峨参提取物及其单体成分抗肿瘤活性及作用机理的研究
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摘要
目的：探讨峨参提取物对于HEPG2、MG-63、B16、HELA细胞体外的抗肿瘤活性和促凋亡的作用研究；探讨峨参内酯的急性毒性以及峨参内酯对H22荷瘤小鼠的体内抗肿瘤活性及其分子机制。
方法：应用MTT比色法检测不同浓度峨参提取物(0、2.5、5、10、20、40、80μg/m1)对HEPG2、MG-63、B16、HELA细胞增殖抑制作用,分别计算不同药物对肿瘤细胞生长的抑制率与半数抑制浓度IC5o；采用DAPI染色法、琼脂糖凝胶电泳法(DNA ladder法)检测峨参内酯对HEPG2、MG-63、B16、HELA细胞凋亡形态学观察；计算峨参内酯对昆明小鼠的半数致死量LDs0采用腹水型肝癌细胞株建立小鼠移植肿瘤模型,待造模成功后随机将小鼠分为7组,分别以峨参内酯高剂量、中剂量、低剂量组、峨参多糖组以及替加氟组作为治疗组,模型组及空白组给予0.5%CMC-Na作为对照。治疗结束后,观察小鼠的一般状态,胸腺指数、脾脏指数、药物的抑瘤率及不同药物组对小鼠的生命延长率,通过免疫组化技术初步检测给予不同剂量的肿瘤组织中bax、 bcl-2、P53的表达情况,探讨峨参抗肿瘤的分子机制。
结果：1、峨参石油醚提取物对HEPG2、HELA细胞的抑制作用十分明显,经计算得出,峨参石油醚提取物对HEPG2、HELA细胞的IC50值分别为36.53μg/ml和18.25μg/ml,对于MG-63和B16细胞抑制作用较小；峨参氯仿提取物对HEPG2、HELA两种肿瘤细胞的IC50值分别为40.62μg/ml和45.66μg/ml,对于MG-63和B16细胞抑制作用较小；峨参乙酸乙酯提取物和峨参多糖几乎无体外抗肿瘤活性；用峨参内酯作用于四种肿瘤细胞48h后,对四种肿瘤细胞均具有显著的抑制作用,经计算得出,峨参内酯对HEPG2、MG-63、B16、HELA四种肿瘤细胞的IC50值分别为12.24μg/m1、14.37μg/ml、43.25μg/ml和18.72μg/ml,且随着药物浓度的增长,药物对肿瘤细胞的抑制率增长也十分显著,在药物浓度为到80gg/ml时,抑制率均达到了70%以上；而异峨参内酯仅对于B16细胞和MG-63细胞有较显著的抑制作用,对于Hela细胞和HEPG2细胞几乎无抗肿瘤作用,经统计学软件计算得出异峨参内酯对MG-63细胞和B16细胞的IC50值分别为34.68μg/ml和65.72μg/ml;
2、DAPI染色后,荧光显微镜观察到,四种细胞的生长明显受到抑制,有明显的细胞核断裂和固缩,且随着峨参内酯浓度的增加,细胞凋亡现象更加明显,而对照组(0μg/ml)细胞完整,未见任何细胞凋亡迹象；DNA ladder检测细胞凋亡实验中,用不同浓度峨参内酯处理细胞后,药物组均产生不同程度的细胞凋亡特征性DNA梯带；
3、通过对昆明小鼠给予峨参内酯的急性毒性实验实验表明,峨参内酯虽然具有较高的抗肿瘤活性,但是其急性毒性也较强,计算得出峨参内酯的LDso为151.52mg/kg,其95%可信区间为93.28mg/kg-252.94mg/kg;
4、峨参内酯高剂量、中剂量、低剂量组以及替加氟组对H22小鼠肝癌实体瘤的抑制率分别为54.39%、63.74%、56.14%、53.80%；通过免疫组化技术初步检测给予不同药物剂量的肿瘤组织中bax、bcl-2、P53的表达情况,初步研究表明,与模型组相比,峨参内酯能显著提高小鼠的生命延长率,峨参内酯能够上调bax的表达,降低bcl-2/bax比值,从而起到诱导肝癌细胞H22凋亡的作用,而各剂量组p53虽然均有一定的表达,但是各实验组之间无显著差异。
结论：峨参内酯对于HEPG2、MG-63、B16、HELA四种肿瘤细胞具有显著的体内外抗肿瘤活性,可以延长H22肝癌小鼠的生存期,其促进凋亡的途径之一可能为上调bax的表达,降低bcl-2/bax比值,从而起到诱导肿瘤细胞凋亡的作用。
Objective To investigate the effect of the extracts of Anthriscus sylvestris (L.)Hoffm on inhibiting proliferation and inducing apoptosis HEPG2, MG-63, B16, HELA cells in vitro; To explore the acute toxicity of the Anthriscus lactone and the anti-tumor activity and molecular mechanism in H22tumor-bearing mice in vivo.
