【作者】 娄金丽；

【导师】 邱全瑛； 林洪生；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2004， 博士

【摘要】 肿瘤转移是一个多因素参与、多步骤的复杂过程,其中肿瘤细胞与血管内皮细胞的粘附、肿瘤新生血管生成是肿瘤血道转移的关键步骤。本着中医治疗肿瘤“扶正与祛邪相结合”的基本原则,以“扶正培本、清热解毒”为治疗法则,根据临床实践经验和前人研究的结果,选用具有抗肺癌作用的威麦宁、抗多种肿瘤作用的小檗碱和具有免疫调节作用的黄芪多糖等三种成分组成中药复方,在整体、细胞、分子及基因水平上研究它的抗肺癌细胞增殖及抗肺癌转移作用,并深入探讨其作用的分子机制,为抗肿瘤转移药物的研制和开发及临床抗肿瘤转移治疗提供实验和理论依据。 本研究分三部分: 1)复方威麦宁各组成药体外抗肿瘤作用的研究:为抗肺癌药物——复方威麦宁的药物组方提供实验依据。 2)复方威麦宁抗肺癌体内实验:①复方威麦宁对小鼠 Lewis 肺癌移植瘤的抑制作用:建立小鼠 Lewis 肺癌移植瘤模型,观察用药后小鼠体重和抑瘤率的变化,并检测药物对肿瘤细胞周期的影响;②复方威麦宁抗小鼠 Lewis 肺癌移植瘤自发性肺转移作用及其机制研究:观察复方威麦宁对小鼠 Lewis 肺癌移植瘤自发性肺转移灶形成的影响,并检测药物对瘤细胞粘附分子表达、瘤组织血管生成的影响等。 3)复方威麦宁抗肺癌体外实验:①复方威麦宁对 PG、PAa 肺癌细胞增殖的抑制作用,并深入探讨其对肿瘤细胞周期的影响;②复方威麦宁体外抗肿瘤转移的机制研究:检测复方威麦宁对高转移肺癌细胞(PG)与血管内皮细胞的粘附作用,观察其对 PG 细胞与 HUVEC 表面粘附分子表达的影响及其对肿瘤新生血管生成作用的影响等。 整体动物实验分为六组:生理盐水组(NS)、小檗碱组(Ber)、威麦宁低剂量组(WML)、威麦宁高剂量组(WMH)、复方威麦宁组(FWN)和环磷酰胺组(CTX)。 体外培养 PG 细胞分为五组:空白对照组(Control)、小檗碱组(Ber)、威麦宁组(WMN)、复方威麦宁低剂量组(FWL)和复方威麦宁高剂量组(FWH)。 体外培养 HUVEC 细胞分为六组:空白对照组(Control)、TNF-α对照组(TNF-α)或 bFGF 对照组(bFGF)、小檗碱组(Ber)、威麦宁组(WMN)、复方威麦宁低剂量组(FWL)和复方威麦宁中剂量组(FWM)。实验结果如下:1 复方威麦宁各组成药体外抗肿瘤作用 采用 MTT 法检测复方威麦宁各组成药对所选用的 PG、PAa、A529、BEL-7402、MGC-803 和 B16 等六种肿瘤细胞体外增殖作用,结果显示:小檗碱(20、40μg/ml)、<WP=5>2 复方威麦宁抗肺癌作用及其机制研究威麦宁(125、250μg/ml)对所选用的六种肿瘤细胞具有不同程度的抑制作用,与对照组相比差异显著(P<0.01),而黄芪多糖(10、20μg/ml)的抑制作用不明显。2 复方威麦宁抗肺癌体内实验 2.1 复方威麦宁对小鼠 Lewis 肺癌移植瘤的抑制作用及其机制研究 建立小鼠Lewis肺癌移植瘤模型,灌胃给药观察复方威麦宁对小鼠Lewis肺癌移植瘤生长的影响。结果显示:口服威麦宁 120-200 mg/kg d对小鼠Lewis肺癌的抑制率为 33.59%-40.63%,口服小檗碱 20g/kg抑瘤率为 15.09%,而口服复方威麦宁的抑瘤率提高到 47.66%,说明此三种药合用有协同抑瘤作用。流式细胞仪检测,复方威麦宁可使瘤细胞阻滞在G0-G1期,与生理盐水对照组相比差异显著(P<0.01)。免疫组化法和Western blotting 技术检测,复方威麦宁可显著降低瘤细胞Cyclin D1的表达(P<0.01)。结果提示:复方威麦宁可能通过降低细胞周期蛋白Cyclin D1的表达,使小鼠Lewis肺癌细胞阻滞在G0-G1期,发挥抗肿瘤作用。2.2 复方威麦宁抗小鼠 Lewis 肺癌移植瘤自发性肺转移作用及其机制研究2.2.1 复方威麦宁对自发性肺转移灶形成的影响 复方威麦宁组小鼠肺转移灶小而少,肺转移发生率为 60%,与对照组相比差异显著(P<0.01),说明复方威麦宁对荷 Lewis 肺癌小鼠肺转移有明显抑制作用。2.2.2 复方威麦宁抗小鼠 Lewis 肺癌移植瘤自发性肺转移作用机制2.2.2.1 复方威麦宁对瘤组织粘附分子表达的影响 免疫组化、RT-PCR、荧光定量 PCR 等方法检测复方威麦宁对瘤组织粘附分子表达的影响,结果表明:复方威麦宁能明显提高瘤组织细胞 E-cadherin 蛋白阳性表达率(55.6%),与 NS 组(33.3%)相比 P<0.01;显著提高瘤组织细胞 E-cadherin mRNA的表达量(P<0.01);同时复方威麦宁能明显降低瘤组织细胞 CD44 蛋白表达水平(P<0.01);降低瘤组织细胞 CD44 mRNA 和 CD44V6 mRNA 表达水平(P<0.01,P<0.05)。结果提示:复方威麦宁抗自发性肺转移作用可能与其增加瘤组织细胞 E-cadherin 的表达和抑制 CD44、CD44V6 的表达有关。2.2.2.2 复方威麦宁对瘤组织血管生成的影响 瘤组织经 HE 染色后镜下观察,可见 NS 组肿瘤细胞呈片状或巢状分布且生长旺盛,癌巢间微血管丰富,复方威麦宁组瘤组织及其周围微血管减少。应用抗 CD34 抗体对瘤组织进行免疫组化法检测,复方威麦宁能显著抑制瘤组织微血管的形成(P<0.01),与瘤组织 HE 染色形态学观察结果相一致。结果提示:复方威麦宁抗自发性肺转移作用可能与其抑制瘤组织血管生成有关。3 复方威麦宁抗肺癌体外实验3.1 复方威麦宁对 PG、PAa 肺癌细胞增殖的抑制作用 MTT法检测结果证明复方威?更多还原

