淫羊藿苷逆转耐氨甲喋呤肺癌A549细胞转移表型的研究
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摘要
第一部分:氨甲蝶呤耐药A549细胞株的建立及其生物学特征
目的:诱导并建立耐氨甲蝶呤对映体的A549细胞株并观察耐药细胞系(L-(+)-MTX/A549、D-(-)-MTX/A549)的生物学特性。
方法:以MTX对映体为诱导剂,采用浓度递增结合低剂量持续诱导方法诱导A549细胞株,建立MTX不同对映体耐药细胞系;倒置相差显微镜观察细胞形态变化; MTT法绘制细胞生长曲线;MTT法检测耐药细胞株的耐药指数;流式细胞仪检测细胞周期和细胞的分裂增殖能力。
结果:采用浓度递增结合低剂量持续诱导方法,建立了MTX不同对映体对A549细胞的耐药细胞株,L-(+)-MTX/A549、D-(-)-MTX/A549耐药指数分别为6.0的和20.2。倒置相差显微镜观察细胞形态发生了改变;细胞生长曲线显示D-(-)-MTX/A549的增值略慢于亲本细胞,而L-(+)-MTX/A549的增值最慢;流式细胞仪检测细胞周期结果显示L-(+)-MTX/A549、D-(-)-MTX/A549耐药细胞株S期细胞数量减少(P < 0. 05),G0 / G1期细胞增多( P < 0. 05);CFSE检测A549、L-(+)-MTX/A549、D-(-)-MTX/A549的MFI分别为6.08±0.55、7.72±0.30、6.90±0.18。两对映体细胞株间有明显手性差异。
结论:本研究建立了MTX两种对映体耐药细胞株并观察了耐药株细胞的生物学特性,为进一步研究氨甲蝶呤两种对映体的耐药机制提供了一种实验模型。
第二部分:淫羊藿苷逆转耐氨甲喋呤肺癌A549细胞转移表型的研究
目的:研究中药淫羊藿苷(icariine,ICA)作用MTX肺癌A549耐药细胞后对细胞转移表型的影响,初步探讨ICA逆转A549/MTX耐药细胞转移表型的作用机制及对肺癌的治疗价值。
方法:采用MTT法检测ICA对A549/MTX耐药细胞的半数抑制浓度(IC50);采用双层软琼脂克隆形成实验检测A549/MTX组、A549/MTX+ICA组细胞的克隆形成率并观察其集落形态;细胞划痕实验检测A549/MTX组、A549/MTX+ICA组细胞的迁移能力;Transwell小室实验检测细胞侵袭能力变化;RT-PCR检测A549/MTX细胞和A549/MTX +ICA细胞中c-myc、nm23-H1基因mRNA水平的变化。
结果:MTT结果显示无毒剂量的ICA与MTX联合应用后A549/MTX细胞的IC50值为35.50±1.85μmol/L,比单独应用MTX(同等剂量)后A549/MTX细胞的IC50值(52.17±2.25μmol/L)有了一定程度的下降;软琼脂实验发现A549/MTX+ICA组细胞克隆形成率为0.94±0.088,小于A549/MTX组细胞的1.56±1.065(P<0.05);划痕实验显示72 h后A549/MTX组细胞的迁移能力大于A549/MTX+ICA组细胞(P<0.05);Transwell小室实验显示A549/MTX组细胞的穿膜细胞数明显多于A549/MTX+ICA组细胞(P<0.05),说明A549/MTX+ICA组细胞的侵袭浸润能力小于A549/MTX组细胞;A549/MTX+ICA组细胞相对于A549/MTX组细胞,nm23-h1的mRNA表达水平明显升高,c-myc的mRNA表达明显减少。
结论:中药ICA具有逆转A549/MTX耐药细胞转移表型的作用。
Part A: Establishment of Methotrexate-resistant A549Cell Line of Human NSCCL and Its Biological Characteristics
Objective: To establish methotrexate(MTX)-resistant human NSCCL A549 cell line derived from the A549 cell line and observe its biological characters.
Methods: A549 cells were exposed to intermittently and progressively increasing concentration of MTX. MTX-resistant human NSCCL A549 cell line was established.The morphology was observed by inverted phase contrast microscope, The sensitivity of the A549/MTX cell line to MTX was measured by cycotoxicity test. The cell growth curve was determined by MTT assay.
