Characterization of Polymeric Solutions as Injectable Vehicles for Hydroxyapatite Microspheres

A polymeric solution and a reinforcement phase can work as an injectable material to fill up bone defects. However, the properties of the solution should be suitable to enable the transport of that extra phase. Additionally, the use of biocompatible materials is a requirement for tissue regeneration. Thus, we intended to optimize a biocompatible polymeric solution able to carry hydroxyapatite microspheres into bone defects using an orthopedic injectable device. To achieve that goal, polymers usually regarded as biocompatible were selected, namely sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and Na-alginate (ALG). The rheological properties of the polymeric solutions at different concentrations were assessed by viscosimetry before and after moist heat sterilization. In order to correlate rheological properties with injectability, solutions were tested using an orthopedic device applied for minimal invasive surgeries. Among the three polymers, ALG solutions presented the most suitable properties for our goal and a non-sterile ALG 6% solution was successfully used to perform preliminary injection tests of hydroxyapatite microspheres. Sterile ALG 7.25% solution was found to closely match non-sterile ALG 6% properties and it was selected as the optimal vehicle. Finally, sterile ALG 7.25% physical stability was studied at different temperatures over a 3-month period. It was observed that its rheological properties presented minor changes when stored at 25°C or at 4°C.     

生物可降解微球作为HPV-E7 蛋白免疫佐剂的研究

目的:对聚乳酸聚羟基乙酸(PGLA)作为疫苗运输载体进行免疫学评价。方法:用复乳法制备PLGA微球,通过表面吸附人乳头状瘤病毒(HPV)E7蛋白制备成聚乳酸聚羟基乙酸(PGLA)微球,考察粒径分布情况及体外释放水平,通过皮下免疫注射途径免疫C57BL/6小鼠,用间接ELISA法检测免疫鼠血清中的抗体水平,由此评价PLGA微球疫苗运输载体的佐剂效应。。结果:复乳法制备的PLGA微球表面光滑,大小均匀,包封率20.1%,注射小鼠6周后(第2周加强免疫1次),微球疫苗诱导产生的IgG1抗体水平较同剂量的铝佐剂组和溴化二甲基双十八胺(DDA)组明显升高,(平均滴度分别为3805、1270、2262);微球疫苗诱导产生IgG2b的抗体水平明显高于铝佐剂组,略低于DDA组,(平均滴度分别为1131、475、2653)而IgG2c的抗体量高于铝佐剂组和DDA组(平均滴度分别为150、36、106)。结论:人乳头状瘤病毒E7蛋白聚乳酸羟基乙酸微球作为疫苗输送体系可以明显的提高抗原的免疫原性。     

生物可降解微球作为HPV-E7蛋白免疫佐剂的研究

目的：对聚乳酸聚羟基乙酸（PGLA）作为疫苗运输载体进行免疫学评价。方法：用复乳法制备PLGA微球,通过表面吸附人乳头状瘤病毒（HPV）E7蛋白制备成聚乳酸聚羟基乙酸（PGLA）微球,考察粒径分布情况及体外释放水平,通过皮下免疫注射途径免疫C57BL/6小鼠,用间接ELISA法检测免疫鼠血清中的抗体水平,由此评价PLGA微球疫苗运输载体的佐剂效应。。结果：复乳法制备的PLGA微球表面光滑,大小均匀,包封率20.1%,注射小鼠6周后（第2周加强免疫1次）,微球疫苗诱导产生的IgG1抗体水平较同剂量的铝佐剂组和溴化二甲基双十八胺（DDA）组明显升高,（平均滴度分别为3805、1270、2262）;微球疫苗诱导产生IgG2b的抗体水平明显高于铝佐剂组,略低于DDA组,（平均滴度分别为1131、475、2653）而IgG2c的抗体量高于铝佐剂组和DDA组（平均滴度分别为150、36、106）。结论：人乳头状瘤病毒E7蛋白聚乳酸羟基乙酸微球作为疫苗输送体系可以明显的提高抗原的免疫原性。     

