【作者】 董兰凤；

【导师】 吕占军；

【作者基本信息】 河北医科大学 ， 免疫学， 2004， 博士

【摘要】 恶性肿瘤一直是威胁人类生命健康的主要疾患。全世界每年死于癌症的患者有数百万人。虽然人们提出了包括手术治疗、放疗、化疗等多种治疗方法，但是由于受到适应症、副作用等因素的限制，对恶性肿瘤的治疗效果仍不理想。因此，采用多种途径并用，着眼于靶向治疗肿瘤的综合治疗方式，对肿瘤的治疗更具潜力。祖国医学对于癥瘕（肿瘤）的治疗一贯采取扶正和祛邪并用的原则， 即在杀灭肿瘤细胞的同时，也要提高机体的免疫功能，增强机体自身对疾病的抵御能力。化疗药阿霉素能将其分子嵌入DNA中，抑制核酸的合成，从而可以杀灭多种肿瘤，但同时也损伤机体正常组织。如何使阿霉素在肿瘤靶部位集中，减低其对机体正常组织的损伤，是发挥其治疗作用的关键。本研究以阿霉素靶向肿瘤治疗为中心，配合应用中药附子多糖或细胞因子rhIL-2，目的是在应用化疗药杀伤肿瘤的同时，设法保护和增强机体的免疫功能，从而达到更好的抗肿瘤效果。肿瘤发生后，主要表现为机体的免疫功能下降（T细胞，NK细胞功能降低），T细胞的增殖情况改变、细胞因子的产生与表达变化, 癌基因、抑癌基因的表达变化等。所以本研究以荷瘤小鼠为模型，在整体水平，从抑瘤率、生命延长率、淋巴细胞增殖、NK细胞杀伤活性、细胞因子表达、癌基因表达、诱导肿瘤细胞凋亡及组织病理学等方面探讨和评价了附子多糖、rhIL-2与阿霉素不同靶向制剂联合应用的抗肿瘤效果和抗肿瘤机制。同时观察了阿霉素的体内不同组织间的药代动力学变化。目的是寻找最佳的抗肿瘤综合治疗途径，有效地控制和杀灭肿瘤，并且最大程度地降低副作用。目的：观察附子多糖和附子酸性多糖对荷瘤小鼠肿瘤的抑制作用，探讨其抗肿瘤机制；观察附子多糖、rhIL-2与阿霉素白蛋白磁微球或阿霉素脂质体靶向治疗的抗肿瘤协同作用，研究综合治疗的抗肿瘤机制，确定最<WP=6>佳的综合治疗方式。方法：（1）药物制备：采用水提醇沉法提取附子多糖和附子酸性多糖，灌胃给药或腹腔注射给药；采用改良法制备阿霉素白蛋白磁微球，尾静脉注射给药；逆相蒸发法制备阿霉素脂质体、阿霉素长循环脂质体和阿霉素长循环热敏脂质体，尾静脉注射给药；（2）抑瘤实验：建立荷瘤小鼠模型，以荷瘤小鼠的肿瘤重量为指标观察药物的抗肿瘤活性并计算抑瘤率；以荷瘤小鼠的存活天数计算生存时间和生命延长率；（3）药代动力学测定：以高效液相色谱法（HPLC）测定静脉给药后24h内游离阿霉素以及各种阿霉素脂质体的药代动力学规律及组织学分布特征，观察阿霉素在小鼠体内的组织分布以及对肿瘤、心脏等不同组织的影响。（4）抗肿瘤机制分析：以乳酸脱氢酶释放法（LDH）测定NK细胞活性，MTT法测定淋巴细胞转化率，流式细胞术检测肿瘤细胞凋亡、肿瘤细胞的增殖指数及p53、Fas、FasL和Caspase-3表达，RT-PCR法测定脾细胞IL-2mRNA及IL-12mRNA的表达，制作病理切片观察肿瘤、心脏、肝脏和肾脏的组织病理学变化，探讨抗肿瘤机制。结果：（1）附子多糖和附子酸性多糖对H22荷瘤小鼠肿瘤有显著的抑制作用,灌胃给药的抑瘤率分别达到54.93%和52.03%,腹腔注射给药的抑瘤率分别达到50.80%和60.16%%。附子多糖灌胃给药对S180荷瘤小鼠的肿瘤也有明显的抑制作用, 抑瘤率达45.30%；两种多糖均显著延长了H22荷瘤小鼠的存活时间，其中附子多糖灌胃给药，使H22荷瘤小鼠的生命延长率达121.35%；两种多糖通过两种途径给药，均显著提高了H22荷瘤小鼠的NK细胞活性（p<0.01 or p<0.05），其中附子多糖中剂量灌胃给药的作用最佳。附子多糖灌胃给药，明显提高了H22和S180荷瘤小鼠的淋巴细胞转化率，而附子酸性多糖灌胃给药，仅仅显著提高了S180荷瘤小鼠的淋巴细胞转化率（p<0.01 or p<0.05）；两种多糖均使H22荷瘤小鼠的肿瘤细胞凋亡率明显增加（p<0.05）。电镜观察可见肿瘤细胞凋亡，表现为核异染色质边缘化，染色质浓缩，碎裂，形成凋亡小体。（2）阿霉素白蛋白磁微球外加磁场靶向治疗与非磁场靶向治疗比较，显著增强了抑瘤作用，抑瘤率分别为52.42%和29.03%。靶向治疗提高了荷瘤小鼠的NK细胞活性和淋巴细胞转化率（p<0.01 or p<0.05）；降低了肿瘤细胞的增殖指数，提高了肿瘤细胞凋亡率以及Fas、FasL的表达；增加了脾脏淋巴细胞细胞IL-2mRNA及IL-12mRNA的表达（p<0.01 or p<0.05）；病理学观察显示，阿霉素非<WP=7>靶向组心肌细胞肌横纹不清晰，而阿霉素白蛋白磁微球磁场靶向组，心肌细胞完整，细胞核清楚，心肌肌横纹清晰可见，肿瘤组织中可见更多的肿瘤细胞坏死、破碎；给药后肝脏、肾脏组织未见明显异常变化。（3）附子多糖与阿霉素白蛋白磁微球外加磁场靶向联合应用，进一步提高了抑瘤作用，抑瘤率高达62.10%；同时更提高了荷瘤小鼠的NK细胞活性和淋巴细胞转化率，增加了肿瘤细胞凋亡率以及Fas、FasL的表达，并促进脾脏淋巴细胞细胞高表达IL-2mRNA及IL-12mRNA（p<0.01 or p<0.05）；肿瘤组织内有多数淋巴细胞浸润并可见单核巨噬细胞。（4）阿霉素白蛋白磁微球磁场靶向治疗的同时，肿瘤内注射rhIL-2，与肿瘤内注射NS相比较，抑瘤率显著提高（分别为61.29%和46.77%）。肿瘤内注射rhIL-2的同时，阿霉素白蛋白磁微球磁场靶向与非磁场?更多还原

