丝素微球的制备方法研究进展

丝素蛋白因其特殊的物理化学性质,为制备不同形态的材料提供了依据;丝素蛋白也因其良好的生物相容性、生物降解性为生物材料应用提供了重要的临床选择。本文综述了国内外学者对丝素微球的制备方法的探究,包括乳化法、喷雾干燥法、层流射流技术以及自组装方法,并比较了各种制备方法的优缺点。此外,本文还介绍了丝素微球在药物缓释领域中的应用进展,并提出了几个针对制备丝素微球亟需解决的问题。     

Characteristics of microspheres formed in PCR with bacterial genomic DNA or plasmid DNA as templates

Earlier, we discovered that, along with linear DNA fragments, nano- and microparticles of DNA and their aggregates are formed in the PCR with yeast genomic DNA used as a template and gene-specific or partially complementary primers. The size of the microparticles (microspheres) varied in the range of 0.5 to 3–4 μm. Only thermostable KlenTaq polymerase but not Taq polymerase could effectively generate microspheres. In this work, we demonstrate that KlenTaq polymerase can produce microspheres of variable size (1 to 7 μm in diameter) if genomic DNA of the bacterium Acidithiobacillus ferrooxidans and partially complementary primers are present in the PCR mixture. Conditions for generation of DNA microparticles in PCR with Taq-polymerase and bacterial genomic DNA as template were also elaborated. It was also found that mainly large microspheres of up to 7 μm accumulated in PCR with plasmid DNAs used as templates and gene-specific primers in the presence of KlenTaq polymerase or mixtures of KlenTaq and Pfu polymerases. Besides, small aggregates, as well as linear branched structures and three-dimensional conglomerates of fused microspheres, were also revealed in the PCR mixtures. UV absorption spectra of native DNA microspheres and microspheres that had undergone heating at 93°C were registered. The key role of Mg²⁺ cations in the formation and stabilization of the microsphere structure was established.     

Multiplexed microsphere diagnostic tools in gene expression applications: factors and futures

Microarrays have received significant attention in recent years as scientists have firstly identified factors that can produce reduced confidence in gene expression data obtained on these platforms, and secondly sought to establish laboratory practices and a set of standards by which data are reported with integrity. Microsphere-based assays represent a new generation of diagnostics in this field capable of providing substantial quantitative and qualitative information from gene expression profiling. However, for gene expression profiling, this type of platform is still in the demonstration phase, with issues arising from comparative studies in the literature not yet identified. It is desirable to identify potential parameters that are established as important in controlling the information derived from microsphere-based hybridizations to quantify gene expression. As these evolve, a standard set of parameters will be established that are required to be provided when data are submitted for publication. Here we initiate this process by identifying a number of parameters we have found to be important in microsphere-based assays designed for the quantification of low abundant genes which are variable between studies.     

碳酸氢钠+载药微球经肝动脉化疗栓塞治疗中晚期肝癌的临床效果

目的:探讨碳酸氢钠+载药微球经肝动脉化疗栓塞治疗中晚期肝癌的临床效果及安全性。方法:选择2015年2月到2018年6月在我院诊治的中晚期肝癌患者78例,根据随机数字表法将其均分为两组,每组各39例。对照组给予载药微球经肝动脉化疗栓塞治疗,实验组给予碳酸氢钠+载药微球经肝动脉化疗栓塞治疗,比较两组的临床疗效,治疗前后CD3~+CD4~+、CD3~+CD8~+T细胞比例的变化,治疗期间不良反应的发生情况及预后。结果:治疗后,实验组与对照组的治疗总有效率分别为74.4%和43.6%,实验组显著高于对照组(P0.05)。实验组治疗期间的发热、腹痛、腹胀、呕吐等不良反应发生情况与对照组的对比差异无统计学意义(P0.05)。两组治疗前后CD3CD4~+T比例对比差异无统计学意义(P0.05),两组治疗后的CD3~+CD8~+T比例显著低于治疗前,且实验组明显低于对照组(P0.05)。治疗后,实验组的躯体功能、心理功能、社会功能、共性症状及副作用评分都低于对照组(P0.05)。结论:碳酸氢钠+载药微球经肝动脉化疗栓塞治疗中晚期肝癌能提高治疗效果,改善机体的免疫功能,提高患者的生活质量,且不会增加不良反应。     