Sustained-Release Delivery of Octreotide from Biodegradable Polymeric Microspheres

The study reports on the drug release behavior of a potent synthetic somatostatin analogue, octreotide acetate, from biocompatible and biodegradable microspheres composed of poly-lactic-co-glycolic acid (PLGA) following a single intramuscular depot injection. The serum octreotide levels of three Oakwood Laboratories formulations and one Sandostatin LAR^® formulation were compared. Three formulations of octreotide acetate-loaded PLGA microspheres were prepared by a solvent extraction and evaporation procedure using PLGA polymers with different molecular weights. The in vivo drug release study was conducted in male Sprague–Dawley rats. Blood samples were taken at predetermined time points for up to 70 days. Drug serum concentrations were quantified using a radioimmunoassay procedure consisting of radiolabeled octreotide. The three octreotide PLGA microsphere formulations and Sandostatin LAR^® all showed a two-phase drug release profile (i.e., bimodal). The peak serum drug concentration of octreotide was reached in 30 min for all formulations followed by a decline after 6 h. Following this initial burst and decline, a second-release phase occurred after 3 days. This second-release phase exhibited sustained-release behavior, as the drug serum levels were discernible between days 7 and 42. Using pharmacokinetic computer simulations, it was estimated that the steady-state octreotide serum drug levels would be predicted to fall in the range of 40–130 pg/10 μL and 20–100 pg/10 μL following repeat dosing of the Oakwood formulations and Sandostatin LAR^® every 28 days and every 42 days at a dose of 3 mg/rat, respectively.     

蛋白质特异性断裂试剂研究进展

蛋白质特异性断裂试剂是近年来发展起来的一些具有特异性断裂肽键功能的化学工具．这些断裂试剂可分为两类,一类通过氧化断裂机制实现蛋白空间结构特异性切割,另一类通过水解断裂机制实现序列特异性切割．蛋白质特异性断裂试剂在蛋白质序列测定,蛋白质的结构与功能研究,蛋白质与核酸相互作用研究以及新型化学治疗药物的合成等方面有着广阔的应用前景．     

Biocompatible Anionic Polymeric Microspheres as Priming Delivery System for Effetive HIV/AIDS Tat-Based Vaccines

Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6P_cy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4⁺ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4⁺ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.     

蛋白转导域研究进展

蛋白转导域(PTDs)是小分子多肽,又称为细胞渗透性蛋白(CPP)或转膜序列(MTS),它可以不依赖于经典的细胞内吞,将多种大分子物质导入细胞内。因此,PTDs被认为是一种理想的运载工具,在将蛋白和其他分子导入活细胞的研究中有着广泛的应用前景。本文着重综述PTDs的跨膜转运机制及其应用等方面的研究进展。     

肌动蛋白集束调控因子及G蛋白信号转导通路

细胞骨架由微丝、微管及中等纤维组成受不同蛋白因子调控以不同方式组装成不同直径的纤维 ,遍布于一切细胞 ,决定细胞的形状 ,赋予其抗压强度 ,对细胞器及大分子进行空间组织 ,实现胞内的能量转换。在肌动蛋白 (ａｃｔｉｎ)组装成张力纤维和张力纤维解离成肌动蛋白单体过程中有多种蛋白因子参与调控 ,从而使细胞骨架处于一个生理的动态平衡中 ,执行和完成不同的生化反应。在众多的调控蛋白中 ,肌动蛋白集束调控蛋白因子 (ａｃｔｉｎｂｕｎｄｌｉｎｇｐｒｏｔｅｉｎ)不仅参与肌动蛋白结构调节 ,还与细胞内信号传导有密切关系。已发现的肌动蛋…     

白藜芦醇分子印迹聚合物微球的制备及特性评价(英文)

以聚苯乙烯微球为种球,白藜芦醇为模板分子,采用单步溶胀聚合法在N,N-二甲基甲酰胺体系中制备了单分散分子印迹聚合物微球。用扫描电镜对微球的结构和形貌进行了表征,并研究了微球的制备条件和吸附特性。微球的凹陷可有效地增加微球的比表面积和结合位点,从而提高了模板分子的结合速率及微球的印迹容量。     

