【作者】 张承科；

【导师】 闫兵；

【作者基本信息】 山东大学 ， 药物化学， 2014， 博士

【摘要】 第一部分二氧化钛纳米材料对小鼠哺乳期的毒性研究近年来,纳米技术逐渐成为推动世界各国经济发展的主要驱动力之一。在未来新型纳米材料和纳米技术有望广泛应用于信息、环保、能源、生物医学、制造等领域,为上述领域带来技术变革。因此,其重要程度受到了越来越广泛的关注,世界各国纷纷制定了符合本国利益的纳米科技发展计划。近年来,随着纳米技术从基础研究领域向应用研究领域和相关产业的转化,一些纳米商品已经投放市场。自2006年以来,世界范围内已经有1317种纳米产品出现在消费品市场。纳米技术在环保、生物医学等与人们健康相关领域的应用日益广泛,如在环保领域,纳米技术用于清除水中有毒、难降解的有机污染物,治理空气污染,在生物医学方面实现重大疾病的早期诊断以及低毒高效的靶向治疗等。这就加大了人类接触不同种类纳米材料的可能性。然而,纳米材料对环境是否存在危害?与人体接触是否会产生难以预期的不良后果?在纳米尺度下,物质具有小尺寸效应、表面效应、量子尺寸效应和宏观量子隧道效应。这就使得纳米材料与人体接触后可能产生与常规大尺寸物质不同的生物效应。纳米材料的尺寸、化学组成、表面修饰以及暴露途径等都是影响纳米材料生物效应的重要因素。在这种背景下诞生的纳米毒理学为纳米材料的安全使用提供了依据,为纳米科技的发展起到指引作用。根据已有报道,二氧化钛纳米材料可以对脑、肺、肝脏造成损伤,甚至有致癌的可能。二氧化钛纳米材料能够通过嗅神经突触进入嗅球并迁移至大脑,分布于大脑的不同部位,并且可以在脑部滞留,诱发脑部氧化应激和炎症反应水平增高,导致小鼠嗅球和海马神经元形态发生改变；还可以引起肝细胞坏死、凋亡和纤维化。哺乳是生殖过程的延续。人类的哺乳期持续时间较长,母乳为婴儿提供其健康发育所需的营养素,并含有一些能保护婴儿免受儿童常见病侵袭的抗体。女性哺乳期间的用药可能会在母代、子代两方面产生影响。一方面药物本身对乳汁产生有影响；另外通过乳汁传递的药物对处于哺乳期的新生儿也会有毒副作用。近来随着人们接触纳米产品日益增多,纳米颗粒对哺乳期女性以及哺乳期婴儿有无毒性已引起学术界的关注和重视。有研究报道,C60和二氧化钛纳米材料随乳汁而传递给仔鼠,并且在仔鼠体内发生了吸收。但上述两项研究并未系统阐述纳米材料对处于哺乳期的母鼠有无毒性,以及纳米材料如何通过乳汁传递的机制。由于二氧化钛纳米材料的良好应用前景以及纳米材料对哺乳期女性毒性的未知,本论文第一部分选取两种不同粒径的二氧化钛纳米材料(8nm和50nm)为研究对象,以哺乳期母鼠为模型,系统评价了二氧化钛纳米材料对处于哺乳期的母鼠及仔鼠哺乳期发育的毒性。并围绕乳腺展开研究,阐述了纳米材料通过乳汁传递给仔鼠的机制。研究结果揭示了纳米材料对哺乳期女性的潜在健康影响,另外对哺乳期女性的用药安全也具有指导意义。实验首先通过尾静脉方式对处于哺乳期的母鼠暴露8nm和50nm的二氧化钛纳米材料,在随后的体内分布研究中发现二氧化钛纳米材料在乳腺中有分布。但在材料暴露期间以及暴露后对母鼠都没有引起明显的系统毒性。暴露组母鼠的主要脏器系数以及组织病理学与对照组相比无显著性差异。但聚集在乳腺中的二氧化钛纳米材料能引起乳腺组织发生氧化应激,病理学结果显示乳腺上皮细胞脱落,高剂量暴露组的母鼠乳腺组织出现脂肪化。其中高剂量暴露8nm二氧化钛纳米材料对乳腺组织损伤更严重,造成的上皮细胞脱落数更多,部分血管充血。类似的病理学变化也同样出现在50nm二氧化钛纳米材料暴露组,但相对而言其造成的病理学改变较轻微。同时,随暴露剂量增加,构成乳腺上皮细胞之间的紧密连接蛋白ZO-1和occludin下调,说明氧化应激也破坏了乳腺上皮细胞之间的紧密连接,这直接导致纳米材料经过上皮细胞之间松散的紧密连接通道随乳汁传递。上皮细胞脱落和脂肪化都可能造成乳腺组织中乳汁蛋白基因表达量下调或者乳汁中蛋白含量下降。对此我们选择了乳汁中4个有重要生理功能的蛋白,分别是p-酪蛋白,a-乳清蛋白,乳铁传递蛋白,表皮生长因子。经过乳腺组织实时荧光定量PCR和乳汁蛋白印迹实验的验证,这4个蛋白表达量、含量与对照组比都未发生改变,其原因可能是乳腺损伤程度较小。而仔鼠在哺乳期的发育指标以及生存率都未受到影响。第二部分噻唑烷酮化合物组合协同抑制耐药肺癌细胞机制研究肺癌是全世界发病率和死亡率最高的恶性肿瘤。据统计,肺癌的五年生存率只有16.3%。癌症治疗手段包括手术治疗、放射治疗和化学治疗,其中化学治疗占有非常重要的地位。肿瘤细胞耐药性的产生及化疗药物引发的毒副作用是肿瘤化疗失败的两个主要原因。近来,研发单一靶点的抗癌药物是减小药物毒副作用的策略之一,比如格列卫(Gleevec),特罗凯(Tarceva),易瑞沙(Iressa)皆是单靶点药物。然而,单一靶点的药物在化疗过程中逐渐显露出劣势,比如治疗效率逐渐下降、严重的副作用以及肿瘤细胞的耐药性。因此,多层次、多靶点的治疗策略在肿瘤治疗中显示出了较大的潜力。这种治疗策略优势为多组分药物或者多靶点药物能发挥协同作用,共同抑制与肿瘤生长相关的信号通路。同时作用于多个靶点的药物组合物在肿瘤缩小和肿瘤细胞抑制方面,能更高效的克服多药耐药性,且有较低的副作用。噻唑烷酮类化合物能够选择性地有效杀死对紫杉醇产生耐药性的肺癌细胞,而对人正常的成纤维细胞无严重杀伤作用。我们前期筛选了一个含100种化合物组合的化合物库,获得了一个含有27,107,167和254四种化合物的组合物(命名为M4),与单个化合物相比较,M4能够明显抑制对紫杉醇有耐药性的非小细胞肺癌H460/TaxR细胞生长。实验表明,M4化合物能调控细胞周期检验点蛋白,包括p53,p21,磷酸化cdc25C(Ser216)的表达升高；组蛋白H4及a-tubulin高度乙酰化；Akt磷酸化；Caspase激活；细胞色素C释放,线粒体膜电粒降低。这些结果表明,通过靶向不同的靶点,组成M4的单个化合物发挥协同作用,共同抑制肿瘤细胞的生长。本课题在前期工作的基础上进一步研究,发现(1)M4所包含的27、107、167和254四种化合物之间具有协同抗癌作用；(2)联合使用HDAC抑制剂和微管聚合抑制剂是治疗癌症的有效方法之一。更多还原

【Abstract】 In recent years, nanotechnology is becoming one of the main driving forces of promoting the economic development in the world. It has already significantly affected modern society in many areas, such as bio-medicine, energy, electronics, transportation, architecture and the environment. Therefore, the importance of nanotechnology has been acquired increasing attention. Countries around the world have developed their nanotechnology development plan in line with national interests. Acompanying a number of nano-products have already on the market, nanotechnology is being shifted from basic research to applied research and to industrial transformation. Since2006, there have been1317kinds of nano products appeared in worldwide consumer market.Nanotechnology have increasingly wide application prospects which is