固定化微球降解土壤中PAEs效果及影响因素

旨为研究土壤邻苯二甲酸酯污染修复中,固定化微球降解土壤中邻苯二甲酸酯的效果及影响因素。以海藻酸钠为载体,采用包埋法对课题组前期提取的微小杆菌进行固定化,比较固定化微球和游离菌降解土壤中邻苯二甲酸酯(Phthalates esters,PAEs)的效果及pH、温度、重金属、无机盐等对降解菌降解目标物的影响。结果显示:(1)在土壤环境相同条件下,固定化微球对邻苯二甲酸二甲酯(Dimethyl ortho-phthalate,DMP)、邻苯二甲酸二正丁酯(Di-n-butyl ortho-phthalate,DnBP)和邻苯二甲酸二(2-乙基己)酯(Bis(2-ethylhexyl)ortho-phthalate,DEHP)的降解效果高于游离菌,DMP在7 d可降解完全,DnBP在10 d内可降解完全,DEHP在20 d降解率63.73%;而游离菌则在15 d内完全降解DMP,20 d内完全降解DnBP,DEHP在20 d降解率48.77%;(2)不同pH值时,固定化微球对DMP、DnBP、DEHP的降解率均高于游离菌,pH9时,固定化微球对于DMP、DnBP、DEHP的降解率最高分别为96.81%、89.39%、58.35%;(3)不同温度,固定化微球对DMP、DnBP、DEHP的降解率也均高于游离菌,温度为30℃时,固定化微球对于DMP、DnBP、DEHP的降解效率达到最高,分别为96.27%、89.19%、59.01%;(4)重金属使游离菌对DMP、DnBP、DEHP降解率下降较多,而使固定化微球对DMP、DnBP的降解率仅下降了16.35%、9.95%,DEHP不仅没有降低,反而增加2.49%,说明重金属对游离菌起到很强的抑制作用,但对于固定化微球的降解效果影响较小;(5)盐碱条件下,中性盐极大降低了游离菌和固定化微球降解DMP、DnBP、DEHP的降解能力,碱性盐和混合盐对降解菌影响较小,且增强了固定化微球对DnBP、DEHP的降解能力。固定化微球降解PAEs效果明显高于游离菌,对外界环境有更好的适应能力,且对重金属、无机盐污染环境有一定的抵御能力。     

Facile synthesis of magnetic metal organic frameworks for the enrichment of low‐abundance peptides for MALDI‐TOF MS analysis

In this work, core‐shell magnetic metal organic framework (MOF) microspheres were successfully synthesized by coating magnetite particles with mercaptoacetic acid and subsequent reactions with ethanol solutions of Cu(OAc)₂ and benzene‐1,3,5‐tricarboxylic acid (designated as H₃btc) alternately. The resulting Fe₃O₄@[Cu₃(btc)₂] possess strong magnetic responsiveness. We applied the novel nanocomposites in the enrichment of low‐concentration standard peptides, peptides in MYO and BSA tryptic digests and in human urine in combination with MALDI‐TOF MS analysis for the first time. In addition, the Cu₃(btc)₂ MOF shells exhibit strong affinity to peptides, thus providing a rapid and convenient approach to the concentration of low‐abundance peptides. Notably, peptides at an extremely low concentration of 10 pM could be detected by MALDI‐TOF MS after enrichment with the magnetic MOF composites. In brief, the facile synthesis and efficient enrichment process of the Fe₃O₄@[Cu₃(btc)₂] microspheres make them promising candidates for the isolation of peptides in even complex biological environments.     

Cholesterol biosensors based on oxygen sensing alginate-silica microspheres

Cholesterol determination in body is important in diagnosis of diseases like coronary heart disease, arteriosclerosis, diabetes, and obstructive jaundice. This research aims at developing fluorimetric cholesterol biosensors based on self-assembled mesoporous alginate-silica (Algilica) microspheres. For preparing the biosensor, Pt-(II)-octaethylporphine (PtOEP; oxygen sensitive metalloporphyrin) dye has been loaded in the Algilica microspheres using the solvent-mediated precipitation method. Cholesterol oxidase (ChOx) was then covalently conjugated to PtOEP/Algilica microspheres using EDC and NHS reagents. PtOEP dye and enzyme encapsulation, activity and stability were then analyzed. Layer-by-layer self-assembly was finally performed using PAH and PSS polyelectrolytes to minimize leaching of the biosensor components. The prepared biosensor exhibited linearity over a range of 0.77-2.5 mM O(2) (K(SV) : 0.097/mM of O(2) ) obtained using from Stern-Volmer plots. The biosensor response to standard cholesterol displayed a linear analytical range from 1.25 to 10 mM of cholesterol with regression coefficient of 0.996 (1.25-3.75 mM), 0.976 (1.25-6 mM), and 0.959 (1.25-10 mM) and response time of 10 min. Thus, the prepared cholesterol biosensor shows great potential in the diagnosis of hypercholesterolemia.     

阳离子β-环糊精聚合物微球的合成与表征

以β-环糊精(β-CD)为原料,环氧氯丙烷(ECH)为交联剂,通过反相乳液合成了β-环糊精聚合物(β-CDP)微球,通过醚化剂N-(2,3-环氧丙基)三甲基氯化铵(GTA)与β-CDP微球反应制得了阳离子型β-CDP微球。采用响应曲面试验,以取代度和反应效率的综合评分为指标,得出了阳离子型微球的最佳醚化工艺,分别使用扫描电子显微镜、激光粒度分布仪、红外光谱仪和综合热分析仪进行了表征,结果表明:GTA已被接枝到β-CDP微球上,且阳离子型β-CDP微球热稳定性良好;β-CDP微球1 g、GTA的质量0.0197 g,反应温度为47.78℃,反应时间为4.06 h,产品平均取代度28.3%,平均反应效率77.9%,综合得分17.56。     

左旋多巴甲酯缓释微球的研究

目的：由于长期服用左旋多巴治疗帕金森病,其药物浓度波动刺激易引起异动症,本实验旨在制备突释小,药物释放浓度稳定的左旋多巴甲酯微球制剂。方法：将左旋多巴甲酯用复乳法包裹于PLGA微球内,采用C18反相色谱研究药物包封率和体外释放行为。结果：通过调节药物浓度和不同高分子组合筛选出突释小,包封率高且缓慢释放的处方。结论：左旋多巴甲酯包裹于PLGA能实现理想的缓释效果,降低药物浓度波动,为后期药效学实验提供基础。     

Engineering a bilayered hydrogel to control ASC differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM). Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.