Results: The MTX-resistant human A549 cell line was established. The A549/MTX resistance index was 6.0. Inverted phase contrast microscope showed the chang of A549/MTX. Cell growth curve demonstrated that the proliferation abilities of A549/MTX cell was decreased significantly compared with that of A549.
Conclusion: We establish MTX-resistant human A549 cell line and observe its biological characteristics. It provides a new experimental model for further studying the mechanism of MTX-resistance.
Part B: Icariin reversed metastatic phenotype of methotrexate-resistant lung cancer A549 cells
Objective:To investigate the effect of traditional Chinese medicine (TCM) icariin(ICA) on metastatic phenotype of methotrexate (MTX)–resistant lung cancer A549 cells and elucidate the action mechanism and therapeutic value of ICA.
Methods:The half inhibition concentration (IC50 ) value of ICA in inhibiting the growth of A549/MTX cells was measured by MTT assay.The colony formation rate and the morphology of cell cluster of A549/MTX and ICA-treated A549/MTX cells were determined by douber-layer soft agar colony formation assay.The migration abilities of A549/MTX and ICA-treated A549/MTX cells were evaluated by cell scratch assay.The invasion ability of cells was tested by using Transwell chamber assay.The change of mRNA levels of nm23-h1 and c-myc genes were detected by Semi-quantitative RT-PCR.
Results:MTT assay showed that the IC50 value of non-toxic ICA plus MTX was reduced compared with that induced by equal dose of MTX (35.50±1.85μmol/L vs 52.17±2.25μmol/L). The colony formation rate of ICA-treated A549/MTX cells was 0.94±0.088, less than that of A549/MTX cells (1.56±1.065,P<0.05). Cell scratching assay demonstrated that the migration capability of A549/MTX cells was stronger than that of ICA-treated A549/MTX cells at 72h (P<0.05). Transwell experiment revealed that more A549/MTX cells passed through artificial basement membrane than ICA-treated A549/MTX cells (P<0.05),indicating that the invasion capbility of ICA-treated A549/MTX cells was weaker than that of A549/MTX cells. Semi-quantitative RT-PCR showed that the expression of nm23-h1 mRNA of ICA-treated A549/MTX cells increased in compare with that of A549/MTX cells,on the contray, an decreasing tendency in the expression of c-myc mRNA was observed after treated with ICA.
Conclusion: TCM ICA can reverse the metastatic phenotype of MTX–resistant A549 cells.
目的:诱导并建立耐氨甲蝶呤对映体的A549细胞株并观察耐药细胞系(L-(+)-MTX/A549、D-(-)-MTX/A549)的生物学特性。
方法:以MTX对映体为诱导剂,采用浓度递增结合低剂量持续诱导方法诱导A549细胞株,建立MTX不同对映体耐药细胞系;倒置相差显微镜观察细胞形态变化; MTT法绘制细胞生长曲线;MTT法检测耐药细胞株的耐药指数;流式细胞仪检测细胞周期和细胞的分裂增殖能力。
结果:采用浓度递增结合低剂量持续诱导方法,建立了MTX不同对映体对A549细胞的耐药细胞株,L-(+)-MTX/A549、D-(-)-MTX/A549耐药指数分别为6.0的和20.2。倒置相差显微镜观察细胞形态发生了改变;细胞生长曲线显示D-(-)-MTX/A549的增值略慢于亲本细胞,而L-(+)-MTX/A549的增值最慢;流式细胞仪检测细胞周期结果显示L-(+)-MTX/A549、D-(-)-MTX/A549耐药细胞株S期细胞数量减少(P < 0. 05),G0 / G1期细胞增多( P < 0. 05);CFSE检测A549、L-(+)-MTX/A549、D-(-)-MTX/A549的MFI分别为6.08±0.55、7.72±0.30、6.90±0.18。两对映体细胞株间有明显手性差异。
结论:本研究建立了MTX两种对映体耐药细胞株并观察了耐药株细胞的生物学特性,为进一步研究氨甲蝶呤两种对映体的耐药机制提供了一种实验模型。
第二部分:淫羊藿苷逆转耐氨甲喋呤肺癌A549细胞转移表型的研究
目的:研究中药淫羊藿苷(icariine,ICA)作用MTX肺癌A549耐药细胞后对细胞转移表型的影响,初步探讨ICA逆转A549/MTX耐药细胞转移表型的作用机制及对肺癌的治疗价值。
方法:采用MTT法检测ICA对A549/MTX耐药细胞的半数抑制浓度(IC50);采用双层软琼脂克隆形成实验检测A549/MTX组、A549/MTX+ICA组细胞的克隆形成率并观察其集落形态;细胞划痕实验检测A549/MTX组、A549/MTX+ICA组细胞的迁移能力;Transwell小室实验检测细胞侵袭能力变化;RT-PCR检测A549/MTX细胞和A549/MTX +ICA细胞中c-myc、nm23-H1基因mRNA水平的变化。