【Abstract】 Tumor is one of the main diseases that are threat to human health. Millions of people die from tumors every year. Although we have found different methods including surgery, radiation-therapy and chemotherapy to treat tumor disease, but the treatment effects are not perfect and also induces a multiplicity of side-effects. Therefore, it is necessary to find more efficient ways to control and cure tumors. One of the main methods is targeting therapy, but combining it with different therapeutic methods shows more potential. According to the Traditional Chinese Medicine Methodology, we use both “Fuzheng” and “Quxie” treatment methods combined on tumor. That means, while trying to eradicate the tumor cells, we should avoid injury the body immune function in-order to ensure the anti-tumor ability. Adriamycin is a chemotherapy drug that can eradicate many varieties of tumors when inserted into the tumor cell DNA molecule structure which inhibits the nucleic acid synthesizing. The key step in antitumor therapy is how to concentrate adriamycin only on tumor site while reducing the body side-effects. In this research, we use Monkshood Polysaccharide or rhIL-2 combined with different adriamycin targeting therapy agents to study the cooperative antitumor effects.Tumors cause decline in immune responses，which is detrimental to T cell and the NK cell functions。Tumors induce changes on the T cell proliferation, the cytokine production and expressions, the oncogenes expressions. In this study, we used tumor-bearing mice as the model, observed the antitumor effect (inhibitory rate), the life prolongation rate, the lymphocyte proliferation, the NK cells killing activity, the cytokine and oncogenes expressions, the activation-induced tumor cells apoptosis, the tissue pathology of different organs and so forth and so on, in-order to <WP=11>compare the antitumor effects of different treatment methods and understand their antitumor mechanism, to find more effective antitumor multipurpose methods. Objective: To investigate the antitumor effect of Monkshood polysaccharide (MPS) and acid Monkshood polysaccharide (AMPS) on mice transplanted with the tumor cell lines H22 and S180, explore their antitumor mechanism; To observe the cooperative inhibitory effects of Monkshood polysacchsride (MPS) or rhIL-2 with adriamycin magnetic albumin microspheres (ADM-MAM) or Adriamycin Liposome targeting therapy on established murine adenocarcinoma (H22) tumor, study the antitumor mechanism of multipurpose methods. Methods: (1) Drugs preparation: The MPS and AMPS were obtained from Monkshood by water extraction and alcohol precipitation. The mice with H22 and S180 tumor were given MPS or AMPS by oral peros (op) or by intraperitoneal injection (ip); The ADM-MAM was prepared by an improved method and was given to H22 tumor-bearing mice by tail vein injection; The adriamycin liposome (AL), adriamycin long circulating liposome (ALCL) and adriamycin long circulating temprerature-sensitive liposome (ALTSL) were prepared by reverse-phase evaporation method and were given to H22 tumor-bearing mice by tail vein injection. (2) Antitumor experiment: We used tumor-bearing mice as a modle: the antitumor activity was observed using the tumor weight as index, the life prolongation rate was observed according to the tumor-bearing mice survival days; (3) Pharmacokinetics determination: The tissue distribution of adriamycin in different categories of liposome was determined by HPLC method with in 24h after injection, the adriamycin concentration in heart and tumor were obseved. (4) Analysis on antitumor mechanism: The activity of natural killer (NK) cells and the lymphocyte transformation capacity were examined by the LDH and MTT methods respectively, the apoptosis of tumor cells and the expressions of p53, Fas, Fas-L and Caspase-3 were examined by flow cytometry (FCM), the expressions of splenic cell IL-2mRNA and <WP=12>IL-12mRNA were determined by RT-PCR, the tumor, heart, liver, kidney tissue pathology slices were made by HE coloration method. R更多还原