RNA病毒利用外泌体促进病毒感染的研究进展

外泌体是一种由细胞主动向胞外分泌的囊泡类小体,因其能在细胞间传递蛋白、脂类和核酸等分子,而被认为是一种新的重要的细胞间通讯方式。RNA病毒,如HIV-1、HCV等,作为一类重要的病原体,一直影响着全人类的健康。近来的研究发现,病毒能够利用外泌体的某些相关功能促进其复制与传播。然而,对外泌体与病毒感染的相关研究才刚刚起步,尚有很多方面并未被详细认知,所要研究的内容还有很多。本文主要总结了外泌体在一些RNA病毒感染中的促进作用及其可能的机制,以期让大家了解RNA病毒与外泌体之间已有的相互关系。     

外泌体在小RNA基因治疗中的研究进展

小RNA,包括小干扰RNA以及微小RNA,已成为多种疾病的潜在治疗药物。目前,小RNA的运输载体主要是病毒或者合成试剂。然而这类载体往往毒性高且特异性低。外泌体是由内源细胞分泌出来的天然纳米材料,本身能够穿越生物膜并在细胞间传递小RNA。以外泌体为基础的小RNA递送作为一种新的转运方式,能够克服低效率,低特异性以及免疫反应等缺陷,有望成为新型载体。本文简要论述了以外泌体为载体的小RNA递送系统在临床治疗研究中的前沿进展。     

外泌体源性miRNAs在肺癌发生发展中的作用

外泌体(exosomes)是细胞分泌的纳米级细胞外囊泡.外泌体通过释放其内的生物活性大分子,比如微小RNA(microRNA,miRNA)到受体细胞,从而介导细胞间交流通讯. MiRNAs作为一类主要在转录后水平负向调控靶mRNAs的非编码RNAs,其在外泌体中含量最为丰富.在肺癌中,miRNAs经肿瘤细胞分泌的外泌体转运释放而发挥重要的作用.本文主要讨论了外泌体源性miRNAs在肺癌发生发展的各个阶段,包括血管生成、细胞增殖、侵袭转移、免疫逃逸、耐药等方面的作用,以及其在作为新型肺癌诊断和预后标志物方面的临床价值.     

外泌体环状RNA在泌尿系统肿瘤中的研究进展

外泌体是一种包含了复杂RNA和蛋白质的膜性囊泡，其主要来源于细胞内溶酶体微粒内陷形成的多囊泡体，经多囊泡体外膜与细胞膜融合后释放到胞外基质中。外泌体在肿瘤微环境中介导细胞间通讯，其功能取决于来源的细胞类型。环状RNA是一类由前体mRNA反向剪接生成的非编码RNA，在外泌体中富集且稳定表达。外泌体环状RNA在疾病中发挥了重要的调控作用，其作为肿瘤标志物和治疗靶点的临床应用前景与价值现已成为研究热点。本文就外泌体环状RNA在泌尿系统肿瘤中的研究进展作一综述。     

不同体液来源外泌体分离鉴定方法评估

外泌体是细胞分泌的磷脂双分子层胞外囊泡,作为载体在细胞间发挥着物质传递和信息交流的功能.外泌体存在于多种不同体液中,在疾病诊断和药物载体方面具有良好的应用前景.由于外泌体纳米级别的大小和异质性,以及体液复杂的组成,使得体液来源外泌体的分离尤为困难.目前,体液来源外泌体分离有6种常用方法:超速离心法、沉淀法、分子筛色谱层...     

外泌体环状RNA在肿瘤微环境中的研究进展

外泌体是细胞分泌的30～150 nm的细胞外囊泡,在肿瘤微环境(tumor microenvironment,TME)中介导细胞间通讯.环状RNA (circular RNA,circRNAs)是一类由前体mRNA (precursor mRNA,pre-mRNA)反向剪接生成的非编码RNA(non-coding RNA,ncRNA),在外泌体中富集且表达稳定.本文主要讨论外泌体起源和circRNAs在外泌体中的分选调控机制,阐述外泌体circRNAs在肿瘤微环境各个阶段中的作用与机制,包括血管生成、EMT、耐药等.最后,本文探讨外泌体circRNAs作为肿瘤标志物和治疗靶点的临床应用前景与价值.     