结果:MTT结果显示无毒剂量的ICA与MTX联合应用后A549/MTX细胞的IC50值为35.50±1.85μmol/L,比单独应用MTX(同等剂量)后A549/MTX细胞的IC50值(52.17±2.25μmol/L)有了一定程度的下降;软琼脂实验发现A549/MTX+ICA组细胞克隆形成率为0.94±0.088,小于A549/MTX组细胞的1.56±1.065(P<0.05);划痕实验显示72 h后A549/MTX组细胞的迁移能力大于A549/MTX+ICA组细胞(P<0.05);Transwell小室实验显示A549/MTX组细胞的穿膜细胞数明显多于A549/MTX+ICA组细胞(P<0.05),说明A549/MTX+ICA组细胞的侵袭浸润能力小于A549/MTX组细胞;A549/MTX+ICA组细胞相对于A549/MTX组细胞,nm23-h1的mRNA表达水平明显升高,c-myc的mRNA表达明显减少。
结论:中药ICA具有逆转A549/MTX耐药细胞转移表型的作用。
Part A: Establishment of Methotrexate-resistant A549Cell Line of Human NSCCL and Its Biological Characteristics
Objective: To establish methotrexate(MTX)-resistant human NSCCL A549 cell line derived from the A549 cell line and observe its biological characters.
Methods: A549 cells were exposed to intermittently and progressively increasing concentration of MTX. MTX-resistant human NSCCL A549 cell line was established.The morphology was observed by inverted phase contrast microscope, The sensitivity of the A549/MTX cell line to MTX was measured by cycotoxicity test. The cell growth curve was determined by MTT assay.
Results: The MTX-resistant human A549 cell line was established. The A549/MTX resistance index was 6.0. Inverted phase contrast microscope showed the chang of A549/MTX. Cell growth curve demonstrated that the proliferation abilities of A549/MTX cell was decreased significantly compared with that of A549.
Conclusion: We establish MTX-resistant human A549 cell line and observe its biological characteristics. It provides a new experimental model for further studying the mechanism of MTX-resistance.
Part B: Icariin reversed metastatic phenotype of methotrexate-resistant lung cancer A549 cells
Objective:To investigate the effect of traditional Chinese medicine (TCM) icariin(ICA) on metastatic phenotype of methotrexate (MTX)–resistant lung cancer A549 cells and elucidate the action mechanism and therapeutic value of ICA.
Methods:The half inhibition concentration (IC50 ) value of ICA in inhibiting the growth of A549/MTX cells was measured by MTT assay.The colony formation rate and the morphology of cell cluster of A549/MTX and ICA-treated A549/MTX cells were determined by douber-layer soft agar colony formation assay.The migration abilities of A549/MTX and ICA-treated A549/MTX cells were evaluated by cell scratch assay.The invasion ability of cells was tested by using Transwell chamber assay.The change of mRNA levels of nm23-h1 and c-myc genes were detected by Semi-quantitative RT-PCR.
Results:MTT assay showed that the IC50 value of non-toxic ICA plus MTX was reduced compared with that induced by equal dose of MTX (35.50±1.85μmol/L vs 52.17±2.25μmol/L). The colony formation rate of ICA-treated A549/MTX cells was 0.94±0.088, less than that of A549/MTX cells (1.56±1.065,P<0.05). Cell scratching assay demonstrated that the migration capability of A549/MTX cells was stronger than that of ICA-treated A549/MTX cells at 72h (P<0.05). Transwell experiment revealed that more A549/MTX cells passed through artificial basement membrane than ICA-treated A549/MTX cells (P<0.05),indicating that the invasion capbility of ICA-treated A549/MTX cells was weaker than that of A549/MTX cells. Semi-quantitative RT-PCR showed that the expression of nm23-h1 mRNA of ICA-treated A549/MTX cells increased in compare with that of A549/MTX cells,on the contray, an decreasing tendency in the expression of c-myc mRNA was observed after treated with ICA.
