用于疫苗的免疫增强剂和递送系统

疫苗是抵抗传染病的最重要的预防性手段。在开发有效疫苗的过程中,人们面临的挑战一方面是要鉴定最为相关的免疫原及有效的免疫接种程序,另一方面则是免疫原用不同的能够影响抗体应答亲和性成熟和细胞介导的免疫应答的佐剂配成制剂。因此,有必要开发既能激发天然免疫应答,又能激发适应性免疫应答的有效佐剂和递送系统。主要阐述了不同免疫增强剂以及可利用于新一代疫苗的递送系统的研制进展情况。     

炎性体及其在流感病毒感染过程中的作用

炎性体是胞液中感受危险信号、启动介导下游免疫防御或细胞死亡（pyroptosis）的多分子复合物,是细胞内天然免疫的重要受承信号转导的中介体.炎性体识别流感病毒后诱导先天免疫反应甚至pyroptosis样细胞死亡.流感病毒高尔基体表达的M2蛋白和P2X7、ATP、ROS在炎性体的调节过程中发挥了重要作用,微生物也可以通过激活炎性体调节呼吸道粘膜免疫.炎性体的提出为最优疫苗的设计提供了新的思路.     

以串联式乙肝病毒核心颗粒呈递的基于多重保守抗原的甲型流感病毒病毒样粒子候选疫苗

现有的甲型流感病毒(IAV)疫苗是靶向病毒的不同部分,不同季节可能是不一样的。疫苗的设计依赖于预测的优势流感毒株,而当疫苗和流行毒株之间不匹配时功效欠佳。此外,目前方法制得的疫苗对有可能引起大流行病的新兴流感毒株只提供有限的保护。有一种解决方案是设计靶向流感病毒保守的蛋白结构区域的疫苗,这些结构域随着时间的推移基本保持不变,并可能存于在新兴变体中。     

在体电脉冲基因导入技术研究进展

基因治疗是把DNA或RNA等外源物质输送到靶细胞,以改善由于基因缺陷和变异引起的疾病,达到治疗目的。有效的基因治疗依赖于外源基因高效而稳定的表达以及有效载体介导的基因输送。电脉冲介导的基因导入技术以其高效率导入得到了广泛的应用,现就在体电脉冲基因导入技术的概况、装置、机理及应用作一综述。     

B型流感病毒血凝素的表达及免疫原性测定北大核心CSCD

B型流感病毒是引起季节性流感的原因之一,严重时会造成重大疾病或死亡。为了检测B型流感病毒2个疫苗候选毒株的血凝素(hemagglutinin,HA)蛋白胞外段在哺乳动物细胞中的表达及在小鼠体内的免疫原性,本研究将带有三聚体标签的HA胞外段(HA-ectodomain,HA-ecto)序列及神经氨酸酶(neuraminidase,NA)全长编码框经密码子优化后构建至pCAGGS载体中,通过线性聚乙烯亚胺将pCAGGS-HA-ecto与pCAGGS-NA共转染293T细胞。收集转染后96h的上清,通过镍离子亲和层析及分子筛层析获得三聚体形式的HA-ecto蛋白,然后将HA-ecto三聚体蛋白免疫小鼠,进行酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)及血凝抑制实验(hemagglutination inhibition,HAI)检测HA-ecto蛋白诱导小鼠后产生的抗体水平。纯化结果显示,通过哺乳动物细胞表达系统能够得到分泌型表达的三聚体HA-ecto蛋白。ELISA及HAI结果显示,三聚体HA-ecto蛋白二次免疫小鼠后,能诱导小鼠产生较高水平的同源和异源交叉抗体。以上结果表明,哺乳动物细胞表达的B型流感病毒HA蛋白可作为亚单位重组流感疫苗的候选。     