health-related fields with people, such as environmental and biomedical field. For example, nanotechnology is used to clear the toxic and organic contaminants in water, control air pollution and achieve remedy of diseases which combines low toxicity and efficient targeted therapy. Therefore, there has been increased possibility that human exposed to different types of nanomaterials. However, the negative effects of nanomaterials on environment or human body is unknown. Materials, at nanoscale, show quantum size effect, small bulk effect, surface effect and macroscopic quantum tunneling effect. So nanomaterials may present unique effects after their expsoure to human body due to their unique physical/chemical properties. In this context, nanotoxicology was proposed as a new branch of toxicology to address the adverse health effects caused by nanomaterials and development of nanotechnology.Titanium oxide nanoparticels (TNPs) have attracted widespread attention due to its broad application prospect, such as environment, cosmetics and food adititive. However, it has been reported that TNPs can cause injury to brain, lungs, liver and even cancer. TNPs may be taken up directly to the brain from olfactory epithelium to the various parts of brain via the olfactory nerves. It produced the sustained accumulation in the brain tissues which induces brain pathological changes and oxidative stress-mediated responses. TNPs could induce some neurons to turn into filamentous shapes and others into inflammatory cells. It also induces hepatocytes necrosis, apoptosis and fibrosis.Breastfeeding is a continuation of the reproductive process. The lactation period of human lasts long. Milk provides babies the nutrients they need which is beneficial to their health and development. It also contains a number of antibodies that can protect infants from common childhood diseases attacks. Medication of women during lactation period may have negative impact on the parent and the offspring or both. The medication may transfer to pups through milk. Milk producing and the transferred medication may have a negative impact on women and newborns. Recently, people have increased contact opportunity to nano-products. So the safety of nanomaterials to lactating women and infants is worthy of concern and attention. It has been reported that C60and TNPs transfer to pups through milk, while it can also be absorbed by pups. However, the two studies did not systematically expounded whether the nanomaterials have negative impact on lactating dams or the transfer mechanism of nanomaterials.Due to the large-scale application of TNPs as well as the unknown potential negative effects on lactating dams, we select two sizes of TNPs in order to study the toxicological effects on lactating dams and their pups. And we conducted research around the lactating mammary glands (MGs) to elaborate the mechanism of nanomaterials transfer to milk. This mechanism has guiding significance to safety of nanomaterials and medication applicaion during lactation period.The biodistribution of two sizes of TNPs after i.v. exposure to lactating dams shows that TNPs accumulate in lactating MGs. However, the TNPs didn’t cause systemic toxicity to dams during exposure period and postexposure period. The dams’ organ index didn’t show significant difference between the dams exposed to TNPs and the PBS control group. The accumulation of TNPs in MGs cause oxidative stress