胆甾醇基γ-聚谷氨酸负载阿霉素纳米胶束的制备与体内外释药性能评价

笔者制备了胆甾醇基γ-聚谷氨酸负载阿霉素纳米胶束(DOX/NPs),并考察了该载药纳米胶束体系的形态与粒径、载药量、包封率以及体内外释药的特性。结果表明:DOX/NPs的最佳载药量为22.4%,包封率为90.2%,平均粒径为(312.3±7.2)nm,电镜下观察呈现明显的核壳结构。体外释药结果显示,DOX/NPs能延缓阿霉素的释放,并具有p H敏感的释药特性。小鼠体内释药结果表明:阿霉素经包埋后其消除半衰期(t1/2)、药时曲线下面积(AUC)、平均滞留时间(MRT)均明显大于游离阿霉素,达到了药物缓释的目的。     

转铁蛋白修饰磁性纳米粒的制备及其体外抗肿瘤活性研究

目的:本研究旨在构建一种转铁蛋白修饰负载阿霉素(DOX)的磁纳米粒靶向递药系统,以提高阿霉素作用的靶向性。方法:采用化学共沉淀法制备转铁蛋白修饰负载阿霉素的磁性纳米粒(DOX@MNP),采用zeta电位及纳米粒度分析仪测定DOX@MNP的粒径及其zeta电位,透析法评价DOX@MNP的体外释药特征。通过MTT实验,研究DOX@MNP与游离DOX对A549细胞的细胞毒性,通过激光共聚焦显微镜和流式细胞仪观察A549细胞对DOX@MNP与游离DOX的摄取情况。结果:DOX@MNP的释药具有p H依赖性。MTT实验结果显示,DOX@MNP与游离DOX具有相当的细胞毒性;激光共聚焦显微镜和流式细胞仪检测结果显示A549细胞对DOX和DOX@MNP的摄取没有明显差异。结论:本文构建了一种转铁蛋白修饰包载阿霉素的磁纳米粒,体外结果显示其具有与游离DOX相当的细胞毒性,为进一步进行体内实验奠定了基础。     

酸敏阿霉素脂质纳米粒的制备及其体外抗肿瘤活性研究

目的:本研究旨在制备具有被动靶向和酸敏特性的脂质混合纳米粒,以期提高阿霉素(doxorubicin,DOX)的靶向递药效率,降低DOX的毒副作用,提高抗肿瘤活性。方法:采用微乳法制备磷酸钙纳米粒核,薄膜分散法制备脂质混合纳米粒,硫酸铵梯度法包封DOX。采用透射电镜观察外观形态,用zeta电位及纳米粒度分析仪测定纳米粒的粒径及zeta电位,透析法评价阿霉素脂质纳米粒体外释药特征。用MTT方法研究阿霉素脂质混合纳米粒对A549细胞的细胞毒性。采用流式细胞仪和激光共聚焦显微镜观察A549细胞对阿霉素脂质纳米粒的摄取。结果:体外释药结果显示阿霉素脂质纳米粒具有酸敏特性。流式结果说明A549细胞对阿霉素脂质纳米粒的摄取具有明显的时间依赖性,激光共聚焦显示阿霉素脂质纳米粒能将阿霉素递送至细胞核中。结论:阿霉素脂质体对A549细胞有明显的细胞毒性,为进一步进行体内实验提供了基础。     

共载依克立达和阿霉素的PBM-Hz-PEG纳米粒制备与性能研究

摘要 目的：研究不同比例依克立达（ELC）和阿霉素（DOX）的联合抗肿瘤效果,确定最佳联用比例。以生物可降解材料聚苹果酸苄基酯（PBM）为载体包封两种药物,得到一种酸敏感纳米胶束。方法：以L-天冬氨酸为原料通过内酯开环法制备PBM,并以酸敏感的腙键（Hz）连接PEG,得到嵌段聚合物PBM-Hz-PEG,红外光谱和核磁氢谱对其结构进行表征。动态透析法制备纳米胶束,测定纳米胶束的粒度、分散系数（PDI）、临界胶束浓度（CMC）及其载药量（DL）、包封率（EE）。动态透析法模拟胶束的体外释药性能,采用三阴性乳腺癌MDA-MB-231细胞系考察载药纳米胶束的体外细胞毒性。结果：①ELC能够增敏DOX,二者摩尔比为1:3时有最强肿瘤抑制作用。②经红外光谱和核磁共振氢谱表征,嵌段共聚物PBM-Hz-PEG成功合成。③空白纳米胶束的粒径为69.67±11.55 nm,PDI为0.245 ± 0.026,CMC值为3.9 μg?mL^-1;载药纳米胶束粒径略大,粒径在96.92 ~ 113.47 nm之间,ELC和DOX的载药量与投料比一致。④载药纳米胶束在pH 7.4和pH 6.0时的药物释放率曲线和体外细胞毒性试验证实载药胶束具有良好的酸敏特性。结论：ELC和DOX联用有较强的肿瘤抑制作用,PBM是二者的优良载体。该PBM-Hz-PEG纳米胶束载药率高,其特有的酸敏性能够有效降低药物对正常组织的毒副作用,具有肿瘤组织富集释放特性,有望成为一种新型智能释药平台。     