基于非刚体配准的体数据插值重建方法

三维图像的处理和操作需要将一般的断层序列插值成为具有各坐标轴一致的分辨率的体数据,而目前最常用的线性插值方法在层间距较大时会导致图像边缘模糊和出现伪影。Penney根据现有的非刚体匹配方法,提出了利用图像形变场数据的插值算法,大大提高了层间插值的质量。本文对Penney提出的算法进行了两方面的改进,在配准过程中用简单的单射性约束取代了复杂的平滑性约束,用邻域平均算法替代Penney使用的最邻近直线插值方法,并将新算法的实验结果与原算法、线性插值进行了对比,新算法在保持高质量插值的前提下提高了计算速度。该算法可以应用于精度要求比较高的体数据插值重建过程。     

杆状病毒表达系统——有效的VLP构建工具

杆状病毒表达系统是以杆状病毒为外源基因载体,昆虫细胞或活体昆虫为受体的真核表达系统。相对于其他表达系统,杆状病毒表达系统具有特殊的优势:杆状病毒基因组作为表达载体可以容纳更多外源基因;杆状病毒极晚期启动子能有效调控外源蛋白的表达;昆虫细胞作为受体能够对外源蛋白进行加工修饰;杆状病毒通常只感染节肢动物,不会对人畜构成危害。因此,该系统越来越受到人们的重视,并已应用于亚单位疫苗的研发与生产,特别其对于构建病毒样颗粒,即由一种或多种病毒结构蛋白自行装配而成且不含病毒基因组的蛋白颗粒,具有不可比拟的优势。对此做详细评述并展望病毒样颗粒疫苗的发展趋势。     

制药者的新宠——昆虫细胞反应系统

制药者们为昆虫细胞反应器所痴迷,因为该系统在基因治疗,疫苗生产等诸多方面都有其独特的优势。昆虫是地球上生物多样性最高的种群之一。虽然在非专业人士看来,它们显得比较原始,但事实上它们的确是非常复杂的。昆虫是多细胞生物,他们有许多特化的器官和系统,其中包括高度进化的神经系统,所以近几十年来一直是生物学研究的热点。     

组胺H2受体拮抗剂对骨髓造血重建的影响

用组胺H_2受体拮抗剂(甲氰咪胍或呋喃硝胺)处理正常和亚致死量γ-射线照射小鼠,探讨正常体内造血和再生骨髓中造血重建与组胺受体的关系。发现非毒性剂量的甲氰咪胍对正常小鼠骨髓多能造血干细胞(CFU-s)无抑制作用,但可抑制小鼠体内粒单系祖细胞(CFU-GM)的生长和亚致死量照射后CFU-s产率的恢复。组胺可能与骨髓的再生有关,组胺H_2受体拮抗剂可抑制骨髓的造血重建。     

Delivery of Foreign Substances to Cells Mediated by Fusion-Active Reconstituted Influenza Virus Envelopes (Virosomes)

Abstract

This paper presents a survey of the properties and applications of reconstituted influenza virus envelopes (virosomes). Influenza virosomes can be reconstituted from the original viral membrane lipids and spike glycoproteins, after solubilization of intact virus with octaethyleneglycol monododecyl ether (C₁₂E₈) and removal of this detergent with a hydrophobic resin (BioBeads SM-2). These virosomes are functionally active, i.e their membrane fusion activity closely mimics the well-defined low-pH-dependent membrane fusion activity of the intact virus, which is solely mediated by the viral hemagglutinin (HA). By virtue of their fusion activity, virosomes represent a powerful carrier system for cellular delivery of foreign substances, encapsulated in their aqueous interior or co-reconstituted in their membranes. Delivery of an encapsulated, water-soluble, compound is illustrated with data on the toxin gelonin. Protein synthesis in BHK-21 cells in culture is efficiently inhibited when gelonin-containing virosomes fuse from within endosomes, after internalization via receptor-mediated endocytosis, or are induced to fuse with the plasma membrane by a transient lowering of the pH in the medium. The results indicate that delivery is quite efficient; as much as 6 × 10³ molecules of gelonin can readily be delivered to the cytoplasm of a single cell by fusion with gelonin-containing virosomes.     

