mittal A, ELMETS C A, KATIYAR S K. Dietary feeding of proanthocyanidins from grape seeds prevents photocarcinogenesis in SKH-1 hairlessmice:relationship to decreasedfat and lipid peroxidation[J]. Carcinogenesis,2003,24:1379-1388
[77]Mantena S K, KATIYAR S K.Grape seed proanthocyanidins inhibit UVradiation-induced oxidative stressand activation of MAPK and NF-kappa B ignaling in human epidermal keratinocytes[J].Free RadicBiol Med,2006,40:1603-1614
[78]Mccarty M F.Polyphenol-mediated inhibition of AP-1 transactivating activity may slow cancer growth by impeding angiogen-esisand tumor in vasiveness[J].MedHypotheses,1998,50:511-514
[79]Sharma S D,Meeran S M,Katiyar S K.Dietary grape seed proanthocyanidins inhibit UVB-induced oxidative stress and activation of mitogen-activate dproteinkinases and nuclear factor-jBsignalingin in vivo SKH-1 hairlessmice [J].MolCancerTher,2007(6):995-1005
[80]Bode A M,Dong Z.Mitogen-activated protein kinase activation in UVinduced signal transduction[J].Sci STKE RE,2000,32:675-681
[81]Meeran S M, Katiyar S K. Cell cycle control as a basis for cancer chemoprevention through dietary agents[J]:FrontBiosci,2008,13:2191-2202
[82]Kluck R M, Bossy-Wetzel E, Green D R, et al.The release of cytochrome c frommitochondria:a primary site for Bcl-2regulation of apoptosis [J]. Science,1997,275:1132-1136
[83]Sedlak T W, Oltvai Z N, Yang E. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax[J].Proc Natl Acad Sci USA,1995,92:7834-7838
[84]Meeran S M, Katiyar S K. Grape seed proanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterations in Cdki-Cdk-cyclin cascade, and caspase-3 activationvia loss of mitochondrial membrane potential[J]. Exp Dermatol,2007,16:405-415
[85]Roy A M, Baliga M S, Elmets C A. Grape seed proanthocyanidins induce apoptosis through p53, Bax, and Caspase-3 pathways[J].Neoplasia,2005 (7):24-36
[86]Agarwal C, Singh R P, Dhanalashhmi S. Anti-angiogenic efficacy of grape seed extract in endothelial cells[J]. Oncol Rep,2004(11):681-685
[87]Strek M, Gorlach S, Podsedek A. Procyanidin oligomers from Japanese quince (Chaenomelesjaponica) fruit inhibitactivity of MMP-2 andMMP-9 metalloproteinases[J]. Agric Food Chem,2007,16:6447-6452
[88]Matchett M D, Mackinnon S L, Sweeney M L. Blueberry flavonoids inhibit matrix metalloproteinase activity in DU145 humanprostate cancer cells[J]. Biochem Cell Biol, 2005,83:637-643
[89]Vayalil P K, Mittal A, Katiyar S K. Proanthocyanidins from grape seeds inhibit expression of matrix metalloproteinases in human prostate carcinoma cells which is associated with the inhibition of activation of MAPK and NF-κB[J]. Carcinogenesis,2004,25:987-995
[90]司徒镇强,吴军正.细胞培养[M].西安：世界图书出版西安公司,2007：200
[91]杜宏,张娜,高霞,等.莲房原花青素对人肝癌细胞HepG2生长及凋亡的作用[J].实用医学杂志,2008,24(6)：891-893
[92]涂水平,吴裕忻.细胞凋亡与胃肠肿瘤[J].内镜,1999,3(1)：61-64
[93]Davison K,Mann K,Miller W. Arsenic trioxide:mechanisms of action[J]. Semin Hematol,2002,39(2 Suppl 1):3-7
[94]Maeda H, Hori S, Nishitoh H, et al. Tumor growth inhibition by arsenic trioxide (As2O3)in the orthotopic metastasis model of androgen-independent prostate cancer [J]. Cancer Res,2001,61(14):5432-5440
[95]陈立军,靳秋月,王瑞珉,等.芸香苷诱导K562细胞凋亡机制[J].中草药,2006,37(5)：738-741
