【作者】 肖义军；

【导师】 陈元仲；

【作者基本信息】 福建医科大学 ， 内科学， 2007， 博士

【摘要】 恶性肿瘤严重困扰人类健康。由于目前临床常用抗肿瘤药物大多存在严重的毒副作用以及肿瘤细胞耐药性的问题,不断寻找新的高效低毒的抗肿瘤药物一直是抗肿瘤研究的重要课题。从植物中寻找天然抗肿瘤活性成分是研究抗肿瘤药物的一条重要途径,目前临床上常用的抗肿瘤药物如紫杉醇、长春新碱、喜树碱、足叶乙苷、三尖杉酯碱均来源于植物。除了从植物中分离单一结构的药物成分外,传统的植物药物近几年来在德国、法国、意大利和瑞士等欧洲国家也得到了科学界的承认和应用。由于肿瘤发生发展的多因性,针对肿瘤发生发展过程中的多种分子靶标、多种信号途径进行肿瘤的联合治疗可能会比单一药物成分的治疗有更好的治疗效果。而植物药的特点正是药效物质多成分、多途径、多靶点的整体作用。将抗肿瘤植物药作为传统化疗药物的增效减毒剂应用于恶性肿瘤的治疗是今后抗肿瘤研究的重要方向之一。Nispex是我们首次从寄主为夹竹桃的红花桑寄生叶中提取分离的主要含多酚类成分一种抗肿瘤植物提取物。文章分四个部分对其制备方法、成分、体内外抗白血病细胞株HL-60的效果、抑瘤机制、抑瘤作用的广谱性等进行了研究,还研究对比了不同寄主来源红花桑寄生提取物的体外抑瘤效果。第一部分为Nispex的制备方法和成分分析。Nispex系寄主为夹竹桃的红花桑寄生叶80%乙醇提取物经聚酰胺柱层析纯化所得,按药材重量计得率约为3%。采用颜色鉴定反应、高效液相-质谱联用分析和资料对比分析,Nispex中主要含槲皮素、槲皮苷、广寄生苷等黄酮类成分和与夹竹桃成分类似的强心苷类成分以及生物碱类成分。以芦丁为对照品测得的黄酮含量为72.37%。第二部分为本文的重点研究部分。测定了Nispex对小鼠的近似LD50,在此基础上以人急性髓系白血病细胞株HL-60为模型,研究了Nispex的体内外抑瘤效果及其与多柔比星联用的体内抑瘤效果,并探讨了其可能的抑瘤作用机制。用MTT法和集落形成实验检测了Nispex对HL-60细胞增殖的抑制活性。Nispex显著地抑制HL-60细胞增殖,表现出明显的量效关系和时效关系。药物作用48 h的IC50值为0.54μg/ml。集落形成实验表明Nispex对肿瘤增殖细胞群作用更为敏感。Nispex对正常人骨髓单个核细胞无抑制作用。体内抑瘤效果研究以HL-60裸鼠移植瘤为模型,连续给药14 d,以相对瘤体积和瘤重的变化来评估药物的抑瘤效果。Nispex 10 mg/kg/d瘤内给药按相对体积计算的抑制率(肿瘤生长抑制率)为59.4%,按瘤重计算的抑制率(抑瘤率)为45.3%(P<0.05);10 mg/kg/d腹腔给药的肿瘤生长抑制率为18.3%,抑瘤率为15.5%(P>0.05);30 mg/kg/d腹腔给药肿瘤生长抑制率为60.6%,抑瘤率为31.2%(P<0.05);Nispex 10 mg/kg/d腹腔给药与多柔比星15 mg/kg(尾静脉一次性给药)联用的肿瘤生长抑制率为94.4%,抑瘤率为92.9%(P<0.01)。进一步研究了Nispex与多柔比星联用的体内协同抑瘤效果,以药物相互作用指数(CDI)来评价药物联合作用疗效,判断标准:CDI>1,CDI=1和CDI<1分别表示两药拮抗,相加和协同,当CDI<0.7(P<0.05)时,表示协同作用非常显著。Nispex 10 mg/kg/d腹腔给药(连续给药9 d)与15 mg/kg多柔比星(尾静脉一次性给药,下同)联用对HL-60移植瘤有较好的抑制作用,抑瘤率89.0%,CDI为0.43(P<0.01),协同作用非常显著;与10 mg/kg的多柔比星联用抑瘤率为56.9%,CDI值为0.80(P<0.05),协同作用显著;与5 mg/kg的多柔比星联用抑瘤率为8.0%,CDI为1.51。病理检查各主要脏器无明显异常。Nispex对小鼠的近似LD50为126.81 mg/kg,主要表现为心脏毒性。采用AO/EB荧光染色、AnnexinⅤ-FITC/PI双标记流式细胞术检测、DNA倍体分析及细胞周期分析、TdT酶介导的原位缺口标记法和细胞DNA片段化检测证明Nispex对肿瘤细胞的杀伤作用主要表现为诱导细胞凋亡,研究结果还显示Nispex抑制HL-60细胞增殖主要是通过将细胞周期阻滞于G0/G1期。EMSA分析、Western Blot分析和免疫荧光分析显示Nispex是一种天然NF-κB抑制剂。Nispex的抗肿瘤活性可能与其抑制肿瘤细胞核中NF-κB的异常激活及抑制肿瘤细胞分泌VEGF有关。第三部分研究了Nispex的广谱抗肿瘤作用。Nispex对小鼠肉瘤细胞株S180、小鼠黑色素瘤细胞株B16等4种鼠源肿瘤细胞株和小鼠胚胎成纤维细胞株3T3等均无细胞毒活性。而Nispex对所检测的人小细胞肺癌细胞株NCI-H446、人宫颈癌细胞株Hela、人T淋巴细胞白血病Jurkat、人急性早幼粒白血病细胞株NB4、人慢性粒细胞白血病细胞株K562、人T淋巴细胞白血病细胞株Molt4、人鼻咽癌细胞株CNE和人骨髓瘤细胞株U266等8种人源肿瘤细胞株均有较强的细胞毒作用,抑制细胞增殖和诱导细胞凋亡,表现出明显的量效关系和时效关系,其作用72 h时的IC50值在0.1~2.5μg/ml之间。对原代培养的慢性粒细胞白血病细胞也有良好的抑制作用,其作用72 h时的IC50值为2.48μg/ml。Nispex对有轻微致瘤性的人胚肾成纤维细胞株293T有抑制细胞增殖的作用,但未发现存在明显的量效关系,即使高达50μg/ml的浓度也不明显诱导其凋亡。细胞集落形成实验表明,与HL-60中的情况一样,Nispex对NB4和Jurkat中的增殖细胞群作用更为敏感。第四部分以培养的HL-60细胞为模型,比较了寄主分别为夹竹桃、桑树、无患子和桂花的红花桑寄生提取物的体外抑瘤效果。夹竹桃寄生抑制肿瘤细胞增殖的效果最好,桑树寄生抑制肿瘤细胞增殖的效果次之,无患子寄生又次之,桂花寄生在检测的浓度范围内基本无抑制作用。以上的研究证明,寄主为夹竹桃的红花桑寄生叶提取物Nispex是一种有效的抗肿瘤植物提取物,对人源肿瘤细胞有较好的选择性。与肿瘤化疗药物多柔比星联用有很好的协同效果。其抗肿瘤作用可能与其抑制肿瘤细胞核中异常激活的NF-κB活性和抑制肿瘤细胞分泌VEGF相关。更多还原

