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1、福建医科大学 硕士学位论文 黄癸素联合用药的抗肿瘤疗效及抗肿瘤转移和抑制仓鼠颊癌作 用的研究 姓名：王希 申请学位级别：硕士 专业： 指导教师：林菁 1 黄癸素联合用药的抗肿瘤疗效及抗肿瘤转移和黄癸素联合用药的抗肿瘤疗效及抗肿瘤转移和 抑制仓鼠颊癌作用的研究抑制仓鼠颊癌作用的研究 中中 文文 摘摘 要要 目的：本研究观察黄癸素联合用药的体内和体外抗肿瘤作用；黄癸素抗肿瘤 转移作用及其机制；黄癸素对化学诱导的仓鼠颊癌的防治作用。 方法： （1）应用 MTT 法检测黄癸素联合临床常用药对体外培养肿瘤细胞增 殖的抑制作用。 （2）应用小鼠移植性实体瘤 H22、S180 的治疗试验观察黄癸素 联合 5
2、-FU 的体内抗肿瘤活性。 （3）通过琼脂糖凝胶电泳法测定黄癸素对 SW480 细胞 DNA 拓扑异构酶活性的影响。 （4）细胞-基质粘附实验检测黄癸素对 B16 细胞粘附能力的影响。（5） 划痕实验检测黄癸素对 B16 细胞迁移能力的影响。（6） 应用 Transwell 小室重建基底膜侵袭实验分析黄癸素对 B16 细胞趋化能力的影 响 。 （7） 免疫 S-P 法测定黄癸素对肿瘤细胞转移相关因子 CD44V6 表达的影响。 （8）明胶酶谱法测定黄癸素对肿瘤细胞分泌基质金属酶 MMP-2、MMP-9 表达 和活性的影响。 （9） 黄癸素对 B16 细胞 C57BL/6 小鼠实验性肺转移的影响
3、。 （10） 黄癸素对 H22 细胞小鼠实验性肺转移的影响。 （11）建立 DMBA 诱导的仓鼠颊 囊癌变的动物模型，于 6 周、9 周和 12 周系统观察黄癸素治疗组和对照组的局 部病变情况，实验结束进行病理学检查。 结果： 一、黄癸素联合用药的抗肿瘤疗效及相关机制 黄癸素联用羟喜树碱对 SGC-7901、SW116、HepG2、SW480 细胞具有显著 的协同抑制作用，与 5-FU 联用对 MGC-803、SGC-7901、SW116 细胞具有协同 抑制作用；与 5-FU 合用可显著抑制小鼠 H22、S180 实体瘤的在体生长；黄癸素 对拓扑异构酶都、的活性均有抑制作用。 二、黄癸素抗肿瘤
4、转移作用及其机制初探 黄癸素可抑制 B16 细胞与基质及内皮细胞的粘附，抑制作用呈浓度依赖性。 划痕实验中，在原始致伤区的基础上，用药组细胞在 Fn 上的迁移均受到明显抑 制。Transwell 小室，药物与细胞作用 48h 后，细胞穿越基底膜的能力显著低于 2 对照组，细胞侵袭数与药物浓度成反比。通过黄癸素作用的肿瘤细胞培养上清所 表达的 MMP-2、MMP-9 活性较对照组有显著的降低。在体试验显示黄癸素有一 定程度抑制 C57BL/6 小鼠黑色素瘤肺转移、小鼠 H22 肝癌肺转移的作用。 三、黄癸素对化学诱导仓鼠颊癌的抑制作用 黄癸素 36、54、72mg/kg 分别作用于 DMBA 诱
5、导的仓鼠，第 9 周开始高剂 量即有抑制病变发生的作用， 第 12 周病理检查见其颊粘膜的不典型增生发生率、 鳞癌发生率均有不同程度降低，54、72mg/kg 组仓鼠颊粘膜的不良增生总发生率 分别为 66.6%、53.3%，比模型组（86.6%）分别降低了 20%和 33.3%。 结论：黄癸素与 5-FU、羟喜树碱对体外培养的多种肿瘤细胞具有协同抑制 作用，可能与其对拓扑异构酶都、活性的抑制作用有关。在体试验表明黄癸 素与 5-FU 合用可显著抑制小鼠 H22、S180 实体瘤的在体生长。细胞试验显示黄 癸素可降低 B16 细胞的粘附、 侵袭和迁移能力。 在体试验显示黄癸素可抑制 B16 黑色
6、素肿瘤和 H22 肝癌的肺转移。黄癸素对化学诱导仓鼠颊癌有抑制作用。 关键词： 黄癸素 协同作用 拓扑异构酶 肿瘤转移 CD44V6 MMP 仓 鼠 颊癌模型 3 The synergistic antitumor effects of berberine -hydroxy-decanoylethyl sulfonate with other chemotherapeutic drugs and its antimetastatic activity as well as the inhibition on buccal cancer development in hamster Abstra
7、ct Objective：To explore the synergistic antitumor effects of HB（berberine -hydroxy -decanoylethyl sulfonate）with other chemotherapeutic drugs in vivo and in vitro， and its correlative mechanism；investigate the inhibition of HB on tumor metastasis and its mechanism；the inhibition of HB is to be evalu
8、ated on tumor development by buccal cancer hamster model. Methods：(1) MTT assay was employed to determine the cytotoxicity of HB combination with clinical chemotherapeutic drugs in tumor cells culture in vitro， such as HepG2, MGC80-3, SW480 cells. (2) HB was administrated to treat mice transplanted