Conclusion: TCM ICA can reverse the metastatic phenotype of MTX–resistant A549 cells.
引文
1.Jolivet J, Cowan KH, Curt GA, et al. The pharmacology and clinical use of methotrexate[J]. N Engl J Med. 1983; 309(18): 1094-104.
2.Schilsky RL. Methotrexate: an effective agent for treating cancer and building careers. The polyglutamate era[J]. Stem Cells. 1996; 14(1): 29-32.
3.Evans WE, Relling MV, Rodman JH, et al. Conventional compared with individualized chemotherapy for childhood acute lymphoblastic leukemia. N Engl J Med. 1998; 338(8):499-505.
4.Ackland SP, Schilsky RL.. High-dose methotrexate: a critical reappraisal[J]. J Clin Oncol. 1987; 5(12):2017–31.
5.Evans WE, Crom WR, Abromowitch M, et al. Clinical pharmacodynamics of high-dose methotrexate in acute lymphoblastic leukemia: identification of a relation between concentration and effect[J]. N Engl J Med. 1986; 314(8):471-7.
6.Niemeyer CM, Gelber RD, Tarbell NJ, et al. Low-dose versus high-dose methotrexate during remission induction in childhood acute lymphoblastic leukemia (Protocol 81-01 update) [J]. Blood. 1991; 78(10):2514-9.
7.陈静,顾龙君,应大明,等.急性白血病氨甲蝶呤耐药及化疗个体化初探[J].中华血液学杂志.. 2002; 23(1): 35-6.
8.Cesana GC, Romano F, Piacentini G, et al.Low-dose interleukin-2 administered pre-operatively to patients with gastric cancer activates peripheral and peritumoral lymphocytes but does not affect prognosis[J]. Ann Surg Oncol. 2007;14(4):1295-1304.
9.徐珊,朱利群,罗莉,等.人绒毛膜癌JAR/VP16多药耐药细胞株的建立及相关基因表达检测[J].南京医科大学学报(自然科学版).2006,26(7):485-90
10.Snow K,Judd W. Characterisation of adriamycin- and amsacrine-resistant human leukaemic T cell lines[J].Br J Cancer. 1991;63(1):17-28
11.Beck JF,Brugger D,Brischwein K,et al.Anticancer drug-mediated induction of multidrug resistance-associated genes and protein kinase C isozymes in the T-lymphoblastoid cell line CCF-CEM and in the blasts from patients with acute lymphoblastic leukemias[J].Jpn J Cancer Res. 2001;92(8):896-903
12 .Goldie JH,Coldman AJ.Application of theoretical models to chemotherapy protocol design[J].Cancer Treat Rep.1986;70(1):127-131
13.王建军,赵晖,林峰,等.耐甲氨蝶呤人骨肉瘤细胞株建立及其生物学特性[J].肿瘤.2007;27(8):623-7
14.陈公琰,杨朝阳,洪璇,等.人肺腺癌耐药细胞模型的建立及生物学特性的初步鉴定[J].中华医学杂志.2007;87(13):924-6
15.Perrotton T, Trompier D, Chang XB,et al.(R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1[J]. J Biol Chem. 2007 ;282(43):31542-8.
16.Shen S, He Y, Zeng S.Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers[J]. Chirality. 2007;19(6):485-90
1 SCHILSKY R L. Methotrexate:an effective agent for treating cancer and building careers.The polyglutamate era[J]Oncologist,1996,14(1):29-32.
2 YIN X X,CHEN Z Q,LIU Z J,etal. Icariine stimulates proliferation and differentia- tion of human osteoblasts by increasing production of bone morphogenetic protein
2[J].Chin Med J(Engl),2007,120(3):204-210.
3陶绍能,何晓东,董林,等.氨甲喋呤对映体耐药A549细胞株的建立及其生物学特征[J].肿瘤防治研究, 2009,36(4):273-277.
4谢娟平,孙文基.淫羊藿属植物药物化学成分及药理研究进展[J].海峡医学, 2006,18:17-21.
5 ZHAO Y,ZHANG L,GUI Z Y,et al. Studies on the immuno modulatory action of Icariin[J]. Clin Tradit Herb Drugs,1996,27(3):669-672.