Methods To detect the inhibitive effect of different concentration of the extracts of Anthriscus sylvestris (L.)Hoffm on proliferation of HEPG2, MG-63, B16, HELA cells in vitro after48h by MTT Assay method. And to calculate the IC50value of the petrol extract of the extracts of Anthriscus sylvestris (L.)Hoffm. To detect the cell apoptosis character of HEPG2, MG-63, B16, HELA cells by treating with different doses of the petrol extract of Anthriscus sylvestris (L.)Hoffm by the DAPI method and DNA ladder method. And to calculated the IC50value of the Anthriscus lactone; To establish the anmimal model by inoculating the H22cell line in the left oxter asepticly, and then devided the mice into7groups randomly. took the Anthriscus lactone high-dose, medium-dose and low-dose group, Anthriscus polysaccharide group and Tegafur group as the treatment group, model group and blank group were treated with0.5%CMC-Na as the control, then gave the mice drugs daily and weighed once a day respectively, after12days,7mice in each group were killed. Then to take out the tumor, thymus and spleen, and weighed them to calculate the mortality, inhibition rate, thymus index, spleen index, and the rate elongation of life. To detect the necrosis of tumor and expression of bax、bcl-2、P53by HE and immunohistochemistry method.
Results1、The extracts of chloroform, petroleum of Anthriscus sylvestris (L.)Hoffm all had the better inhibitive effect on the proliferation of HEPG2、HELA cells, the IC50value were36.53μg/ml and18.25μg/ml on HEPG2、HELA cells by The extracts of petroleum. The IC50value were40.62μg/ml and45.66μg/ml on HEPG2、HELA cells by The extracts of chloroform. Anthriscus ethyl acetate extract and Anthriscus polysaccharide almost have no anti-tumor activity in vitro. The Anthriscus lactone had better inhibitive effect on the proliferation of the four tumor cells. The IC50value were12.24μg/ml、14.37μg/ml、43.25μg/ml and18.72μg/ml for HEPG2、MG-63、B16、HELA cells respectively. The rate of inhibitive effect was related with the concentration, and the rate of inhibitiv effect reached70%at the80μg/ml.The isoan-thricin had significantly inhibitive effect or the proliferation of B16cells and MG-63cells, and the IC50value were34.68μg/ml and65.72μg/ml for B16cells and MG-63cells respectively.
2、By DAPI stained, then to observe the impact of different concentrations of Anthriscu lactone on HEPG2, MG-63, B16, HELA cells by gel electrophoresis method. These fou tumor cells'growth was suppressed, including the cell collapse, fragmentation of cel nucleus, pyknosis and so on. And higher concentrations, more obvious. The cells in the control group were intact. At different concentrations treated, The DNA apopsis electrophoresis lines were observed by DNA ladder method.
3、The acute toxicity test of Anthriscus lactone used Kunming mice. The test showed the Anthriscus lactone althougth had higher anti-tumor activity, but also had higher acute toxicity. the LD50value was151.52mg/kg, the95%confidence interval was93.28mg/kg-252.94mg/kg.
4、The inhibitive rate of high-dose group, medium-dose group, low-dose group, and the Tegafur group were54.39%、63.74%、56.14%、53.80%respectively. To detect the bax,bcl-2and P53expression in different group's tumor tissues by immunohistochemical method. The preliminary study showed Anthriscus lactone can significantly improve the lives of mice and can upregulate the expression of bax and reduce the ratio of bcl-2and bax So Anthriscus lactone had the effect of inducing the H22cells apoptosis. Although each group's p53had certain expression, but there was no significant differences.
Conclusions The Anthriscus lactone had significant anti-tumor activity on HEPG2、 MG-63、B16、HELA cells in vivo. And can significantly improve the lives of mice which have H22tumor cells. Its one of the ways to promote apoptosis may be related with bax expression raised and the ratio of bcl-2and bax reduced.
方法：应用MTT比色法检测不同浓度峨参提取物(0、2.5、5、10、20、40、80μg/m1)对HEPG2、MG-63、B16、HELA细胞增殖抑制作用,分别计算不同药物对肿瘤细胞生长的抑制率与半数抑制浓度IC5o；采用DAPI染色法、琼脂糖凝胶电泳法(DNA ladder法)检测峨参内酯对HEPG2、MG-63、B16、HELA细胞凋亡形态学观察；计算峨参内酯对昆明小鼠的半数致死量LDs0采用腹水型肝癌细胞株建立小鼠移植肿瘤模型,待造模成功后随机将小鼠分为7组,分别以峨参内酯高剂量、中剂量、低剂量组、峨参多糖组以及替加氟组作为治疗组,模型组及空白组给予0.5%CMC-Na作为对照。治疗结束后,观察小鼠的一般状态,胸腺指数、脾脏指数、药物的抑瘤率及不同药物组对小鼠的生命延长率,通过免疫组化技术初步检测给予不同剂量的肿瘤组织中bax、 bcl-2、P53的表达情况,探讨峨参抗肿瘤的分子机制。