【Abstract】 Therioma is one of the most serious diseases that troubled human beings health greatly. The treatment of cancer with chemotherapeutic agents and radiation has two major problems: time-dependent development of tumor resistance to therapy (chemoresistance and radioresistance) and nonspecific toxicity toward normal cells. So, the search for new agents that effectively kill cancer cells, but that have minimal or no side effects on normal cells is always the aim of anticancer drug researchers. About 70% of the therapeutic drugs in use today are derived from plants. Paclitaxel, vincristine, camptothecin, etoposide and harringtonine are typical anticancer drugs that generally used in clinical therapy derived from plants. Except these individual component drugs, traditional herbal medicinal products have been commonly accepted and applied in European Union countries such as Germany, France, Switzerland and Italy et al. The herbalists have access to hundreds of years of observational data on the anticancer activity of many herbs. Laboratory studies are confirming the knowledge that is already documented in traditional texts.The induction, promotion and progression of cancer is a multistepprocess that involves biochemical interactions from the level of the genes, through cell-signaling pathways, intercellular communication mechanisms, supply of nutrients, channels for metastases, and a host immune response. An integrative approach for managing a patient with cancer should target the multiple biochemical and physiological pathways that support tumor development while minimizing normal tissue toxicity. Traditional herbal medicinal products always contain a range of complex organic chemicals that may have synergistic activity. Given the multiple effects of these agents, their future use for cancer therapy probably lies in synergistic combination. During active cancer therapy, they should generally be evaluated in combination with chemotherapy and radiation. In this role, they may act as biological response modifiers potentially enhancing the efficacy of the so-called conventional therapies and protecting from therapy-associated toxicities.Research of the last few years has shown that many plant products exhibit chemopreventive effect on carcinogenesis. Many plant-derived polyphenols have been studied intently for their potential chemopreventive properties and are pharmacologically safe. Recent research has suggested that these plant polyphenols might be used to sensitize tumor cells to chemotherapeutic agents and radiation therapy by inhibiting pathways that lead to treatment resistance. These agents have also been found to be protective from therapy-associated toxicities.Nispex is an extract mainly composed by polyphenols of Scurrula parasitica L., a Chinese medicinal herb, which parasitized on Nernium indicum Mill. . It inhibits human cancer cells proliferation and induces human cancer cells apoptosis, but not murine cancer cells.In this