6 DING L,LIANG X G,ZHU D Y,etal. Icariin promotes expression of PGC-1alpha, PPARalpha, and NRF-1 during cardiomyocyte differentiation of murine embryonic stem cells in vitro[J].Acta Pharmacol Sin,2007,28(10): 1541-1549.
7.董林,何晓东,陶绍能,等.甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究[J].肿瘤,2009,28(5):404-408.
8鄂征.组织培养与分子细胞技术[M].北京:北京出版社,1995:151-161.
9钱立平,范钰,陈坚,等. RNAi沉默PRL-3基因对大肠癌细胞侵袭的抑制[J]世界华人消化杂志,2008, 16(7): 767-770.
10 SIEG D J,HAUCK C R,SCHLAEPFER D D.Required role of focal adhesion kinase(FAK) forintegrin- stimulated cell migration[J].CellSci,1999,112(16): 2677-2691.
11张建,徐永健,熊维宁,等.转染IkBα基因抑制NF-kB活性对A549细胞侵袭行为的影响[J].癌症,2008,27(7):710-715.
12 M arcu KB,Bo ssone SA ,Patel AJ. Myc function and regulation[J].Annu Rev Biochem,1992,61: 809-814
13 Amin G,Wangner A I,Hav N. Sequence2specific transcrip tionalactivation by myc and repression by Max[J].Mo l Cell Bio,1993,13: 383-393
14 Noguchi M,H irohashi S,Hara F,et al. Heterogenousamp lification of myc fam- ily oncogenes in small cell lungcarcinoma[J].Cancer,1990,66: 2053-2059
15 Steeg PS,Bevilacqua G,Kopper L,et al. Evidence for a novelgene associated with low tumor metastatic potential[J].Natl Caner,Inst,1998,80: 200-206
16 Po stel EH,Berberich SJ,F lint SJ,et al. Human c-myc transcription factor PuF identified as nm23-h1 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis[J]. Science,1993,261 (7) : 478-483
1谢娟平,孙文基.淫羊藿属植物化学成分及药理研究进展[J].海峡药学,2006; 18: 17-21
2葛林阜,董政军,姜国胜,等.淫羊藿苷对急性早幼粒白血病细胞端粒酶活性的影响[J].中国肿瘤生物治疗杂志,2002,9(1) : 36-38.
3 Zhao Y,Zhang L,Cui ZY,et al. Studies on the immuno modulatory action of Icariin[J]. Chin Tradit Herb Drugs,1996,27 ( 3) :669-672.
4王天然,邢善田,周金黄.淫羊藿苷促进抗体生成的作用[J].药学通报,1987, 9 :5331-5336
5李书桐,李铁军,郑钦岳,等.淫羊藿苷对小鼠体外脾脏细胞增殖及产生集落刺激因子样活性的影响[J ].第二军医大学学报,1995,16 (4) :3401-3405
6葛林阜,董政军,姜国胜,等.淫羊藿苷对小鼠免疫调节作用的研究[J].中华实用医学,2001,3 (3) :251-254
7毕可红,张玉昆,葛林阜,等.淫羊藿苷对受照小鼠免疫与造血功能的影响[J].中国辐射卫生,2001,10 (2) :1041-1046
8李晓冰,金东庆,郭良君,等.淫羊藿苷在辐射小鼠免疫学功能恢复中作用的研究[J].中国辐射卫生,2002,11(3) :1711-1714
9赵勇,张玲,王芸,等1淫羊藿苷的体外免疫调节作用研究[J ].中草药,1996, 27 (11) :6691-6695
10曹颖瑛,郑钦岳,张国庆,等.淫羊藿苷促进小鼠脾细胞IL-3 mRNA及IL–6 mRNA的表达[J].第二军医大学学报,1998,19 (2) :1991-1996
11 Liu TH ,Wang BX ,Wang Y , et al.Effect of icariin and it s metabolites on the production of cytokines by THP -1 cells[J].Acta Pharmaceutica Sinica,2000, 35(4) :2451-2457
12李晓燕,张玲,王芸,等.淫羊藿苷逆转转化生长因子对LA K、CD3AK细胞的免疫抑制作用[J].中国免疫学杂志,2000,16 (5) :266-271
13赵勇,张玲,崔正言等.淫羊藿甙对HL-60细胞增殖与分化的影响[J].中国药理学通报,1996,12(1):52-54
14赵勇,崔正言,张玲,等.淫羊藿甙对人急性早幼粒白血病细胞分化的影响[J]中华肿瘤杂志,1997,19(1):53-55.