[96]季宇彬,高世勇,张秀娟.羊栖菜多糖诱导肿瘤细胞凋亡的研究[J].中国中药杂志,2004,29(3)：245-247
[97]曾文军,王柳均.细胞凋亡的流式细胞仪检测技术研究进展[J].医学文选,2005,24(3)：425～428
[98]杜晓芬,谢笔钧,等.莲房原花青素对人口腔表皮样癌(KB)细胞生长及形态的影响[J].现代口腔医学杂志,2005,19(4)：384-386
[99]季宇彬,高世勇.海嘧啶体内外抗肿瘤作用研究[J].中国药学杂志,2005,40(23)：1788-1793
[100]曹文涛,廖爱军,曾斌,等.白藜芦醇对胃癌SGC-7901细胞VEGF表达的影响[J].现代生物医学进展,2006,6(10)：52-53
[101]姜国均,周帮会,王萍等.线粒体在细胞凋亡中的作用[J].动物医学进展,2003,24(5)：36-38
[102]黄文芳,曾娅莉,刘华等.辛伐他汀通过线粒体途径诱导K562细胞凋亡[J].沈阳药科大学学报,2007,24(12)：727-729
[103]Wang J, Yu Y, Hashimoto F, et al. Baicalein induces apoptosis through ROS-mediated mitochondrial dysfunction pathway in HL-60 cells[J]. Int J Mol Med,2004,14(4):627-632
[104]Orrenius S, Zhivotovsk B, Nicotera P. Regulation of cell death:the calcium-apoptosis link[J]. Nat Rev Mol Cell Biol.2003,4:552-565
[105]陈奇.中药药理研究方法学[M].第二版.北京：人民卫生出版社,2006：107
[106]于慧茹.桦木酸抗肿瘤作用及其机制的研究[D].哈尔滨商业大学硕士论文,2006：17-18
[107]徐淑云,卞如濂,陈修.药理实验方法学[M].第三版.北京：人民卫生出版社,2001：228～1822
[108]韩锐.抗癌药物研究与实验技术[M].第一版.北京：中国协和医科大学北京医科大学联合出版社,1997,4：375-400
[109]周思朗,屈艳妮,张健,等.SRB法与MTT法细胞计数应用比较[J].中国现代医学杂志,2005,15(17)：2615-2617
[110]冯春琼,马文丽,宋艳斌,等.细胞凋亡的MTT染色法检测[J].第一军医大学学报,2002,3：21-24
[111]赵永同,朱峰.凋亡的分子机理[J].生命科学,1996,8(3)：8
[112]Kluza L,Lansiaux A,Wattez N,er al.Apoptotic response of HL-60 human leukemia cells to the antitumor drug TAS2103[J].Cancer Res,2000,60(15):4077
[113]Hall PA. Assessing apoptosis:a critical survey[J]. Endocr Relat Cancer,1999,6(l):3-8
[114]唐国华,贺修胜.流式细胞仪检测细胞DNA含量技术的标化与控制[J].中国医师杂志,2004,增刊：278
[115]孙俊.烷化剂引起的DNA损伤与细胞周期和细胞凋亡的关系[J].中国药学杂志,1997,32(7)：393
[116]周春喜,梁学颖,李宁,等.凋亡细胞线粒体膜电位的流式细胞术分析[J].2003,19(5)：431-432
[117]郭静,蒲咏梅,张东才.钙离子信号与细胞凋亡[J].生物物理学报,2005,1：112-114
[118]郭荣球,李亚林.钙稳态失衡与细胞凋亡的研究现状[J].邵阳学院学报(自然科学版),2005,2：77-79
[119]厉为良.细胞内pH,钙离子与细胞凋亡[J].国外医学.生理.病理科学与临床分册,1998,2：82-84
[120]张艳芬,范雪晖.细胞内外Ca2+对细胞凋亡的影响[J].新乡医学院学报,2003,3：139-141
[121]Hiroshi Y, Yamada, Gary J. Inhibition of Tripl/S8/hsugl,a component of the human 19 sproteasome, enhances mitotic apoptosis inducesd by sprindle poisons[J]. Mol Cancer Ther,2006,5(1):29-38
[122]Moammir H, Raj K, Nihal A. Cancer chemoprevention by resveratrol:In vitro and in vivo studies and the underlying mechanisms[J]. International Journal of Oncology,2003,23:17-28
[123]Zhen-Hua Ma,Qing-Yong Ma.Resveratrol:A medical drug for acute pancreatitis[J]. World Journal of Gastroenterology 2005,11(21):3171-3174
[124]Aggarwal BB, Shishodia S, Takada Y, et al. Curcumin suppresses the paclitaxel-induced nuclear factor-kappa B pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice[J]. Clin Cancer Res 2005,11:7490-7498
[125]Timothy SZ,Stephane H,Keisuke K,et al. Deficiency in caspase-9 or casepase-3 induces compensatory caspase activation [J].Nature Medicine,2000,6:1241-1247
[126]Kadenbach B, Arnold S, Lee I, et al. The possible role of cytochrome coxidase in stress-induced apoptosis and degenerative diseases[J]. Biochim Biophys Acta,2004,1655(1-3):400-408
[127]Richard Chikara Koya, Hisakazu Fujita, Shigeomi Shimizu, et al. GelsolinInhibits Apoptosis by Blocking Mitochondrial Membrane Potential Loss and Cytochrome c Release [J]. The Journal Of Biology Chemistry,2000,275(20):15343-15349
[128]庞飒,龚兴国.细胞内信号分子传导的研究进展[J].生物化学与生物物理进展,2000,27(1)：24