生物可降解聚合物纳米粒给药载体

生物可降解聚合物纳米粒用于给药载体具有广阔的前景。本文综述了生物可降解聚合物纳米粒给药载体领域的最新进展 :包括纳米粒表面修饰特性、药物释放、载多肽和蛋白质等生物大分子药物传输中的潜在应用。     

Targeted Intracellular Delivery of Resveratrol to Glioblastoma Cells Using Apolipoprotein E-Containing Reconstituted HDL as a Nanovehicle

The objective of this study is to transport and deliver resveratrol to intracellular sites using apolipoprotein E3 (apoE3). Reconstituted high-density lipoprotein (rHDL) bearing resveratrol (rHDL/res) was prepared using phospholipids and the low-density lipoprotein receptor (LDLr)-binding domain of apoE3. Biophysical characterization revealed that resveratrol was partitioned into the phospholipid bilayer of discoidal rHDL/res particles (~19 nm diameter). Co-immunoprecipitation studies indicated that the LDLr-binding ability of apoE3 was retained. Cellular uptake of resveratrol to intracellular sites was evaluated in glioblastoma A-172 cells by direct fluorescence using chemically synthesized NBD-labeled resveratrol (res/NBD) embedded in rHDL/res. Competition and inhibition studies indicate that the uptake is by receptor mediated endocytosis via the LDLr, with co-localization of apoE3 and res/NBD in late endosomes/lysosomes. We propose that rHDL provides an ideal hydrophobic milieu to sequester resveratrol and that rHDL containing apoE3 serves as an effective “nanovehicle” to transport and deliver resveratrol to targeted intracellular sites.     

Evaluation of Polyethylene Oxide Compacts as Gastroretentive Delivery Systems

Compacts containing selected bioadhesive polymers, fillers, and binders were investigated for their potential as a bioadhesive gastroretentive delivery system to deliver water soluble and water insoluble compounds in the stomach. Compacts with 90:10, 75:25, and 60:40 of polyvinylpyrrolidone (PVP) and polyethylene oxide (PEO) were evaluated for swelling, dissolution, bioadhesion, and in vitro gastric retention. Compacts containing higher PEO showed higher swelling (111.13%) and bioadhesion (0.62 ± 0.03 N/cm²), and retained their integrity and adherence onto gastric mucosa for about 9 h under in vitro conditions. In vivo gastroretentive property of compacts were evaluated in Yorkshire cross swines. Compacts containing 58% PVP, 40% PEO and 2% of water soluble or water insoluble marker compounds showed gastroadhesive and retentive properties in vivo. It is concluded that PEO in combination with PVP yields a non disintegrating type bioadhesive dosage form which is suitable for gastroretentive applications. A part of this study has been presented at the Controlled Release Society’s symposium held at Vienna, 2006.     

Solid Lipid Nanoparticles as Delivery Systems for Bioactive Food Components

The inclusion of bioactive compounds, such as carotenoids, omega-3 fatty acids, or phytosterols, is an essential requisite for the production of functional foods designed to improve the long-term health and well-being of consumers worldwide. To incorporate these functional components successfully in a food system, structurally sophisticated encapsulation matrices have to be engineered, which provide maximal physical stability, protect ingredients against chemical degradation, and allow for precise control over the release of encapsulated components during mastication and digestion to maximize adsorption. A novel encapsulation system initially developed in the pharmaceutical industries to deliver lipophilic bioactive compounds is solid lipid nanoparticles (SLN). SLN consist of crystallized nanoemulsions with the dispersed phase being composed of a solid carrier lipid–bioactive ingredient mixture. Contrary to larger colloidal solid lipid particles, specific crystal structures can be “dialed-in” in SLN by using specific surfactant mixtures and ensuring that mean particle sizes are below 100–200 nm. Moreover, in SLN, microphase separations of the bioactive compound from the solidifying lipid matrix can be prevented resulting in an even dispersion of the encapsulated compound in the solid matrix thereby improving chemical and physical stability of the bioactive. In this review article, we will briefly introduce the structure, properties, stability, and manufacturing of solid lipid particles and discuss their emerging use in food science.     