15葛林阜,董政军,姜国胜,等.淫羊藿苷对急性早幼粒白血病细胞端粒酶活性的影响[J]中国肿瘤生物治疗杂志,2002,9(1) : 36-38.
16李贵新,张玲,王芸,等.淫羊藿苷诱导白血病细胞凋亡及其对癌基因表达的影响[J]中华血液学杂志,2002,23 (6) :322-323
17毛海婷,张玲,王芸,温培娥,等.淫羊藿甙对人高转移肺癌细胞膜的影响[J]中药材,1999,12(1):65-68
18 Lee MK ,Choi YJ ,Sung SH , et al 1Antihepatotoxic activity of icariin , a major constit uent of Epimedium koreanum[J]Planta Med,1995,61 (6) :5231-5235
19毛海婷,张玲,王芸等.ICA和PJA对高转移性人肺癌细胞体外侵袭转移能力抑制的研究[J]中国免疫学杂志,2001;17(1):27-29
20 Kuroda M,Mimaki Y,Sashida Y,et al.Flavonol glycosides from Epimedium sagit tatum and t heir neurite outgrowth activit y on PC12 cells[J ]Planta Med,2000,66 (6) :575-562.
21 Zhang YW,Morita I,Shao G,et al.Screening of anti-hypoxia/ reoxygenation agents by an in vitro model.Part1:Natural inhibitors for protein t yrosine kinase activated by hypoxia/ reoxygenation in cult ured human umbilicalvein endot helial cells [J]Planta Med,2000,66(2) :1141-1147
22 Galizia G, Ferraraccio F, Lieto E, etal.DeVita F. p53 downregulation and Metallo thionein overexpression in gastric cancer patients are associated with a poor survival rate[J]Surg Oncol,2006; 93: 241-252
23王敏,刘崇铭,张宝凤.淫羊藿苷扩张脑血管作用的研究[J].沈阳药学院学报,1992 ,8 (4) :2721-2726
24关利新,衣欣,杨履艳,等.淫羊藿苷扩血管作用机制的研究[J].中国药理学通报,1996,12 (4) :3201-3207
25王伟,张涛,赵明镜,等. 5种中药黄酮对血管平滑肌细胞凋亡的交互作用[J].北京中医药大学学报,2000;23(4):18-21
2.Schilsky RL. Methotrexate: an effective agent for treating cancer and building careers. The polyglutamate era[J]. Stem Cells. 1996; 14(1): 29-32.
3.Evans WE, Relling MV, Rodman JH, et al. Conventional compared with individualized chemotherapy for childhood acute lymphoblastic leukemia. N Engl J Med. 1998; 338(8):499-505.
4.Ackland SP, Schilsky RL.. High-dose methotrexate: a critical reappraisal[J]. J Clin Oncol. 1987; 5(12):2017–31.
5.Evans WE, Crom WR, Abromowitch M, et al. Clinical pharmacodynamics of high-dose methotrexate in acute lymphoblastic leukemia: identification of a relation between concentration and effect[J]. N Engl J Med. 1986; 314(8):471-7.
6.Niemeyer CM, Gelber RD, Tarbell NJ, et al. Low-dose versus high-dose methotrexate during remission induction in childhood acute lymphoblastic leukemia (Protocol 81-01 update) [J]. Blood. 1991; 78(10):2514-9.
7.陈静,顾龙君,应大明,等.急性白血病氨甲蝶呤耐药及化疗个体化初探[J].中华血液学杂志.. 2002; 23(1): 35-6.
8.Cesana GC, Romano F, Piacentini G, et al.Low-dose interleukin-2 administered pre-operatively to patients with gastric cancer activates peripheral and peritumoral lymphocytes but does not affect prognosis[J]. Ann Surg Oncol. 2007;14(4):1295-1304.