结果：1、峨参石油醚提取物对HEPG2、HELA细胞的抑制作用十分明显,经计算得出,峨参石油醚提取物对HEPG2、HELA细胞的IC50值分别为36.53μg/ml和18.25μg/ml,对于MG-63和B16细胞抑制作用较小；峨参氯仿提取物对HEPG2、HELA两种肿瘤细胞的IC50值分别为40.62μg/ml和45.66μg/ml,对于MG-63和B16细胞抑制作用较小；峨参乙酸乙酯提取物和峨参多糖几乎无体外抗肿瘤活性；用峨参内酯作用于四种肿瘤细胞48h后,对四种肿瘤细胞均具有显著的抑制作用,经计算得出,峨参内酯对HEPG2、MG-63、B16、HELA四种肿瘤细胞的IC50值分别为12.24μg/m1、14.37μg/ml、43.25μg/ml和18.72μg/ml,且随着药物浓度的增长,药物对肿瘤细胞的抑制率增长也十分显著,在药物浓度为到80gg/ml时,抑制率均达到了70%以上；而异峨参内酯仅对于B16细胞和MG-63细胞有较显著的抑制作用,对于Hela细胞和HEPG2细胞几乎无抗肿瘤作用,经统计学软件计算得出异峨参内酯对MG-63细胞和B16细胞的IC50值分别为34.68μg/ml和65.72μg/ml;
2、DAPI染色后,荧光显微镜观察到,四种细胞的生长明显受到抑制,有明显的细胞核断裂和固缩,且随着峨参内酯浓度的增加,细胞凋亡现象更加明显,而对照组(0μg/ml)细胞完整,未见任何细胞凋亡迹象；DNA ladder检测细胞凋亡实验中,用不同浓度峨参内酯处理细胞后,药物组均产生不同程度的细胞凋亡特征性DNA梯带；
3、通过对昆明小鼠给予峨参内酯的急性毒性实验实验表明,峨参内酯虽然具有较高的抗肿瘤活性,但是其急性毒性也较强,计算得出峨参内酯的LDso为151.52mg/kg,其95%可信区间为93.28mg/kg-252.94mg/kg;
4、峨参内酯高剂量、中剂量、低剂量组以及替加氟组对H22小鼠肝癌实体瘤的抑制率分别为54.39%、63.74%、56.14%、53.80%；通过免疫组化技术初步检测给予不同药物剂量的肿瘤组织中bax、bcl-2、P53的表达情况,初步研究表明,与模型组相比,峨参内酯能显著提高小鼠的生命延长率,峨参内酯能够上调bax的表达,降低bcl-2/bax比值,从而起到诱导肝癌细胞H22凋亡的作用,而各剂量组p53虽然均有一定的表达,但是各实验组之间无显著差异。
结论：峨参内酯对于HEPG2、MG-63、B16、HELA四种肿瘤细胞具有显著的体内外抗肿瘤活性,可以延长H22肝癌小鼠的生存期,其促进凋亡的途径之一可能为上调bax的表达,降低bcl-2/bax比值,从而起到诱导肿瘤细胞凋亡的作用。
Objective To investigate the effect of the extracts of Anthriscus sylvestris (L.)Hoffm on inhibiting proliferation and inducing apoptosis HEPG2, MG-63, B16, HELA cells in vitro; To explore the acute toxicity of the Anthriscus lactone and the anti-tumor activity and molecular mechanism in H22tumor-bearing mice in vivo.
Methods To detect the inhibitive effect of different concentration of the extracts of Anthriscus sylvestris (L.)Hoffm on proliferation of HEPG2, MG-63, B16, HELA cells in vitro after48h by MTT Assay method. And to calculate the IC50value of the petrol extract of the extracts of Anthriscus sylvestris (L.)Hoffm. To detect the cell apoptosis character of HEPG2, MG-63, B16, HELA cells by treating with different doses of the petrol extract of Anthriscus sylvestris (L.)Hoffm by the DAPI method and DNA ladder method. And to calculated the IC50value of the Anthriscus lactone; To establish the anmimal model by inoculating the H22cell line in the left oxter asepticly, and then devided the mice into7groups randomly. took the Anthriscus lactone high-dose, medium-dose and low-dose group, Anthriscus polysaccharide group and Tegafur group as the treatment group, model group and blank group were treated with0.5%CMC-Na as the control, then gave the mice drugs daily and weighed once a day respectively, after12days,7mice in each group were killed. Then to take out the tumor, thymus and spleen, and weighed them to calculate the mortality, inhibition rate, thymus index, spleen index, and the rate elongation of life. To detect the necrosis of tumor and expression of bax、bcl-2、P53by HE and immunohistochemistry method.
Results1、The extracts of chloroform, petroleum of Anthriscus sylvestris (L.)Hoffm all had the better inhibitive effect on the proliferation of HEPG2、HELA cells, the IC50value were36.53μg/ml and18.25μg/ml on HEPG2、HELA cells by The extracts of petroleum. The IC50value were40.62μg/ml and45.66μg/ml on HEPG2、HELA cells by The extracts of chloroform. Anthriscus ethyl acetate extract and Anthriscus polysaccharide almost have no anti-tumor activity in vitro. The Anthriscus lactone had better inhibitive effect on the proliferation of the four tumor cells. The IC50value were12.24μg/ml、14.37μg/ml、43.25μg/ml and18.72μg/ml for HEPG2、MG-63、B16、HELA cells respectively. The rate of inhibitive effect was related with the concentration, and the rate of inhibitiv effect reached70%at the80μg/ml.The isoan-thricin had significantly inhibitive effect or the proliferation of B16cells and MG-63cells, and the IC50value were34.68μg/ml and65.72μg/ml for B16cells and MG-63cells respectively.
2、By DAPI stained, then to observe the impact of different concentrations of Anthriscu lactone on HEPG2, MG-63, B16, HELA cells by gel electrophoresis method. These fou tumor cells'growth was suppressed, including the cell collapse, fragmentation of cel nucleus, pyknosis and so on. And higher concentrations, more obvious. The cells in the control group were intact. At different concentrations treated, The DNA apopsis electrophoresis lines were observed by DNA ladder method.
3、The acute toxicity test of Anthriscus lactone used Kunming mice. The test showed the Anthriscus lactone althougth had higher anti-tumor activity, but also had higher acute toxicity. the LD50value was151.52mg/kg, the95%confidence interval was93.28mg/kg-252.94mg/kg.
