【作者】 王东；

【导师】 王振宇；

【作者基本信息】 哈尔滨工业大学 ， 食品科学与工程， 2015， 硕士

【摘要】 松多酚是源自松树的一种天然产物,具有较好的抗辐射功效,但是由于其本身特有的多羟基结构,极易受到光照、温度、氧气、p H等环境因素的影响而降解甚至形成副产物。微胶囊包埋技术作为新的包埋手段在药物靶向和缓释方面具有巨大潜力。壳聚糖作为一种天然载体材料,本身既有一定的免疫增强功效,又可以对其包载的芯材起到一定的保护和缓释作用。将壳聚糖作为药物载体,包埋松多酚,制备成具有缓释抗辐射功效的微球,既解决了松多酚本身稳定性差的问题,又增强了药物整体的功效性,使得这一复合物载体安全无毒,疗效高,是抗辐射治疗领域中一个新亮点,具有很强的现实意义。本课题采用乳化交联技术,以壳聚糖为壁材,红松多酚为芯材,戊二醛为交联剂,分别以壳聚糖浓度、搅拌速度、交联温度、乳化时间、交联剂用量、交联时间六个条件为单因素,以包埋率和载药量为响应值,检测各因素对松多酚微球包埋率、载药量的影响,并通过电子显微镜拍照观察各因素对松多酚微球形貌的影响。在此基础上,选出四个较明显的影响因素通过Design-expert-7.0.0设计的29组实验统计结果表明松多酚微球的最佳制备工艺关键参数为:壳聚糖浓度为2%,交联剂用量7.19ml,搅拌速度660.98r/min,交联温度41.18℃,乳化时间198.65min,交联时间80min。采用优化条件后通过扫描电镜可观察到最优工艺所得微球的形貌较好,成球率高,球形的大小分布较均匀;并能看到明显的核壳结构;通过激光粒度仪测得微球的均粒径为3um;包埋率和载药量经测定分别是73.57%和7.47%。通过模拟人工胃肠环境,考察其在人体的缓释情况为:松多酚微球在胃的酸性条件下较稳定,能够平稳过渡到达肠道。本课题以19-21g的昆明小鼠为试验对象,随机分为正常对照组、辐射模型组、松多酚给药组(分低、中、高剂量三组)、松多酚微球给药组(分低、中、高剂量三组),每组20只,雌雄各半,先对这些昆明小鼠连续对应灌胃14天,接着以60Co-γ射线为辐射源,采用6.0Gy剂量辐射除正常对照组外的各组小鼠,24h后处死,检测各项指标,结果发现:1)与正常对照组比较,模型组、松多酚给药组和松多酚微球给药组中雌、雄小鼠的各免疫器官均受到不同程度的损伤,血清SOD活性降低、MDA水平不同程度增加,骨髓细胞DNA含量下降,脾脏T淋巴细胞增殖能力减弱,骨髓细胞微核数量增加,单核-巨噬细胞的吞噬能力降低,说明60Co-γ射线会通过多种途径对小鼠的免疫系统造成损伤;2)与模型组对比,松多酚给药组和松多酚微球给药组中雌、雄小鼠的各免疫器官指数随着给药剂量的增加而提升,血清SOD活性也随着给药剂量的增加而升高、MDA水平随给药剂量的增加而减少,骨髓细胞DNA含量、脾脏T淋巴细胞增殖能力和单核-巨噬细胞的吞噬能力均伴随给药剂量的加大而提升,同时,给药剂量加大会使骨髓细胞微核数量减少,说明松多酚和松多酚微球均能够通过多种途径对60Co-γ射线导致的小鼠机体各免疫系统损伤起预防作用;3)与松多酚低、中、高三个给药剂量对比,松多酚微球低、中、高给药组中雌、雄小鼠的免疫器官指数相对应均提升,血清SOD活性更高、MDA水平减少,骨髓细胞DNA含量、脾脏B淋巴细胞增殖能力和单核-巨噬细胞的吞噬能力均增强,同时,骨髓细胞微核数量减少,这说明了松多酚微球对60Co-γ射线的辐射防护效果要高于松多酚。通过动物实验中各项指标的表征,说明松多酚和松多酚微球主要是通过以下途径保护小鼠免受辐射损伤:一、直接清除机体在辐射应激时产生的有害自由基;二、提高内源性抗氧化物酶活性而增强机体的抗氧化能力、免疫能力和抗应激能力;三、增强机体自身免疫力,促进免疫细胞增殖而降低机体损伤。此外,我们发现在动物实验的各项检测指标中,雌性小鼠和雄性小鼠的结果差异性较显著:在脏器免疫器官指数、血清SOD和MDA、脾脏淋巴细胞增殖能力、单核-巨噬细胞吞噬能力和骨髓DNA含量的检测中,除应激本身带来的损伤外,雌性小鼠显示出更强的抗辐射能力,而在骨髓微核与方面,雄性小鼠则占有较强的优势。这说明不同性别的小鼠在对抗相同的应激环境,其机体会出现差异化表达,这不仅与不同性别所特有的不同激素及其受体有关,可能还与某些重要免疫细胞在不同性别的极化表现有关,而其中的关键差异蛋白表达有待于进一步研究。更多还原

