β-榄香烯—姜黄素复方脂质体的制备与联用多西他赛抗肺癌作用的研究

发布时间：2018-08-04 13:02

【摘要】：肺癌是目前患病率、死亡率均较高的一种癌症,肺癌的治疗手段多为传统的放化疗以及手术切除,这些方式往往在杀伤肿瘤细胞的同时也损伤了正常组织,对患者身体造成极大的伤害。中药作为我国临床治疗肺癌的主要手段之一,在临床上与化疗药物合用以证明可减毒增效、延长生存期和生存质量。中药莪术是姜科植物蓬莪术、广西莪术和温郁金的干燥根茎。味辛、苦、温,归肝、脾经,具有行气破血、消积止痛之功。β-榄香烯和姜黄素分别是莪术中挥发油类成分和姜黄素类成分的代表,这两种药物单独对癌症的研究很多,本课题将β-榄香烯和姜黄素配伍应用,探究两药抑制肺癌细胞生长的协同作用。进而与临床抗癌一线药物多西他赛联用,共同探究中西药对肺癌细胞的协同抑制作用。脂质体由磷脂组成,在水中磷脂分子亲水头部插入到水中,疏水尾部则伸向空气,搅动后形成双层磷脂分子的球形囊泡,可将药物包封于双分子层内提高药物的稳定性同时降低药物的毒性。脂质体在体内会被巨噬细胞吞噬,集中在肺部等器官,具有靶向性、细胞亲和性和组织相容性。由于药物包封在脂质体内部,在靶组织释放缓慢,具有一定的缓释性,可以弥补β-榄香烯和姜黄素在体内代谢过快的情况。因此将β-榄香烯和姜黄素制成复方脂质体,以达到靶向、缓释等效用。雾化吸入给药对于治疗肺部疾病有很好的疗效,可以将药物直接递送到靶器官,可进一步提高靶向性,同时避免肝脏首过效应。中医理论中提到"肺为娇脏",是对肺的生理病理特征的概括。肺部具有大量肺泡,肺泡是由单层上皮细胞构成的半球状囊泡,周围还有许多毛细血管,非常利于药物的吸收,但同时也容易受到刺激。因此雾化吸入给药的安全性需要保证,本文对脂质体的雾化吸入刺激性做了初步评价。1.β-榄香烯和姜黄素最佳配伍比例的确定首先建立了稳定的人肺癌细胞A549细胞和鼠肺腺癌细胞LLC细胞的体外传代和培养方法。MTT法的原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为不溶性的蓝紫色结晶—甲佨(Formazan)并沉积在细胞中,而死细胞无此功能。DMSO能溶解细胞中的甲佨,并通过酶联免疫检测仪测定其吸光值,即可间接反映活细胞数量。通过MTT法研究不同浓度β-榄香烯和姜黄素单独及配伍对两种细胞的生长抑制作用。采用金氏公式(q=(EA+B)/(EA+EB-EA × EB))来评价药物联用的协同作用,q值越大,协同作用越明显。最终通过两药与多西他赛联用后协同作用的大小确定β-榄香烯和姜黄素的最佳浓度配伍比例为1:4。当β-榄香烯浓度为10 mg·L-1、姜黄素浓度为40 mg·L-1、多西他赛浓度为800 mg.L-1时,三种药物联用对LLC细胞的抑制率可达87.44%。2.β-榄香烯-姜黄素复方脂质体的制备与评价以确定的最佳配伍比例将β-榄香烯和姜黄素制成复方脂质体。首先建立复方脂质体中β-榄香烯和姜黄素的含量测定方法。通过单因素考察筛选出三因素三水平,以β-榄香烯和姜黄素的包封率和载药量为指标,利用效应面法进行优化处方。采用Design-Expert.8.05b软件,利用Box-Behnken法优选出最佳处方为姜黄素20 mg、β-榄香烯5 mg、卵磷脂666.7 mg、胆固醇133.3 mg,最终采用pH7.0的磷酸缓冲盐10 mL旋转水化。验证实验结果显示β-榄香烯包封率为(97.86±1.53)%、姜黄素包封率为(97.71±1.53)%,总载药量为(0.1925±0.006)%。制得的脂质体外观呈黄色,分散均匀无沉淀,微观形态呈球形,大小均匀。复方脂质体粒径为(232.0±6.4)nm,电位为(0.71±0.1)mV。通过细胞实验将复方脂质体和溶液剂分别与多西他赛联用,对比结果显示复方脂质体在较高浓度多西他赛时对LLC细胞的生长抑制率显著高于溶液剂(P0.05)。3.脂质体雾化吸入刺激性的研究由于制得的β-榄香烯-姜黄素复方脂质体载药量较小,考虑采用雾化吸入给药方式。为考察脂质体雾化吸入后对肺部的刺激性,设计大鼠雾化吸入实验,采用自主研发的雾化吸入装置(专利号:ZL201520442646.5)按临床折算剂量对大鼠进行雾化吸入给药。以生理盐水为对照,比较β-榄香烯和姜黄素脂质体对大鼠的肺部刺激性,通过肺部组织HE染色切片和肺泡灌洗液进行BCA总蛋白的含量和乳酸脱氢酶活力进行评价。结果显示,无论是病理切片的观察还是肺泡灌洗液的分析结果均显示β-榄香烯和姜黄素脂质体组与生理盐水组无显著性差异(P0.05)。初步表明两药脂质体对大鼠的肺部无显著刺激性,为后期研究奠定了基础。
[Abstract]:Lung cancer is a kind of cancer with high morbidity and mortality, and the treatment of lung cancer is mostly traditional radiotherapy and chemotherapy and surgical excision. These methods often damage the normal tissue while killing the tumor cells, and cause great harm to the patient's body. It is used in bed with chemotherapeutic drugs to prove that it can reduce toxicity and increase efficiency and prolong life and quality of life. The rhizome of zedoary zedoary turmeric, Guangxi zedoary turmeric and Wen Yujin are the dried rhizomes of zedoary turmeric and zedoary. It is a kind of volatile oil component and curcumin in zedoary. The two drugs, represented by these drugs, have a lot of research on cancer alone. This subject applies the combination of beta elemene and curcumin to explore the synergistic effect of two drugs to inhibit the growth of lung cancer cells. Then, the synergistic inhibition effect of Chinese and Western drugs on lung cancer cells is explored together with the clinical anticancer drug, docetaxel. Liposomes are from the phospholipid group. In the water, the phospholipid molecules are inserted into the water and the hydrophobic tail is extended to the air and stirs into the spherical vesicles of the bilayer phospholipid molecules. The drug can be encapsulated in the double molecular layer to improve the stability of the drug and reduce the toxicity of the drug. The liposomes will be phagocytic in the body of the giant macrophage in the body and are concentrated in the lungs and other organs. The release of beta elemene and curcumin in the body is slow, which can make up for the rapid metabolism of beta elemene and curcumin in the body. Therefore, beta elemene and curcumin are made into compound liposomes to achieve target and slow release. Aerosol inhalation is used. It has a good effect on the treatment of lung disease. It can be delivered directly to the target organ, which can further improve the targeting and avoid the first effect of the liver. The theory of Chinese medicine refers to "the lungs are dirty", which is a summary of the physiological and pathological characteristics of the lung. The lungs have a large number of alveoli, and the alveoli are hemispherical vesicles