9、solid tumors H22 and S180 with 5-FU. (3) The inhibitory effects on topoisomerases was measured by supercoiled DNA relaxation assay.(4) Cell-matrix adhesion assay was used to study the adhesive ability of B16 cells. (5) The effects of HB on B16 cellsmigration on Fibronection was observed by erasion t
10、race test. (6) Transwell chamber assay was applied to measure the effect of HB on invasion of B16 cells. (7) Detect the expression of CD44V6 in tumor cell by immunohistochemical technique( S - P method) (8) The serious activity of MMP-2 and-9 of B16 was measured by gelatin zymography. (9) Test the i
11、nfluence of HB on B16 cells of C57BL/6 mice experimental pulmonary metastasis. (10) Test the influence of HB on H22 cells of mice experimental pulmonary metastasis. (11) Through establishing the purpose of using hamster cheek model induced by DMBA, observe cancerous pathological changes in six weeks
12、, 9 weeks and 12 weeks to HB group and the modle 4 group .Probe into the effect of HB on dimethyI-benzanthracene DMBA-induced buccal macosa premalignant lesion in hamsters. Results: HB acted synergistically with HCPT to significantly inhibit in vitro culture cells （SGC-7901、SW116、HepG2、SW480）prolife
13、ration. HB and 5FU collaboratively could also inhibit those in vitro culture cells，and inhibit the growth of H22 and S180 solid tumors in mice. HB inhibited the activities of topoisomerase I and II. According to MTT method, different concentration of HB showed dose dependent inhibitory effects The r
14、esults showed that HB could inhibit B16 cells from moving on Fibronection effectively by scratches experiments. After 48 h, the invasion of B16 cells was less than those in control groups, and the number of invasive cells was dose dependent. The activity levels of both MMP 2 and MMP 9 in B16 treated
15、 with HB were significantly low than control. To a certain extent, HB could influence murine established pulmonary metastases of H22 hepatoma and B16 melanoma .Compare to the modle group, the incidence of buccal lesion and cancer induced by DMBA in hamster was significantly lower in the group treated with HB in the dose of 54 and 72mg/kg. Conclusion: HB have synergistic action with 5-FU（in vivo and in vitro） or HCPT（in vitro） on suppressing tumor proliferation. HB can inhibit topoisomerase activity. HB can restrain the adhesion and sport ability