还原可降解聚磺酸甜菜碱型纳米水凝胶的制备及载药研究

目的:制备与表征还原可降解的聚磺酸甜菜碱型纳米水凝胶,利用该纳米递药系统包载阿霉素(DOX)并初步评价其抗肿瘤性能。方法:利用回流沉淀聚合的方法合成含二硫键的聚磺酸甜菜碱甲基丙烯酸酯(PSBMA)纳米水凝胶及不含二硫键的PSBMA纳米凝胶(nd-PSBMA);通过粒度仪和透射电镜考察两种纳米水凝胶的粒径、形态以及稳定性;通过考察谷胱甘肽(GSH)对纳米凝胶溶液相对浊度的影响以评价还原环境对两种纳米凝胶的还原可降解性;利用纳米凝胶包载阿霉素(DOX),考察载药凝胶在GSH中的释药行为,并初步评价其对A549肿瘤细胞的杀伤作用。结果:以N, N'-双丙烯酰胱胺为交联剂制备了含二硫键的PSBMA纳米凝胶,其粒径在180～200 nm;同时以N, N'-双丙烯酰胺为交联剂制备了不含二硫键的n-PSBMA纳米凝胶。两种纳米凝胶与小鼠血清共孵育7天水合粒径仍无明显变化,表明磺酸甜菜碱型纳米凝胶具有良好的抗蛋白吸附能力。此外,PSBMA纳米凝胶在GSH溶液中迅速地降解,且降解速度与GSH浓度呈正相关;而nd-PSBMA纳米凝胶在GSH溶液中几乎不降解。载DOX的PSBMA纳米凝胶可在GSH作用下快速的释放药物而载DOX的nd-PSBMA纳米凝胶在GSH作用下缓慢的释放药物;体外细胞实验显示空白纳米凝胶和载药nd-PSBMA对A549细胞无明显毒性作用,但载DOX的PSBMA纳米凝胶可高效地杀死A549肿瘤细胞,其药效与游离DOX相仿。结论:还原可降解的PSBMA纳米水凝胶有望成为智能型控释药物载体。     

D-甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用

聚合物胶束作为药物载体具有良好的稳定性和生物相容性,提高疏水性药物溶解性等优势,是一类很有应用潜力的药物传输系统。本研究以合成的共价键连D-甘露糖的双亲性聚合物分子(PGMA-Mannose)为药物载体,包载抗癌药物阿霉素(DOX)制备具有甘露糖受体靶向性和pH敏感药物释放特性的新型载药聚合物胶束。利用激光共聚焦显微镜和MTT细胞毒性评价方法对载药胶束的细胞内吞摄取和毒性进行评价。实验结果表明,载药胶束能特异性识别人乳腺癌细胞MDA-MB-231表面过度表达的甘露糖受体,被癌细胞大量摄取并在细胞溶酶体酸性环境内释放药物,而载药胶束在表面甘露糖受体低表达的HEK293细胞中只有少量摄取。与原药DOX相比,该载药胶束对癌细胞的毒性显著提高,而对正常细胞的毒性较低。因此,该PGMA-Mannose聚合物胶束有望成为一种新型的靶向药物输送系统应用于癌症的治疗。     

熊去氧胆酸对阿霉素诱导的H9c2心肌细胞的影响及机制研究

目的:探讨熊去氧胆酸(UDCA)对阿霉素(DOX)诱导的H9c2心肌细胞损伤的影响及机制。方法:体外培养H9c2细胞,1μM DOX和不同浓度UDCA处理H9c2,CCK-8法测定细胞活力;实时定量聚合酶链反应检测心肌细胞凋亡分子Bax及炎症因子IL-1β、IL-6的表达;Western blotting检测UDCA对DOX诱导的心肌细胞凋亡相关蛋白Bax、Bcl2、Caspase3表达水平变化。结果:与对照组相比,DOX组心肌细胞活力减弱;炎症因子IL-1β,IL-6表达上调;促凋亡分子Bax和cleaved Caspase3表达增多;抑制凋亡蛋白Bcl2下调(P<0.05)。与DOX组相比,UDCA+DOX组显著恢复心肌细胞活力;炎症因子IL-1β、IL-6表达下调;促凋亡分子Bax、cleaved Caspase3下调;抑制凋亡蛋白Bcl2表达上调(P<0.05)。结论:UDCA能缓解DOX诱导的H9c2心肌细胞损伤,其机制可能与抑制炎症及凋亡有关。本研究为阿霉素心肌毒性的防治提供新的实验基础及理论依据。     