Thylakoid Membrane Protein Kinase Activity as a Signal Transduction Pathway in Chloroplasts

In plants external stimuli are perceived through a cascade of signals and signal transduction pathways. Protein phosphorylation and de-phosphorylation is one of the most important transduction paths for the perception of signals in plants. The highest concentrations of plant phospho-proteins are located in chloroplasts. This facilitates the protection of thylakoid membranes from stress-induced damage and augments adaptive strategies in plants. In this review, the protein kinases associated with phosphorylation of thylakoid membrane protein, and the adaptive changes in thylakoid membrane architecture and developmental cues are given. The presence of membrane bound kinases in thylakoid membranes have evolutionary implications for the signal transduction pathways and the photosynthetic gene expression for thylakoid membrane protein dynamics. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thylakoid Membrane Protein Kinase Activity as a Signal Transduction Pathway in Chloroplasts

Photosynthetica - In plants external stimuli are perceived through a cascade of signals and signal transduction pathways. Protein phosphorylation and de-phosphorylation is one of the most important...     

Revised Role of Glycosaminoglycans in TAT Protein Transduction Domain-mediated Cellular Transduction

Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.     

Protein Clusters in Phosphotyrosine Signal Transduction

Signal transduction systems based on tyrosine phosphorylation are central to cell–cell communication in multicellular organisms. Typically, in such a system, the signal is initiated by activating tyrosine kinases associated with transmembrane receptors, which induces tyrosine phosphorylation of the receptor and/or associated proteins. The phosphorylated tyrosines then serve as docking sites for the binding of various downstream effector proteins. It has long been observed that the cooperative association of the receptors and effectors produces higher-order protein assemblies (clusters) following signal activation in virtually all phosphotyrosine signal transduction systems. However, mechanistic studies on how such clustering processes affect signal transduction outcomes have only emerged recently. Here we review current progress in decoding the biophysical consequences of clustering on the behavior of the system, and how clustering affects how these receptors process information.     

尺寸均一的壳聚糖微球的制备及其作为胰岛素控释载体的研究

采用新型微孔膜乳化技术制备了载胰岛素的壳聚糖微球。研究表明,要制备粒径均一的壳聚糖微球,必须将亲水性膜修饰成疏水性;制得的微球粒径和所采用的膜孔径之间存在很好的线性关系,使得微球粒径可控;以胰岛素为模型药物,主要考察了交联剂用量和交联时间对微球表面形态、药物包埋率和微球体外释药特性的影响。结果表明当氨基与醛基的摩尔比为1∶0.7、交联时间为1h时,所得载药微球的包埋率最高,随着戊二醛用量的增加和交联时间的延长,药物体外释放速率减慢。     

TAT蛋白转导肽介导的秀丽线虫体内外源蛋白的跨膜转导研究

TAT蛋白转导肽是HIV-1病毒编码的一段富含碱性氨基酸序列的多肽,能够高效介导多种外源生物大分子通过多种膜性结构,如细胞质膜和血脑屏障等。为探索TAT蛋白转导肽介导的秀丽线虫体内外源蛋白跨膜转导作用,以EGFP为报告基因结合常规分子克隆技术构建了原核表达载体pET28b-EGFP和pET28-TAT-EGFP,继而利用诱导剂IPTG(终浓度1mmol/L)诱导表达了靶蛋白并结合荧光显微观察、SDS-PAGE和Western blot等鉴定技术获得表达靶蛋白的大肠杆菌BL21(DE3)细胞,最后将其涂布到含有Kana⁺的LB固体培养基上直接饲喂野生型N2株系线虫,利用荧光显微镜观察绿色荧光信号在线虫体内的分布。结果证明,TAT-EGFP融合蛋白较之于EGFP可高效、可溶性表达,而且通过直接饲喂秀丽线虫表达靶蛋白的大肠杆菌48小时后,TAT-EGFP荧光信号明显分布于线虫肠壁细胞,而EGFP荧光信号则分布在秀丽线虫肠腔,空载体对照组未见任何荧光信号,说明TAT蛋白转导肽能够高效介导外源蛋白在秀丽线虫体内跨膜转导。同时,通过比较空载体对照组与实验组线虫微分干涉图像,未见线虫出现明显的细胞形态变化,说明TAT蛋白转导肽介导的外源蛋白跨膜转导作用是安全的,为在秀丽线虫体内直接研究外源蛋白的功能以及进行蛋白药物的研发提供了重要参考。     