4、The inhibitive rate of high-dose group, medium-dose group, low-dose group, and the Tegafur group were54.39%、63.74%、56.14%、53.80%respectively. To detect the bax,bcl-2and P53expression in different group's tumor tissues by immunohistochemical method. The preliminary study showed Anthriscus lactone can significantly improve the lives of mice and can upregulate the expression of bax and reduce the ratio of bcl-2and bax So Anthriscus lactone had the effect of inducing the H22cells apoptosis. Although each group's p53had certain expression, but there was no significant differences.
Conclusions The Anthriscus lactone had significant anti-tumor activity on HEPG2、 MG-63、B16、HELA cells in vivo. And can significantly improve the lives of mice which have H22tumor cells. Its one of the ways to promote apoptosis may be related with bax expression raised and the ratio of bcl-2and bax reduced.
引文
[1]李冰,孙燕.肿瘤[M].科学普及出版社.1966：5-8.
[2]崔桂敏,马少忠,杨华,等.肿瘤学[M].中国中医药出版社.2009：5-6.
[3]柴玉英,张向阳,李勇.我国人口主要疾病死因构成变化的影响因素[J]中国全科医学.2010,13(9)：1025-1027.
[4]谭永明.从中医角度认识癌症之浅见[J].中外健康文摘.2010,7(23)：336-337.
[5]李庆玲.谈中医药治疗肿瘤的特色及其作用[J].中医药报,2001,29(5)：223-233.
[6]黄修燕,黄自丽,晁愚,等.中药“松友饮”对高转移人肝癌侵袭性及小鼠免疫功能的影响[J].肿瘤.2008,28(6)：489-492.
[7]Huang xY, wang L, Huang zL, et al.Herbal extract"songyouYin"inhibits tumor growth and prolongs survival in nude mice bearing human hepatocellular carcinoma xenograft with high metastatic potential[J]. J callcer Res Clin OncoI.2009,135(9):1245-1255.
[8]汪海英,李福安,魏全嘉.抗肿瘤中药的研究现状[J].青海医学院学报.2008,29(4)：278-282.
[9]钱彦方.活血化瘀中药对肿瘤形成和转移的影响[J].中医杂志.2008,49(10)：942-944.
[10]Kerr Jf etal.Br J Cancer,1972,26:239-257
[11]黄清松,李红枝,张咏莉,等.三七皂苷Rgl抗突变和抗肿瘤研究短篇论著[J].临床和实验医学杂志.2006,5(8)：1124.
[12]吕淑华.黄芪多糖抗肿瘤作用研究进展[J].江西中医学院学报.2009,21(1)：85-87.
[13]闫凤,孟庆雄.常见的动物毒素诱导肿瘤细胞凋亡的分子机制[J].生物学杂志.2010,27(3)：71-74.
[14]柯尊金,丁心喜,董文奎,等.槲皮素对人膀胱癌BIU-87细胞增殖和凋亡的影响[J].实用癌症杂志.2008,23(2)：116-118.
[15]毕黎琦.中药天花粉蛋白对黑色素瘤细胞凋亡及凋亡周期的影响[J].中国中西医结合杂志.2008,18(1)：35.
[16]陈丽荣,郑枫,MC Willingham,等.紫杉醇诱发人乳癌细胞凋亡的机制研究[J].中华肿瘤杂志.2007,19(2)：103-106.
[17]郑元元.凋亡相关基因TRAIL、Bcl-2在非小细胞肺癌中的表达及临床意义研究[D]. 郑州大学硕士学位论文.2012年.
[18]KakarSS, RoyD. Curcumin inhibitsTPA induced expression c-fos,c-jun and c-myc protooncogenes messenger RNAs in mouse skin[J].CancerLett,1994; 87:81-89
[19]吴青,陈燕,李新刚,等.姜黄素影响Caspase-8和Caspase-9诱导淋巴瘤Raji细胞凋亡的研究[J].中国实验血液学杂志.2005,13(4)：624-627.
[20]Cecchinato V, Chiaramonte R, Nizzardo M.Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells[J]. Biochem Pharmacol. 2007,74(11):1568-1574
[21]冯磊,张连芬,严婷,等.中药虎杖中抗癌活性物质研究[J].中药材.2006,29(7)：689-691.
[22]李覃,王伟,陈虹,等.虎杖提取物白藜芦醇诱导肿瘤细胞凋亡的研究进展[J].武警医学院学报.2009,18(10)：886-888.
[23]MIHRAM, ERSTER S, ZAIKAA, et al.p53 has a directapoptogen-ic role at themito-chondria [J].MolCell,2003,11(7):577.
[24]Lin HY, Tang HY, Shih A, et al.Thyroid hormone is a MAPK-de-pendent growth factor for thyroid cancer cells and is anti-apoptotic.[J].Steroids.2007,72(2):180-187.
[25]袁静.檞皮素抗肿瘤作用研究进展[J].国外医学中医中药分册.1996,18(5)：3.
[26]李娜,胡玉红,孙盛梅,等.小檗碱诱导肿瘤细胞凋亡的研究进展[J].黑龙江医药科学.2007,30(1)：102-103.
[27]Kang BY, Chung SW, Cho D, et al. Involvement of p38 mitogen-activated protein kinase in the induction of interleukin-12 p40 production in mouse macrophages by berberine,abenzodioxoloquinolizinc alkaloid[J].Biochem Pharmacol.2002,63 (10):1901-1910
[28]马靖,符乃阳.甘草提取物体外选择性诱导几种人肿瘤细胞凋亡[J].癌症.2001,20(8)：806-811.