9.徐珊,朱利群,罗莉,等.人绒毛膜癌JAR/VP16多药耐药细胞株的建立及相关基因表达检测[J].南京医科大学学报(自然科学版).2006,26(7):485-90
10.Snow K,Judd W. Characterisation of adriamycin- and amsacrine-resistant human leukaemic T cell lines[J].Br J Cancer. 1991;63(1):17-28
11.Beck JF,Brugger D,Brischwein K,et al.Anticancer drug-mediated induction of multidrug resistance-associated genes and protein kinase C isozymes in the T-lymphoblastoid cell line CCF-CEM and in the blasts from patients with acute lymphoblastic leukemias[J].Jpn J Cancer Res. 2001;92(8):896-903
12 .Goldie JH,Coldman AJ.Application of theoretical models to chemotherapy protocol design[J].Cancer Treat Rep.1986;70(1):127-131
13.王建军,赵晖,林峰,等.耐甲氨蝶呤人骨肉瘤细胞株建立及其生物学特性[J].肿瘤.2007;27(8):623-7
14.陈公琰,杨朝阳,洪璇,等.人肺腺癌耐药细胞模型的建立及生物学特性的初步鉴定[J].中华医学杂志.2007;87(13):924-6
15.Perrotton T, Trompier D, Chang XB,et al.(R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1[J]. J Biol Chem. 2007 ;282(43):31542-8.
16.Shen S, He Y, Zeng S.Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers[J]. Chirality. 2007;19(6):485-90
1 SCHILSKY R L. Methotrexate:an effective agent for treating cancer and building careers.The polyglutamate era[J]Oncologist,1996,14(1):29-32.
2 YIN X X,CHEN Z Q,LIU Z J,etal. Icariine stimulates proliferation and differentia- tion of human osteoblasts by increasing production of bone morphogenetic protein
2[J].Chin Med J(Engl),2007,120(3):204-210.
3陶绍能,何晓东,董林,等.氨甲喋呤对映体耐药A549细胞株的建立及其生物学特征[J].肿瘤防治研究, 2009,36(4):273-277.
4谢娟平,孙文基.淫羊藿属植物药物化学成分及药理研究进展[J].海峡医学, 2006,18:17-21.
5 ZHAO Y,ZHANG L,GUI Z Y,et al. Studies on the immuno modulatory action of Icariin[J]. Clin Tradit Herb Drugs,1996,27(3):669-672.
6 DING L,LIANG X G,ZHU D Y,etal. Icariin promotes expression of PGC-1alpha, PPARalpha, and NRF-1 during cardiomyocyte differentiation of murine embryonic stem cells in vitro[J].Acta Pharmacol Sin,2007,28(10): 1541-1549.
7.董林,何晓东,陶绍能,等.甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究[J].肿瘤,2009,28(5):404-408.
8鄂征.组织培养与分子细胞技术[M].北京:北京出版社,1995:151-161.
9钱立平,范钰,陈坚,等. RNAi沉默PRL-3基因对大肠癌细胞侵袭的抑制[J]世界华人消化杂志,2008, 16(7): 767-770.
10 SIEG D J,HAUCK C R,SCHLAEPFER D D.Required role of focal adhesion kinase(FAK) forintegrin- stimulated cell migration[J].CellSci,1999,112(16): 2677-2691.
11张建,徐永健,熊维宁,等.转染IkBα基因抑制NF-kB活性对A549细胞侵袭行为的影响[J].癌症,2008,27(7):710-715.
12 M arcu KB,Bo ssone SA ,Patel AJ. Myc function and regulation[J].Annu Rev Biochem,1992,61: 809-814
13 Amin G,Wangner A I,Hav N. Sequence2specific transcrip tionalactivation by myc and repression by Max[J].Mo l Cell Bio,1993,13: 383-393
14 Noguchi M,H irohashi S,Hara F,et al. Heterogenousamp lification of myc fam- ily oncogenes in small cell lungcarcinoma[J].Cancer,1990,66: 2053-2059
15 Steeg PS,Bevilacqua G,Kopper L,et al. Evidence for a novelgene associated with low tumor metastatic potential[J].Natl Caner,Inst,1998,80: 200-206
16 Po stel EH,Berberich SJ,F lint SJ,et al. Human c-myc transcription factor PuF identified as nm23-h1 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis[J]. Science,1993,261 (7) : 478-483
1谢娟平,孙文基.淫羊藿属植物化学成分及药理研究进展[J].海峡药学,2006; 18: 17-21
2葛林阜,董政军,姜国胜,等.淫羊藿苷对急性早幼粒白血病细胞端粒酶活性的影响[J].中国肿瘤生物治疗杂志,2002,9(1) : 36-38.
3 Zhao Y,Zhang L,Cui ZY,et al. Studies on the immuno modulatory action of Icariin[J]. Chin Tradit Herb Drugs,1996,27 ( 3) :669-672.