consisting of single layer epithelial cells. There are many capillaries around it, which are very beneficial to the absorption of drugs, but also easy to be stimulated. Therefore, the safety of inhalation inhalation needs to be ensured. In this paper, a preliminary evaluation of the optimum compatibility of.1. beta elemene and curcumin by atomization inhalation of liposomes is first established to establish a stable human lung cancer cell A549 fine. The principle of.MTT method for the generation and culture of LLC cells in cell and rat lung adenocarcinoma cells is that succinic dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue purple crystals - Formazan and deposit in cells. Dead cells do not have the function.DMSO to dissolve the thyroid gland in the cells and be immunized by enzyme linked immunosorbent assay. The number of living cells could be indirectly reflected by the measuring instrument, and the inhibitory effects of the two cells on the growth of two kinds of cells were studied by the method of q= (EA+B) / (EA+EB-EA x EB). The greater the Q value, the more obvious the synergism was. Finally, through two. The synergistic effect of drug and docetaxel combined to determine the optimum concentration ratio of beta elemene and curcumin was 1:4. when the concentration of beta elemene was 10 mg L-1, the concentration of curcumin was 40 mg. L-1, and the concentration of docetaxel was 800 mg.L-1, and the inhibition rate of three drugs on LLC cells could reach 87.44%.2. beta elemene curcumin compound fat. The preparation and evaluation of plastids to determine the optimum compatibility ratio of beta elemene and curcumin into compound liposomes. First, the content determination method of beta elemene and curcumin in compound liposomes was established. Three factors and three levels were screened out by single factor investigation, and the encapsulation efficiency and drug loading of beta elemene and Jiang Huang were used as indexes. Design-Expert.8.05b software was used to optimize the prescription. The best formula was selected by Design-Expert.8.05b software. The best prescription was curcumin 20 mg, beta elemene 5 mg, lecithin 666.7 mg, cholesterol 133.3 mg, and finally using pH7.0 phosphate buffer salt 10 mL to rotate hydration. The experimental results showed that the encapsulation efficiency of beta elemene was (97.86 + 1.53)%, curcumin. The encapsulation rate was (97.71 + 1.53)% and the total load was (0.1925 + 0.006)%. The liposomes were yellow, dispersed evenly and without precipitation. The micromorphology was spherical and uniform. The particle size of the compound liposome was (232 + 6.4) nm, and the potential was (0.71 + 0.1) mV. combined with docetaxel and the compound liposome and solution agent respectively. The results showed that the growth inhibition rate of compound liposomes at high concentration of docetaxel was significantly higher than that of solution agent (P0.05).3. liposome atomization inhalation. The effect of the prepared beta elemene curcumin compound liposome was smaller and the aerosol inhalation method was considered. The effect of liposome inhalation on the lung after liposome inhalation was investigated. The irritation of the rats was designed by atomization inhalation experiment. The atomization inhalation of rats was carried out by the self developed atomization inhalation device (patent number: ZL201520442646.5). The rats were compared with the physiological saline. The lung irritation of rats was compared with the liposomes of beta elemene and curcumin liposomes, and the lung tissue was stained with HE staining and lung tissue. The total protein content of BCA and the activity of lactate dehydrogenase were evaluated. The results showed that there was no significant difference between the liposomes group and the saline group (P0.05), no matter the observation of pathological section or the analysis of the alveolar lavage fluid (P0.05). The initial step showed that the two drug liposomes were not significant to the lungs of the rats. Irritation has laid a foundation for later research.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R283.6;R285

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