[29]Chia H W, Robert M, Shao C L, et al.Cobra cardiotoxin-inducedcell death in fetal rat cardiomycoytes and cortical neurons:diferent74pathway but similar cell surface target[J]. Toxlcon.2006(46):430-440.
[30]Wu HL, Hsu CY, Liu WH, et al.Berberine-induced apoptosisof human leukemia HL-60 cells is associated with down-regulation of nucleophosm in B23 and telomerase activitv[J].IntJ Cancer.2009,81 (6):923-929.
[31]王凤岐,吴大真.峨眉神效验方[M].科学普及出版社.1982：42.
[32]樊梅英,葛滢,关保华,等.明党参、峨参种群分布格局的比较研究[J].科技通报.2003,19(3)：203.
[33]Institute of Botany. Chinese Academy of Sciences. IconograPhia CornoPhytorum Simcorum[M]. Beijing:Science Press.1987:1055.
[34]马超英.基于细胞凋亡调节基因为靶点的峨参抗肿瘤活性成分筛选及其作用机理研究[J].基础研究.2012,3：21-24.
[35]Hong SM, et al. Immunological adjuvant containing extract from anthriscus sylvestris for activating immune system of host[J]. KR2006032925-A.
[36]Kozawa M, et al. Chem Pharm Bull.1978,23(4):1337-1338.
[37]王岳峰,耿耘,朱利平,等.峨参根化学成分研究[J].安徽农业科学.2009,37(23)：11001-11002.
[38]王淑丽.峨参活性成分的提取、分离与筛选[D].西南交通大学硕士研究生论文.2008年.
[39]刘玛菡.峨参质量标准研究[D].西南交通大学硕士研究生论文.2009年.
[40]Yeonjoong Yong, Soon Young Shin, Young Han Lee, Yoongho Lim, et al. Antitumor activity of deoxypodophyllotoxin isolated from Anthriscus sylvestris:Induction of G2/M cell cycle arrest and caspase-dependent apoptosis.Bioorg[J]. Med Chem. Lett.2009, (19):4367-4371.
[41]马超英,涂显琴,牛顺子等.峨参石油醚提取物对肺癌细胞A549和H460增殖的抑制作用观察[J].河北医药.2011,33(13)：1925—1926.
[42]李莉.峨参益气活血作用的实验研究[D].西南交通大学硕士研究生学位论文.2007年.
[43]Deoxypodophyllotoxin, flavolignan, from Anthriscus sylvestris Hoffm. inhibits migration and MMP-9 via MAPK pathways in TNF-a-induced HASMC S.-J. Suh et al. Vascular Pharmacology.2009,51:13-20.
[44]Korea, Meihua JIN,a Tae Chul MOON,b Zhejiu QUAN,a Eunkyung LEE,c Yun Kyung KIM,a Ju Hae YANG,aSeok-Jong SUH,d Tae Cheon JEONG,a Seung Ho LEE,a Cheorl-Ho KIM,*,d and Hyeun Wook CHANG*,a The Naturally Occurring Flavolignan, Deoxypodo- phyllotoxin, Inhibits Lipopoly saccharide-Induced iNOS Expression through the NF-k B Activation in RAW264.7 Macrophage Cells Biol. Pharm. Bull.2008,31 (7):1312-1315.
[45]耿耘,李莉,魏屹,等.峨参对实验性衰老小鼠作用的实验研究[J].时珍国医国药.2011,22(1)：144-145.
[46]马超英,李莉,耿耘,等.峨参对食醋所致脾虚小鼠的作用观察[J].四川中医.2008,26(9)：12.
[47]李莉,高雅蓉,马超英,等.峨参对血瘀模型大鼠血液流变性的影响[J].江西中医药.2007,38(6)：78-79.
[48]丁昭莉.峨参的毒性及其提取物抗氧化活性和免疫作用观察[D].西南交通大学硕士研究生学位论文.2011年.
[49]涂显琴.峨参提取物抗肿瘤活性的初步研究[D].西南交通大学硕士论文.2012年.
[50]Tang Y, Simoneau AR, Xie J,Shahandeh B, Zi X.Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53. Cancer Prevention Research,2008,1(6):439-451.
[51]谢鑫.超声微泡负载自杀基因HSV1_T_省略洛韦对前列腺癌细胞抑制效应的研究[D].重庆医科大学硕士学位论文.2012年.
[52]李胜.Skp2基因沉默对人肺癌细胞生物学活性的影响及机制研究[D].重庆医科大学博士学位论文.2008年..
[53]Zi X,Simoneau AR.Flavokawain A,a novelchalcone from kava extract,induces apoptosis in bladder cancer cells by involvementof Bax protein-dependent and mitochondria dependent apoptotic pathway and suppresses tumor growth in mice.Cancerf J]. Res 2005, 65:3479-3486.
[54]Tang Y,Li X,Liu Z,Simoneau AR,Xie J,Zi X.Flavokawain B,a kava chalcone,exhibits robust apoptotic mechanisms on androgen receptor negative, hormone-refractory prostate cancer cell lines and reduces tumor growth in a preclinical model[J]. International Journal of Cancer,2010,127(8):1758-1768.
[55]Herrmann M, Lorenz H M, Voll R, et a.l A rapid and simple method for the isolation of apoptotic DNA fragment[J].Nucleic Acids Res,1994,22(24):5506-5507.
[56]Hanahan,D and Weinberg,RA.The hallmarks of cancer[J].Cell,2000,100:57-70.
[57]张鑫.吴茱萸碱诱导人胃癌细胞SGC-7901细胞凋亡[J].哈尔滨商业大学学报自然科学版.2010,5.