and mammary epithelium cell shedding. Hyperplasia and adipocytes was found both in TNP1and TNP2exposure group at dose of6and8mg/kg BW. What noteworthy is that quantification of numbers of shedding epithelial cells showed that8mg/kg BW dose exposure of TNP1induces more shedding epithelial cells than its larger-sized counterpart. It also demostrates that TNP induced milk blood barrier disruption manifested by both cell shedding and loss of tight junctions. Theredore, the accumulation of TNPs in lactating MG disturbs the oxidant/antioxidant equilibrium and subsequent epithelial cells shed with tight junction loss. These results indicate that there is correlation between epithelial cells shedding and tight junction loss of mammary gland and TNP transfer to milk. MG epithelial cell shedding has another consequence that whether nutrient quality of milk will be deteriorated. The four proteins, including β-casein, a-lactalbumin, lactoferrin and epidermal growth factor, were chosen in the reason of their important function in the three aspects. The levels of four proteins, both mRNA and protein, were not meaningfully decreased after exposure to TNPs at8mg/kg BW. These data also suggest that exposure of TNPs does not impair milk secretion of dam, or deteriorate the nutrient quality of milk. Our data also showed that there was no significant difference in the pups that displayed hair growth and eye opening between the TNPs treated group at dose of8mg/kg BW and PBS control group.Part IILung cancer is one of the main causes of death among all diseases. The five-year survival rate of lung cancer is merely16.3%. Cancer treatments include surgery, radiotherapy and chemotherapy. However, drug resistance and the dose-limiting toxicity are key reasons to account for unsuccessful cancer chemotherapy. Discovery of single target-specific drugs has been the focus of cancer drug development. Research in this area has led to the successful drugs such as Gleevec, Tarceva and Iressa. Single-target drugs have gradually showed some disadvantages, such as lack of efficacy, serious side effects, and development of drug resistance. Recently, multi-level and multi-targeting therapies have shown potential applications in cancer treatment. Such therapies, including multi-component drugs or multi-targeting drugs, may produce concerted pharmacological intervention of multiple targets and signaling pathways that drive the growth of tumors. Synergistic action of such drugs may overcome side effects that resulted from high doses of single-target drugs, increase drug selectivity, and offer an opportunity for more precise control of biological systems. Drug combinations that simultaneously impact multiple targets are more effective to overcome MDR and lower side-effects in cancer cell inhibition and tumor shrinkage.The thiazolidinone derivatives are useful anticancer agents with P-gp-evading property and minimal side effects. By screening compound combinations prepared from a thiazolidinone compound library in a drug-resistant variant H460/TaxR NSCLC cell line, a four-compound combination was identified that synergistically inhibit the growth of cancer cells from both lines. We found that (1) thiazolidinone compound combinations that have synergistic inhibitory effects on P-gp overexpressing NSCLC;(2) individual compound in this combination act as either tubulin polymerizationinhibitors or histone deacetylase (HDAC) inhibitors.更多还原