【Abstract】 Pine polyphenol is a kind of natural product derived from Pinus koraiensis, which had a good anti-radiation function. But because of its unique polyhydroxy molecular structures, which is vulnerable to light, high temperature, oxygen, extreme p H and other harmful environmental factors, which could made the functional composition degradate and even turned into toxic byproducts. Microsphere as a new technology of drug embedding, which had great potential in targeting and sustained drug delivery. Chitosan as a kind of natural carrier material, which not only had great anti-radiation function, but also played a protective role and could sustained release core drug. In this study, chitosan as a kind of drug carriers, embedding pine polyphenols, prepared an anti-radiation drug system, which not only solved the problem of poor stability of polyphenols, but also enhanced the effectivity of the drug system, which guaranteed this composite drug delivery system safe, non-toxic and high efficacy. This is a bright new spot in this field, which has a strong practical significance for the research of anti-radiation drugs.In this study we used emulsification cross-linking technology, chitosan was wall material, pine polyphenols was core material, glutaraldehyde was cross-linking agent, and explore the best pine polyphenols microspheres preparation conditions. Then chosed the factor of chitosan concentration, crosslinking time, crosslinking agent amount, stirring speed, crosslinking temperature and emulsifying time as the single factor respectively, set the embeding rate and drug loading rate as response, detect the influence of various factors to the polyphenol microsphere embeding rate and drug loading rate, and observed the microspheres shape and appearance through electron microscope photograph. Based on these, selected four obvious influence factors, Design expert7.0.0 designed 29 group of tests, whose result showed that the best preparation key parameters of polyphenols microspheres were as following: chitosan concentration was 2%, crosslinking agent amount was 7.19 ml, stirring speed was 660.98 r/min, crosslinking temperature was 41.18 ℃, emulsifying time was 198.65 min, crosslinking time was 80 min;After optimized, the morphology of microspheres observed by scanning electron microscopy(SEM) was good, the rate of qualified balls was high, the size of the spherical distribution is uniform; the core-shell structure is obviously; the microspheres particle size is 3um measured by laser granulometer; the embeding rate and drug loading rate were 73.57% and 7.47% respectively. Through simulated human gastric and intestinal conditions, the microsphere release situation was: microsphere under the condition of the acidity stomach is stable, which could transit to intestinal smoothly, and released stably, whose slow release performance proved that the microspheres is successful.This project used KM mouse, which weight 19-21 g. And devided into four groups: the normal control group, radiation model groups, pine polyphenol groups(low, middle and high dose), pine polyphenol microsphere groups(low, middle and high dose), each group had half male and half female. After fed 14 days, we used 60 Co gamma ray as radiation source, 6.0 Gy doses, radiated the each group mice except the normal control group, after 24 h executed, testing all the indexes, the result showed that: 1) compared with normal control group, radiation model group, pine polyphenol group and pine polyphenols microsphere group female and male mice immune organ have different degree of injury, serum SOD activity reduced and MDA level increased, the bone marrow DNA content decreased, the spleen lymphocyte proliferation ability also weakened, and monocyte-macrophage phagocytosis ability had reduced, bone marrow cell micronucleus number increased, illustrated that 60 Co gamma ray damaged the body immune system in mice; 2) compared with model group, pine polyphenol group and pine polyphenols microsphere group female and male mice immune organ index increased with the drug dosage increased, serum SOD activity increased with the increasing of drug dosage, the MDA level reduced with the increasing of drug dosage, bone marrow DNA content, spleen lymphocyte proliferation ability and monocyte-macrophage phagocytosis increased with the increasing of the drug dose, at the same time, the increased dosage could make the bone marrow micronucleus decreased, which showed that pine polyphenols and pine polyphenols microspheres could protect the body immune system from the 60 Co gamma rays damage to mice; 3) Compared with low, medium and high dosage three dose groups of pine polyphenols, low, middle and high dosage group of pine polyphenols microspheres male and female mice immune organ index improved in different degree, serum SOD activity are much higher and MDA level reducd, bone marrow DNA content, the spleen lymphocyte proliferation ability and monocyte-macrophage phagocytosis ability had been enhanced, at the same time, the bone marrow cells micronucleus number reduced, which showed that the protect effect of pine polyphenols microspheres to 60 Co gamma ray radiation is better than pine polyphenols monomer.Various index showed that pine polyphenols and pine polyphenols microspheres achieved the role of protect mice from radiation injury mainly through the following ways: Firstly, removed the harmful free radicals in body, which is producted by radiation stress; Secondly, improved the activity of endogenous antioxidant enzymes and enhance the body’s antioxidant capacity, immunity and anti-stress ability; Thirdly, enhanced the body immunity, promote the immune cell proliferation and reduced the damage.In addition, we found that in the detected indexes of animal experiment, the difference between the result of female mice and male mice is significant: the immune organ index, serum SOD and MDA, spleen lymphocyte proliferation ability, mononuclear macrophage phagocytosis test and marrow DNA content, except the damage brougut by stress itself, female mice showed a stronger ability to resist the radiation damage, while in bone marrow micronucleus, male mice had the strong superiority. This suggested that different gender in fighting the same stress environment, the body would appear differentiation, it’s not only because of different gender, peculiar to the different hormone and its receptor, even some important immune cells in different gender polarization performance are also related, and the key differences between protein expression needed further research.更多还原