[58]代光明,杜先智,梅同华.柴胡皂苷D对肺癌A-549细胞增殖、凋亡的影响及机制探讨[J].西南师范大学学报(自然科学版).2009,34(3)：75-78.
[59]叶明霞,周顺长,孔利佳,任大宏等.S180肉瘤,艾氏腹水瘤肿瘤移植模型建立的研究[J],中国实验动物学杂志,2002；12(1)：14-16.
[60]Carr BrendelVM arkov IC D, Ferrer K, et al Immunity tomur ine breast cancer cells modified to express MUC-1, human breast cancer antigen, in transgenic mice to lerant to human MUC1[J]. Cancer Rss,2000,60(9):2435-2443
[61]董媛.扶正抗癌方对H22腹水型瘤株移植瘤小鼠CyclinD1和Caspase_3表达影响的实验研究[D].陕西中医学院硕士学位论文.2011年.
[62]Komaki R,Milas L,Ro JY,et al.Prognostic biomarker study in pathologically Staged N1 non-small cell lung caneer[J].Int J Radiat Oncol Biolphys,1998,40(4):787-796
[63]张颖丽.一年生膜荚黄芪有效成分与拮抗环磷酰胺毒副作用研究[D].延边大学硕士学位论文.2011年.
[64]姜勇男,李燕,陆虹等.荷瘤小鼠免疫器官及免疫功能的研究[J].延边医学学院学报,1996,19(2)：70-72
[65]栗坤.前花青素和白芷生物碱抗宫颈癌作用及机理研究[D].燕山大学工学博士学位论文.
[66]Qiu DK,Ma X,Peng YS,et al.Sigificance of cyclooxygenase-2 expression in human primary hepatocallular carcinoma[J].World J Gastroenterol,2002,8(5):815-817
[67]刘亚玲,刑欣,刑凌霄,等.杂色曲霉素对小鼠胸腺和脾脏转录因子FoxP3+调节性T淋巴细胞的影响[J].中国药理学与毒理学杂志.2009,6(23)：188-194
[68]高丽,张肇铭,杨官娥.光合细菌制剂对荷瘤小鼠免疫功能的影响及抑瘤作用[J].山西医科大学学报.2004,35(1)：8-9.
[69]郑国兴,范成成,冷波,等.文蛤多肽粉对人癌细胞sMMc-7721的抑制作用及免疫调节作用[J].厦门大学学报.2008,47(2)：135-137.
[70]Komaki R,Milas L,Ro JY,et al.Prognostic biomarker study in pathologically Staged N1 non-small cell lung caneer[J].Int J Radiat Oncol Biolphys,1998,40(4):787-796.
[71]Kbyhi T,Ruan S,Jabbur JR,et al.Differentil p53 phosphoryaltion and activation of apoptosis-pormoting genes Bax and Fas/APO-1 by irradiation and ara-C treatment.Cell Death Differ,1998,5(7):584-591.
[72]Bonrer MM,Brousset P,Pafnner MB,etal.ExPression of apoptosis regulatory proteins of the Bcl-2 family and p53 in Primay reseeted non-small cell lung cancer.Br J Ccer,1999,79(5-6):952-958.
[2]崔桂敏,马少忠,杨华,等.肿瘤学[M].中国中医药出版社.2009：5-6.
[3]柴玉英,张向阳,李勇.我国人口主要疾病死因构成变化的影响因素[J]中国全科医学.2010,13(9)：1025-1027.
[4]谭永明.从中医角度认识癌症之浅见[J].中外健康文摘.2010,7(23)：336-337.
[5]李庆玲.谈中医药治疗肿瘤的特色及其作用[J].中医药报,2001,29(5)：223-233.
[6]黄修燕,黄自丽,晁愚,等.中药“松友饮”对高转移人肝癌侵袭性及小鼠免疫功能的影响[J].肿瘤.2008,28(6)：489-492.
[7]Huang xY, wang L, Huang zL, et al.Herbal extract"songyouYin"inhibits tumor growth and prolongs survival in nude mice bearing human hepatocellular carcinoma xenograft with high metastatic potential[J]. J callcer Res Clin OncoI.2009,135(9):1245-1255.
[8]汪海英,李福安,魏全嘉.抗肿瘤中药的研究现状[J].青海医学院学报.2008,29(4)：278-282.
[9]钱彦方.活血化瘀中药对肿瘤形成和转移的影响[J].中医杂志.2008,49(10)：942-944.
[10]Kerr Jf etal.Br J Cancer,1972,26:239-257
[11]黄清松,李红枝,张咏莉,等.三七皂苷Rgl抗突变和抗肿瘤研究短篇论著[J].临床和实验医学杂志.2006,5(8)：1124.
[12]吕淑华.黄芪多糖抗肿瘤作用研究进展[J].江西中医学院学报.2009,21(1)：85-87.
[13]闫凤,孟庆雄.常见的动物毒素诱导肿瘤细胞凋亡的分子机制[J].生物学杂志.2010,27(3)：71-74.
[14]柯尊金,丁心喜,董文奎,等.槲皮素对人膀胱癌BIU-87细胞增殖和凋亡的影响[J].实用癌症杂志.2008,23(2)：116-118.
[15]毕黎琦.中药天花粉蛋白对黑色素瘤细胞凋亡及凋亡周期的影响[J].中国中西医结合杂志.2008,18(1)：35.
[16]陈丽荣,郑枫,MC Willingham,等.紫杉醇诱发人乳癌细胞凋亡的机制研究[J].中华肿瘤杂志.2007,19(2)：103-106.