doctoral dissertation, we described the preparation method of Nispex, analyzed its components preliminarily, and studied its anticancer effects in vitro and in vivo, its mechanisms of anticancer. The anticancer effects of extracts of Scurrula parasitica L. parasitized on other host plant had compared here, also.PartⅠ: in this part, the preparation method of Nispex was described. Leaves of Scurrula parasitica L. was extracted by 80% ethanol. And the extracts were purified by polyamides column chromatography further, the eluates of 30%, 50%, 70% and 90% ethanol were mixed being called Nispex (means extracts of Scurrula parasitica L. on Nernium indicum Mill.). Colour reactions indicted that Nispex consisted mainly of flavonoids, cardiac glycosides and alkaloids. Nispex contained 72.37% flavonoids, determined by colorimetry with rutin as the reference substance. There were about 30 components in Nispex by LC-MS analysis, including rutin, hyperoside, quercetin, quercitroside, kaempferol, genistin, liquidamboside, epicatechingallate, avicularin, liquiritin, daidzein4, 7-diglucoside, adynerin and oleandrin derivative, odoroside H, uzarigenin, et al.PartⅡ: In this part, the LD50 value of Nispex to mice was detected by 126.81 mg/kg. Pathological examination showed that the major toxicity presented in heart, cardiac muscle cells of the sudden death mice caused by Nispex becoming swollen and disturbance. The anticancer effect of Nispex in vitro was studied by MTT assay and colony forming assay with human acute myeloid leukemia cell line HL-60. Nispex inhibited HL-60 cells proliferation significantly in time and dose dependent manner, the IC50 was 0.54μg/ml when treated 48 hours. Colony forming assay indicted that proliferating-cell population were more sensitive to Nispex. Xenograft of HL-60 was established by subcutaneous implantation of cultured HL-60 cells in BALB/c nude mice. Tumor growth inhibition in these mice was used to evaluate the anticancer activity of drugs in vivo. Mice used in these experiments were randomly divided into six groups: control group, ADR 15 mg/kg group, 30 mg/kg/day Nospex-ip group, 10 mg/kg/day Nospex-ip group, 10 mg/kg/day Nospex-it group, 10 mg/kg/day Nospex-ip combined with ADR 15 mg/kg group. ADR were injected through caudal vein once only at the first day; Nospex treatment, 14 days. Tumor growth inhibition was evaluated by relative tumor volume (RTV) and tumor weight (TW). HL-60 grafts of 30 mg/kg/day Nospex-ip were inhibited by 60.6% according to RTV, 31.2% according to TW(P<0.05); grafts of 10 mg/kg/day Nospex-it were inhibited by 59.4% according to RTV, 45.3% according to TW(P<0.05); and grafts of ADR wre inhibited by 65.2% (RTV), 60.4% (TW), grafts of 10 mg/kg/day Nospex-ip combined with ADR were inhibited by 94.4% according to RTV, 92.9% according to