Evaluation of Three Cell Culture Systems as Substrates for Influenza Virus Assay

The propagation of various influenza virus strains in primary rhesus monkey kidney (RMK), primary hamster kidney (HK), and the MDCK-USD canine kidney (CK) cell cultures was compared. Virus-infected cultures were examined for cytopathic effect (CPE) and by performing the hemadsorption (HAd) technique. The highest HAd titers were found most often in HK, followed by RMK and CK. However, the CK cell line provided the best substrate for detecting CPE. Although influenza B strains tended to grow to higher titers, these strains produced less CPE than did the A strains.     

Monophosphoryl Lipid A‐Adjuvanted Virosomes with Ni‐Chelating Lipids for Attachment of Conserved Viral Proteins as Cross‐Protective Influenza Vaccine

Induction of CD8⁺ cytotoxic T cells (CTLs) to conserved internal influenza antigens, such as nucleoprotein (NP), is a promising strategy for the development of cross‐protective influenza vaccines. However, influenza NP protein alone cannot induce CTL immunity due to its low capacity to activate antigen‐presenting cells (APCs) and get access to the MHC class I antigen processing pathway. To facilitate the generation of NP‐specific CTL immunity the authors develop a novel influenza vaccine consisting of virosomes with the Toll‐like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) and the metal‐ion‐chelating lipid DOGS‐NTA‐Ni incorporated in the membrane. In vitro, virosomes with incorporated MPLA induce stronger activation of APCs than unadjuvanted virosomes. Virosomes modified with DOGS‐NTA‐Ni show high conjugation efficacy for his‐tagged proteins and facilitate efficient uptake of conjugated proteins by APCs. Immunization of mice with MPLA‐adjuvanted virosomes with attached NP results in priming of NP‐specific CTLs while MPLA‐adjuvanted virosomes with admixed NP are inefficient in priming CTLs. Both vaccines induce equally high titers of NP‐specific antibodies. When challenged with heterosubtypic influenza virus, mice immunized with virosomes with attached or admixed NP are protected from severe weight loss. Yet, unexpectedly, they show more weight loss and more severe disease symptoms than mice immunized with MPLA‐virosomes without NP. Taken together, these results indicate that virosomes with conjugated antigen and adjuvant incorporated in the membrane are effective in priming of CTLs and eliciting antigen‐specific antibody responses in vivo. However, for protection from influenza infection NP‐specific immunity appears not to be advantageous.     

Saccharomyces cervisiae as an Efficient Carrier for Delivery of Bioactives: a Review

Food Biophysics - Recently, there has been growing interest in usingnatural vesicles for encapsulation of variousfood-grade ingredients. The structure ofyeast cells, along with their presence in...     

可注射更昔洛韦温敏型原位凝胶剂的研究

目的：构建温敏型三嵌段共聚物,研究其理化性质以及用其制备的可注射更昔洛韦温敏型原位凝胶剂的制剂特性。方法：以聚乙二醇（PEG）作为亲水嵌段,丙交酯（LA）和β- 丁内酯（β-BL）的无规共聚物PBLA 作为疏水嵌段,采用开环聚合法合成温敏型三嵌段共聚物PBLA-PEG-PBLA,并对其理化性质进行表征,考察其溶液的胶凝温度/ 临界凝胶浓度、流变学性质、通针性和溶蚀行为以及以更昔洛韦作为模型药物、用其制得的可注射载药温敏型原位凝胶剂的体外释放特性。结果：合成的PBLA-PEG-PBLA 嵌段共聚物重均分子质量在6 000 左右,多分散系数为1.5 左右;其溶液临界凝胶浓度（g?mL-1）为5%~10%,质量浓度（g?mL-1）在10%~25% 时胶凝温度为31~35 ℃,接近并略低于体温;其凝胶在低温下储能模量与黏度较小,当温度接近相转变温度后两者迅速增大;其载药凝胶剂累计释放量经拟合显示遵循一级动力学方程,并呈扩散释药机制。结论：较低质量浓度[10%~15%（g?mL-1）] 的PBLA-PEG-PBLA 更符合玻璃体注射要求,更适用于制备可注射载药温敏型原位凝胶剂。