[17]郑元元.凋亡相关基因TRAIL、Bcl-2在非小细胞肺癌中的表达及临床意义研究[D]. 郑州大学硕士学位论文.2012年.
[18]KakarSS, RoyD. Curcumin inhibitsTPA induced expression c-fos,c-jun and c-myc protooncogenes messenger RNAs in mouse skin[J].CancerLett,1994; 87:81-89
[19]吴青,陈燕,李新刚,等.姜黄素影响Caspase-8和Caspase-9诱导淋巴瘤Raji细胞凋亡的研究[J].中国实验血液学杂志.2005,13(4)：624-627.
[20]Cecchinato V, Chiaramonte R, Nizzardo M.Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells[J]. Biochem Pharmacol. 2007,74(11):1568-1574
[21]冯磊,张连芬,严婷,等.中药虎杖中抗癌活性物质研究[J].中药材.2006,29(7)：689-691.
[22]李覃,王伟,陈虹,等.虎杖提取物白藜芦醇诱导肿瘤细胞凋亡的研究进展[J].武警医学院学报.2009,18(10)：886-888.
[23]MIHRAM, ERSTER S, ZAIKAA, et al.p53 has a directapoptogen-ic role at themito-chondria [J].MolCell,2003,11(7):577.
[24]Lin HY, Tang HY, Shih A, et al.Thyroid hormone is a MAPK-de-pendent growth factor for thyroid cancer cells and is anti-apoptotic.[J].Steroids.2007,72(2):180-187.
[25]袁静.檞皮素抗肿瘤作用研究进展[J].国外医学中医中药分册.1996,18(5)：3.
[26]李娜,胡玉红,孙盛梅,等.小檗碱诱导肿瘤细胞凋亡的研究进展[J].黑龙江医药科学.2007,30(1)：102-103.
[27]Kang BY, Chung SW, Cho D, et al. Involvement of p38 mitogen-activated protein kinase in the induction of interleukin-12 p40 production in mouse macrophages by berberine,abenzodioxoloquinolizinc alkaloid[J].Biochem Pharmacol.2002,63 (10):1901-1910
[28]马靖,符乃阳.甘草提取物体外选择性诱导几种人肿瘤细胞凋亡[J].癌症.2001,20(8)：806-811.
[29]Chia H W, Robert M, Shao C L, et al.Cobra cardiotoxin-inducedcell death in fetal rat cardiomycoytes and cortical neurons:diferent74pathway but similar cell surface target[J]. Toxlcon.2006(46):430-440.
[30]Wu HL, Hsu CY, Liu WH, et al.Berberine-induced apoptosisof human leukemia HL-60 cells is associated with down-regulation of nucleophosm in B23 and telomerase activitv[J].IntJ Cancer.2009,81 (6):923-929.
[31]王凤岐,吴大真.峨眉神效验方[M].科学普及出版社.1982：42.
[32]樊梅英,葛滢,关保华,等.明党参、峨参种群分布格局的比较研究[J].科技通报.2003,19(3)：203.
[33]Institute of Botany. Chinese Academy of Sciences. IconograPhia CornoPhytorum Simcorum[M]. Beijing:Science Press.1987:1055.
[34]马超英.基于细胞凋亡调节基因为靶点的峨参抗肿瘤活性成分筛选及其作用机理研究[J].基础研究.2012,3：21-24.
[35]Hong SM, et al. Immunological adjuvant containing extract from anthriscus sylvestris for activating immune system of host[J]. KR2006032925-A.
[36]Kozawa M, et al. Chem Pharm Bull.1978,23(4):1337-1338.
[37]王岳峰,耿耘,朱利平,等.峨参根化学成分研究[J].安徽农业科学.2009,37(23)：11001-11002.
[38]王淑丽.峨参活性成分的提取、分离与筛选[D].西南交通大学硕士研究生论文.2008年.
[39]刘玛菡.峨参质量标准研究[D].西南交通大学硕士研究生论文.2009年.
[40]Yeonjoong Yong, Soon Young Shin, Young Han Lee, Yoongho Lim, et al. Antitumor activity of deoxypodophyllotoxin isolated from Anthriscus sylvestris:Induction of G2/M cell cycle arrest and caspase-dependent apoptosis.Bioorg[J]. Med Chem. Lett.2009, (19):4367-4371.
[41]马超英,涂显琴,牛顺子等.峨参石油醚提取物对肺癌细胞A549和H460增殖的抑制作用观察[J].河北医药.2011,33(13)：1925—1926.
[42]李莉.峨参益气活血作用的实验研究[D].西南交通大学硕士研究生学位论文.2007年.
[43]Deoxypodophyllotoxin, flavolignan, from Anthriscus sylvestris Hoffm. inhibits migration and MMP-9 via MAPK pathways in TNF-a-induced HASMC S.-J. Suh et al. Vascular Pharmacology.2009,51:13-20.
[44]Korea, Meihua JIN,a Tae Chul MOON,b Zhejiu QUAN,a Eunkyung LEE,c Yun Kyung KIM,a Ju Hae YANG,aSeok-Jong SUH,d Tae Cheon JEONG,a Seung Ho LEE,a Cheorl-Ho KIM,*,d and Hyeun Wook CHANG*,a The Naturally Occurring Flavolignan, Deoxypodo- phyllotoxin, Inhibits Lipopoly saccharide-Induced iNOS Expression through the NF-k B Activation in RAW264.7 Macrophage Cells Biol. Pharm. Bull.2008,31 (7):1312-1315.