双重响应二硫化钼纳米载药体系的制备及其性能研究

摘要 目的：制备肿瘤微环境响应释放的靶向二硫化钼纳米载药体系,并评价其载药量和释药性能。方法：以水热法合成的MoS₂纳米片为基底,利用MoS₂纳米片上的S空缺位点连接硫辛酸聚乙二醇羧酸,然后通过酰胺反应连接精氨酸-甘氨酸-天冬氨酸（RGD）靶向分子,再连接上交联剂3-(2-吡啶二硫代)丙酸N-琥珀酰亚胺酯（SPDP）,得到药物载体MoS2-PEG-RGD-SPDP（MPRS）,MPRS进一步与巯基化的阿霉素（DOX）反应,形成MPRS-DOX纳米载药体系。通过透射电子显微镜（TEM）,X-射线光电子能谱仪（XPS）以及纳米粒度电位仪对合成的材料进行表征;利用紫外可见分光光度计测试MPRS的载药性能,采用荧光分光光度计考察MPRS-DOX的释药性能。结果：成功合成MPRS-DOX纳米载药体系,其粒径大小在200 nm左右,Zeta电位为+28.2 mV;其载药效率为86.8%,载药量为53.5%。体外释药实验表明,在10 mM 谷胱甘肽（GSH）和pH=5.5的条件下DOX释放量最多。结论：成功制备了粒径合适的MPRS-DOX纳米载药体系,MPRS-DOX具有GSH和pH双重响应性,可实现预期的模拟肿瘤微环境内控制释放药物。这种GSH和pH双重响应的纳米载药体系为新一代刺激响应型纳米载药系统的构建提供了新的思路。     

pH和近红外光双响应的包裹二硫化钼纳米片和阿霉素的金属-有机框架ZIF-8用于肿瘤化学/光热协同治疗

利用金属-有机框架材料ZIF-8包裹二硫化钼(MoS_2)纳米片和阿霉素(DOX)构建一种可通过酸性pH和近红外(NIR)光双触发的肿瘤化学/光热协同治疗体系。首先,通过水热反应和超声处理制备粒径为～100 nm、厚度为0.3～1.4 nm的MoS_2纳米片。然后,通过一步法将可酸降解的金属-有机框架ZIF-8包裹在所制备的MoS_2纳米片上,并同时装载抗肿瘤药物DOX,形成装载DOX的ZIF-8包裹MoS_2纳米复合物(DOX/MoS_2@ZIF-8)。将该纳米复合物应用到肿瘤细胞的化学/光热协同治疗:当处于酸性条件(例如:溶酶体中pH大约为5)和NIR激光(780 nm,2.1 W/cm~2)照射的情况下,DOX/MoS_2@ZIF-8纳米复合物上包裹的ZIF-8金属-有机框架会发生酸降解,释放出所包裹的DOX,细胞质中的DOX可以进入细胞核中诱导细胞凋亡;同时,MoS_2纳米片能够将光能转换为热能,光致高温同样能诱导细胞凋亡,因此,化学/光热协同肿瘤治疗得以实现。细胞存活率试验证明:该DOX/MoS_2@ZIF-8纳米复合物在SMMC-7721细胞上表现出良好的化学/光热协同治疗作用,能够对肿瘤细胞进行高效地杀伤。     

新型γ-聚谷氨酸自组装纳米胶束的制备及用于蛋白载体的研究

对水溶性的γ-聚谷氨酸(γ-PGA)进行了接枝改性,合成了两亲性γ-聚谷氨酸(γ-PGA)接枝衍生物,采用超声探头法制备胆甾醇基γ-PGA自组装胶束,并以卵清蛋白(OVA)作为模型蛋白,研究其载药和释药性能.结果表明,制备的两亲性胆甾醇基γ-PGA自组装胶束平均粒径为299.6+ 27.3nm,粒径的多分散系数较窄(0.17),且具有较低的细胞毒性;其疏水核-亲水壳的纳米微结构对蛋白药物显示了良好载药性能,对OVA载药量可达118.8 μg/mg,包封率33.5％;体外释药结果显示,负载OVA的甾醇基γ-PGA自组装胶束能延缓蛋白的释放,释药速率与介质pH密切相关.     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Cytoskeleton and mitochondrial morphology and function

It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.     

Chemokines and cytokines: axis and allies in asthma and allergy

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。