TAT蛋白转导域：蛋白质治疗的新曙光

TAT蛋白转导域是源自人类免疫缺陷病毒Tat蛋白的一段碱性氨基酸多肽,能够将与之共价连接的多肽、蛋白、核酸等生物大分子快速而高效地转导入细胞内部,在药物转运和疾病治疗等领域有着巨大的应用潜力.TAT蛋白转导域首先通过电荷相互作用吸附于细胞膜,然后通过脂筏介导的巨胞饮作用进入细胞.随着体外研究的不断成熟,应用TAT蛋白转导域治疗人类肿瘤、卒中、炎症等疾病的动物模型也获得了成功,TAT蛋白转导域进入临床指日可待.     

Cell Membrane Diversity in Noncovalent Protein Transduction

Crossing of the plasma membrane for all macromolecules without energy, receptors or any artificial methods was thought to be difficult. Our previous studies demonstrated that arginine-rich intracellular delivery (AID) peptides are able to deliver macromolecules, such as proteins, RNAs and DNAs, into either animal or plant cells. Cellular internalization could be mediated by effective and nontoxic AID peptides in either a covalent or noncovalent protein transduction (NPT) manner. AID peptides were so versatile that the procedure seemed to replace the current artificial transfection methods. However, the utilization of AID peptides has been limited to animal or plant systems so far. None has proposed that AID peptides could work in other species. Here, we select some representative organisms to screen whether NPT mediated by AID peptides works in them. They include cyanobacteria, bacteria, archaea, algae, fungi and yeasts. The results reveal that not all living beings possess this capability of protein transduction. Interestingly, all species of prokaryotes tested, which were thought to be highly diverse from the animal and plant systems, appear to be capable of NPT. The mechanism of AID-mediated NPT in cyanobacteria is in a classical endocytosis- and energy-independent pathway and may involve macropinocytosis. In contrast, green algae and multicellular fungi of the eukaryotes are impermeable to protein passage. Our results bring an interesting clue to the reexamination of the phylogeny of both algae and fungi.     

骨形态发生蛋白的受体及其信号传递过程

近年来已克隆出几种Ⅰ型和Ⅱ型BMP受体.BMP受体属于TGFβ受体超家族的成员,具有丝氨酸/苏氨酸蛋白激酶活性.Ⅰ型与Ⅱ型BMP受体均可与BMP配基结合,但若执行传递信号功能,两受体需首先形成复合物.删除突变和基因剔除研究证明,ⅠA型BMP受体对动物的中胚层发育至关重要.而ⅠB型BMP受体在软骨细胞形成、成骨细胞分化以及程序化细胞死亡方面起主要作用.BMP受体信号传递分子Smad 1和Smad 5也已被克隆和鉴定,它们在BMP受体介导的功能中起重要作用.     

蛋白转导域透膜作用的研究进展

HIV-TAT蛋白转导域（Protein transduction domain,,PTD）是新近发现的一种在蛋白转导过程中能高效穿过生物膜的结构域,源自人类免疫缺陷病毒Tat蛋白的一段碱性氨基酸多肽,能与多肽、蛋白质及DNA等分子连接并跨膜导入绝大部分的组织细胞或透过血脑屏障,转导效率高且对细胞无损伤。TAT-PTD与细胞膜之间的电荷作用,吸附于膜表面,依赖脂筏介导的巨胞饮作用进入细胞。TAT融合蛋白系统是一种极有价值的运载工具,在基础医学研究和临床治疗方面有着广泛的应用前景。