[45]耿耘,李莉,魏屹,等.峨参对实验性衰老小鼠作用的实验研究[J].时珍国医国药.2011,22(1)：144-145.
[46]马超英,李莉,耿耘,等.峨参对食醋所致脾虚小鼠的作用观察[J].四川中医.2008,26(9)：12.
[47]李莉,高雅蓉,马超英,等.峨参对血瘀模型大鼠血液流变性的影响[J].江西中医药.2007,38(6)：78-79.
[48]丁昭莉.峨参的毒性及其提取物抗氧化活性和免疫作用观察[D].西南交通大学硕士研究生学位论文.2011年.
[49]涂显琴.峨参提取物抗肿瘤活性的初步研究[D].西南交通大学硕士论文.2012年.
[50]Tang Y, Simoneau AR, Xie J,Shahandeh B, Zi X.Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53. Cancer Prevention Research,2008,1(6):439-451.
[51]谢鑫.超声微泡负载自杀基因HSV1_T_省略洛韦对前列腺癌细胞抑制效应的研究[D].重庆医科大学硕士学位论文.2012年.
[52]李胜.Skp2基因沉默对人肺癌细胞生物学活性的影响及机制研究[D].重庆医科大学博士学位论文.2008年..
[53]Zi X,Simoneau AR.Flavokawain A,a novelchalcone from kava extract,induces apoptosis in bladder cancer cells by involvementof Bax protein-dependent and mitochondria dependent apoptotic pathway and suppresses tumor growth in mice.Cancerf J]. Res 2005, 65:3479-3486.
[54]Tang Y,Li X,Liu Z,Simoneau AR,Xie J,Zi X.Flavokawain B,a kava chalcone,exhibits robust apoptotic mechanisms on androgen receptor negative, hormone-refractory prostate cancer cell lines and reduces tumor growth in a preclinical model[J]. International Journal of Cancer,2010,127(8):1758-1768.
[55]Herrmann M, Lorenz H M, Voll R, et a.l A rapid and simple method for the isolation of apoptotic DNA fragment[J].Nucleic Acids Res,1994,22(24):5506-5507.
[56]Hanahan,D and Weinberg,RA.The hallmarks of cancer[J].Cell,2000,100:57-70.
[57]张鑫.吴茱萸碱诱导人胃癌细胞SGC-7901细胞凋亡[J].哈尔滨商业大学学报自然科学版.2010,5.
[58]代光明,杜先智,梅同华.柴胡皂苷D对肺癌A-549细胞增殖、凋亡的影响及机制探讨[J].西南师范大学学报(自然科学版).2009,34(3)：75-78.
[59]叶明霞,周顺长,孔利佳,任大宏等.S180肉瘤,艾氏腹水瘤肿瘤移植模型建立的研究[J],中国实验动物学杂志,2002；12(1)：14-16.
[60]Carr BrendelVM arkov IC D, Ferrer K, et al Immunity tomur ine breast cancer cells modified to express MUC-1, human breast cancer antigen, in transgenic mice to lerant to human MUC1[J]. Cancer Rss,2000,60(9):2435-2443
[61]董媛.扶正抗癌方对H22腹水型瘤株移植瘤小鼠CyclinD1和Caspase_3表达影响的实验研究[D].陕西中医学院硕士学位论文.2011年.
[62]Komaki R,Milas L,Ro JY,et al.Prognostic biomarker study in pathologically Staged N1 non-small cell lung caneer[J].Int J Radiat Oncol Biolphys,1998,40(4):787-796
[63]张颖丽.一年生膜荚黄芪有效成分与拮抗环磷酰胺毒副作用研究[D].延边大学硕士学位论文.2011年.
[64]姜勇男,李燕,陆虹等.荷瘤小鼠免疫器官及免疫功能的研究[J].延边医学学院学报,1996,19(2)：70-72
[65]栗坤.前花青素和白芷生物碱抗宫颈癌作用及机理研究[D].燕山大学工学博士学位论文.
[66]Qiu DK,Ma X,Peng YS,et al.Sigificance of cyclooxygenase-2 expression in human primary hepatocallular carcinoma[J].World J Gastroenterol,2002,8(5):815-817
[67]刘亚玲,刑欣,刑凌霄,等.杂色曲霉素对小鼠胸腺和脾脏转录因子FoxP3+调节性T淋巴细胞的影响[J].中国药理学与毒理学杂志.2009,6(23)：188-194
[68]高丽,张肇铭,杨官娥.光合细菌制剂对荷瘤小鼠免疫功能的影响及抑瘤作用[J].山西医科大学学报.2004,35(1)：8-9.
[69]郑国兴,范成成,冷波,等.文蛤多肽粉对人癌细胞sMMc-7721的抑制作用及免疫调节作用[J].厦门大学学报.2008,47(2)：135-137.
[70]Komaki R,Milas L,Ro JY,et al.Prognostic biomarker study in pathologically Staged N1 non-small cell lung caneer[J].Int J Radiat Oncol Biolphys,1998,40(4):787-796.
[71]Kbyhi T,Ruan S,Jabbur JR,et al.Differentil p53 phosphoryaltion and activation of apoptosis-pormoting genes Bax and Fas/APO-1 by irradiation and ara-C treatment.Cell Death Differ,1998,5(7):584-591.
[72]Bonrer MM,Brousset P,Pafnner MB,etal.ExPression of apoptosis regulatory proteins of the Bcl-2 family and p53 in Primay reseeted non-small cell lung cancer.Br J Ccer,1999,79(5-6):952-958.