4王天然,邢善田,周金黄.淫羊藿苷促进抗体生成的作用[J].药学通报,1987, 9 :5331-5336
5李书桐,李铁军,郑钦岳,等.淫羊藿苷对小鼠体外脾脏细胞增殖及产生集落刺激因子样活性的影响[J ].第二军医大学学报,1995,16 (4) :3401-3405
6葛林阜,董政军,姜国胜,等.淫羊藿苷对小鼠免疫调节作用的研究[J].中华实用医学,2001,3 (3) :251-254
7毕可红,张玉昆,葛林阜,等.淫羊藿苷对受照小鼠免疫与造血功能的影响[J].中国辐射卫生,2001,10 (2) :1041-1046
8李晓冰,金东庆,郭良君,等.淫羊藿苷在辐射小鼠免疫学功能恢复中作用的研究[J].中国辐射卫生,2002,11(3) :1711-1714
9赵勇,张玲,王芸,等1淫羊藿苷的体外免疫调节作用研究[J ].中草药,1996, 27 (11) :6691-6695
10曹颖瑛,郑钦岳,张国庆,等.淫羊藿苷促进小鼠脾细胞IL-3 mRNA及IL–6 mRNA的表达[J].第二军医大学学报,1998,19 (2) :1991-1996
11 Liu TH ,Wang BX ,Wang Y , et al.Effect of icariin and it s metabolites on the production of cytokines by THP -1 cells[J].Acta Pharmaceutica Sinica,2000, 35(4) :2451-2457
12李晓燕,张玲,王芸,等.淫羊藿苷逆转转化生长因子对LA K、CD3AK细胞的免疫抑制作用[J].中国免疫学杂志,2000,16 (5) :266-271
13赵勇,张玲,崔正言等.淫羊藿甙对HL-60细胞增殖与分化的影响[J].中国药理学通报,1996,12(1):52-54
14赵勇,崔正言,张玲,等.淫羊藿甙对人急性早幼粒白血病细胞分化的影响[J]中华肿瘤杂志,1997,19(1):53-55.
15葛林阜,董政军,姜国胜,等.淫羊藿苷对急性早幼粒白血病细胞端粒酶活性的影响[J]中国肿瘤生物治疗杂志,2002,9(1) : 36-38.
16李贵新,张玲,王芸,等.淫羊藿苷诱导白血病细胞凋亡及其对癌基因表达的影响[J]中华血液学杂志,2002,23 (6) :322-323
17毛海婷,张玲,王芸,温培娥,等.淫羊藿甙对人高转移肺癌细胞膜的影响[J]中药材,1999,12(1):65-68
18 Lee MK ,Choi YJ ,Sung SH , et al 1Antihepatotoxic activity of icariin , a major constit uent of Epimedium koreanum[J]Planta Med,1995,61 (6) :5231-5235
19毛海婷,张玲,王芸等.ICA和PJA对高转移性人肺癌细胞体外侵袭转移能力抑制的研究[J]中国免疫学杂志,2001;17(1):27-29
20 Kuroda M,Mimaki Y,Sashida Y,et al.Flavonol glycosides from Epimedium sagit tatum and t heir neurite outgrowth activit y on PC12 cells[J ]Planta Med,2000,66 (6) :575-562.
21 Zhang YW,Morita I,Shao G,et al.Screening of anti-hypoxia/ reoxygenation agents by an in vitro model.Part1:Natural inhibitors for protein t yrosine kinase activated by hypoxia/ reoxygenation in cult ured human umbilicalvein endot helial cells [J]Planta Med,2000,66(2) :1141-1147
22 Galizia G, Ferraraccio F, Lieto E, etal.DeVita F. p53 downregulation and Metallo thionein overexpression in gastric cancer patients are associated with a poor survival rate[J]Surg Oncol,2006; 93: 241-252
23王敏,刘崇铭,张宝凤.淫羊藿苷扩张脑血管作用的研究[J].沈阳药学院学报,1992 ,8 (4) :2721-2726
24关利新,衣欣,杨履艳,等.淫羊藿苷扩血管作用机制的研究[J].中国药理学通报,1996,12 (4) :3201-3207
25王伟,张涛,赵明镜,等. 5种中药黄酮对血管平滑肌细胞凋亡的交互作用[J].北京中医药大学学报,2000;23(4):18-21