TW(P<0.01), the coefficient of drug interaction (CDI) was 0.24<0.7, meaning the synergistic effect was very significant. Enhancement of Adriamycin by Nispex was explored further by 10 mg/kg/day Nospex-ip with ADR 15mg/kg, ADR 10 mg/kg and ADR 5 mg/kg. The inhibition of tumor growth by ADR was enhanced significantly combined by Nispex 10 mg/kg/day. CDI was 0.43 when combined with ADR 15 mg/kg, 0.80 when combined with ADR 10mg/kg. Apoptotic HL-60 cells induced by Nispex were detected by staining with AO/EB, flow cytometry analysis, TUNEL assay and DNA fragmentation analysis. The cell cycles were mainly arrested at G0/G1 after treated by Nispex. Nispex was confirmed to be a natural inhibitor of NF-κB pathway by EMSA, Western blot and immunofluorescence assay. VEGF levels were reduced significantly in the supernatant of HL-60 cell cultures treated by Nispex with ELISA .PartⅢ: In this part, MTT assay and colony forming assay was used to detected the inhibition of cell proliferation by Nispex on 8 human cancer cells (human acute lymphoblastic leukemia cell line Molt4, human small cell lung cancer cell line NCI-H446, human myeloma cell line U266, human acute promyelocytic leukemia cell line NB4, human chronic myelocytic cell line K562, human nasopharyngeal carcinoma cell line CNE, human cervical carcinoma cell line Hela, human T cell leukemia cell line Jurkat ) , primary human chronic myelocytic cells, and 5 murine cancer cell lines. Nispex inhibited human cancer cells proliferation significantly in time and dose dependent manner. But Nispex had no cytotoxicity on murine cancer cells. Nispex also inhibited 293T cells (a human embryoic kidney cell line) proliferation, but not showing dose dependent manner, Nispex did not induce 293T cells apoptosis as high as 50.0μg/ml drug concentration.PartⅣ: In this part, MTT assay was used to compare the effects of inhibiting human cancer cells proliferation by extracts of Scurrula parasitica L. from four diffenent host, Nernium indicum Mill., Morus alba L., Opsmanthus fragrans Lours., and Sapindus mulorossi Gaertn. . Extract of Scurrula parasitica L. parasitized on Nernium indicum Mill. was the most effective, and extract of Scurrula parasitica L. parasitized on Morus alba L. taken the second place. Extract of Scurrula parasitica L. parasitized on Opsmanthus fragrans Lours. had no effectiveness even if as high as 50.0μg/ml drug concentration.Conclusion: Nispex, an extract of Scurrula parasitica L. parasitized on Nernium indicum Mill. , has effective anticancer activity in vitro and in vivo; it selectively kills human cancer cells, but not murine cancer cells. Nispex enhanced Adriamycin anticancer effects significantly. Nispex’s anticancer effects may be partially ascribed to the inhibition of activation of NF-kB and suppression of VEGF secretion.更多还原