【Abstract】 The process of tumor metastasis consists of many factors and many steps. Adhesionbetween tumor cells and endothelial cells and tumor angiogenesis are the critical steps of tumorblood metastasis. According to the theory of traditional Chinese medicine, clinical practices andpast investigative results, we chose WeiMaiNing,berberine and HuangQi polysaccharide, whichhas the anti-lung cancer, anti-various tumors and immune regulation function, respectively, andconstituted the Chinese medicine compound prescription(FWN). The effect of FWN onanti-proliferation, anti-metastasis in lung cancer and molecular mechanism were investigated invivo and vitro, so as to provide the academic bases for anti-metastasis drugs researching,development and applying in clinic. This study consists of three parts: 1) The effect of each constitute medicine of FWN on inhibiting tumor cells in vitro, whichprovides the experimental bases for constituting prescription of FWN. 2) Anti-lung cancer experiment in vivo: ① The effect of FWN on inhibiting murine Lewislung carcinoma in vivo: To establish murine Lewis lung carcinoma (3LL) transplant model,observe changes of the body weight and inhibitory rate after FWN treatment, and detect theeffect of FWN on cell cycle.② The effect and molecular mechanism of FWN on suppressingspontaneous lung metastasis in 3LL in vivo: To observe the effect of FWN on spontaneous lungmetastasis focus formation, and detect the effect of FWN on adhesion molecule expression intumor cell and on tumor angiogenesis. 3) Anti-lung cancer experiment in vitro:① The effect of FWN on anti-proliferation inPG,PAa: To investigate the effect on cell cycle. ② The effect and molecular mechanism ofFWN on anti-tumor metastasis in vitro: To observe the effect of FWN on adhesion between highmetastasis lung cancer cell(PG) and endothelial cells, detect the effect of FWN on adhesionmolecule expression in PG, HUVEC and on tumor angiogenesis. The animal experiment was divided randomly into six group: nomal saline solution(NS),Berberine(Ber),WeiMaiNing lower dose(WML),WeiMaiNing higher dose(WMH),compoundprescription of WeiMaiNing(FWN) and Cyclophosphamide(CTX). The cultured PG cell was divided into five group: control, Berberine(Ber),WeiMaiNing(WMN), compound prescription of WeiMaiNing lower dose(FWL) and compoundprescription of WeiMaiNing higher dose(FWH).The cultured HUVEC cell was divided into six<WP=8>英文摘要 5group: It included TNF-α(or bFGF) group besides above five group in PG.The results displayed as follow:1 The effect of each constitute medicine of FWN on inhibiting tumor cells in vitro Berberine(20、40μg/ml)and WeiMaiNing(125、250μg/ml)displayed inhibitory effecton six tumor cells chose at different degree by MTT, but HuangQi polysaccharide had not effecton them.2 Anti-lung cancer experiment in vivo2.1 The effect and molecular mechanism of FWN on inhibiting murine Lewis lung carcinomain vivo Inhibitory rate of WMN taken orally in 3LL was from 33.59% to 40.63% at a dosageranging 120~200mg/kg?d. Inhibitory rate of berberine taken orally was 15.09% at 20g/kg d.Inhibitory rate of FWN taken orally was increased greatly up to 47.66%.The result indicates thatthis three drugs cooperate with each other in inhibitory effect. FWN could block 3LL cells inG0-G1 phase by Flow cytometry(P<0.01). FWN could significantly decrease Cyclin D1expression in 3LL cells by immunohistochemistry staining and Western blotting(P<0.01).2.2 The effect and molecular mechanism of FWN on suppressing spontaneous lung metastasisin 3LL in vivo2.2.1 The effect of FWN on spontaneous lung metastasis focus formation In FWN group, lung metastasis focus was smaller and fewer, formation rate of spontaneouslung metastasis focus was 60%(P<0.01).This indicates that FWN has obviously inhibitoryeffect on lung metastasis in 3LL.2.2.2 The molecular mechanism of FWN on suppressing spontaneous lung metastasis in 3LL2.2.2.1 The更多还原