外泌体在瘢痕中的作用

创面愈合后期瘢痕增生较为常见,而传统的治疗方式存在些许弊端.外泌体作为旁分泌因子参与细胞间的信号传导,并促进血管生成和细胞增殖.外泌体作为无细胞衍生物的研究热点之一,在创面修复领域备受关注.多种干细胞来源的外泌体可通过多种途径抑制瘢痕增生,甚至可成为促进创面无瘢痕修复的治疗工具.未来,外泌体联合生物支架、增强靶向能力、...     

人脐血血浆外泌体提取方法的比较北大核心CSCD

为探寻高效且稳定的提取人脐血血浆外泌体的方法,利用超高速离心法、蔗糖垫密度梯度离心法、改良超速离心法和聚乙二醇(polyethylene glycol, PEG)沉淀法提取人脐血血浆外泌体,并比较4种方法的优劣。利用透射电镜、动态光散射技术观察外泌体的形态、结构及大小;聚氰基丙烯酸正丁酯(bicinchoninic acid, BCA)法测定外泌体蛋白总量;Western blotting检测外泌体表面标志蛋白CD63、HSP70以及外泌体阴性蛋白GM130 (高尔基标志蛋白)的表达。结果表明,与提取外泌体的“金标准”,即超高速离心法相比,蔗糖垫密度梯度离心法稳定性好,获取的外泌体粒径较均一,但操作较复杂,耗时长;改良超速离心法操作较简单,纯度较高;PEG沉淀法提取的外泌体蛋白量最高,操作时间最短,但杂质较多。结果表明,4种方法均能从人脐血血浆中获取外泌体,但在操作时间、纯度、提取量等方面存在一定差异。因此,应根据实验目的和具体要求选择合适的提取人脐血血浆外泌体的方法。     

工程化外泌体介导巨噬细胞清除肿瘤外泌体*

目的:通过膜表面修饰改造技术构建工程化外泌体(engineered exosomes,enExos),并以此介导巨噬细胞特异性清除膜表面富含表皮生长因子受体(epidermal growth factor receptor,EGFR)的肿瘤外泌体。方法:利用表面展示技术获得膜表面展示趋化因子(chemokine 8,CXCL8)的外泌体,同时在其磷脂双分子层上修饰EGFR核酸适配体制备工程化外泌体;纳米颗粒跟踪和纳米粒度电位分析enExos的尺寸、电位;CCK-8试剂盒检测细胞活力;透射电子显微镜观察enExos与高表达EGFR的肿瘤外泌体的特异性结合;荧光成像技术及流式细胞术分析探究enExos靶向趋化巨噬细胞吞噬高表达EGFR的肿瘤外泌体。结果:成功构建膜表面展示EGFR与CXCL8的工程化外泌体,enExos可以特异性识别并捕获高表达EGFR的肿瘤外泌体,同时利用其趋化因子CXCL8特异性靶向巨噬细胞膜表面趋化因子受体CXCR1/CXCR2,刺激巨噬细胞对肿瘤外泌体的捕获及清除。结论:工程化外泌体促进了特定肿瘤外泌体的清除,为后续深入研究工程化外泌体抑制癌症转移的作用奠定基础,并期望为癌症转移治疗提供新的研究方向。     

外泌体及其在肿瘤诊疗中的意义

外泌体(exosome)是由多种活细胞分泌的囊泡小体,其中含有蛋白质和RNA等多种组分。这种机体内普遍存在的纳米级被膜结构能够参与细胞间的物质交换和信息交流,在多种生理和病理过程中发挥重要作用。外泌体在外周血、尿液、唾液、腹水、羊水等体液中具有很高的丰度,而不同组织来源的外泌体在组成和功能方面存在差异,同时这种差异受到细胞外基质和微环境的动态调控。肿瘤来源或肿瘤相关的外泌体是调控肿瘤发生发展的重要机制,对肿瘤外泌体的分析和检测可以辅助肿瘤的早期诊断、疗效评价和预后分析。此外,外泌体及其修饰加工产物还可以作为基因或药物的有效载体,用于肿瘤治疗。关于外泌体的研究是肿瘤学的一个新兴领域,在转化医学的研究模式下,将极大推动肿瘤学研究进展,为肿瘤临床诊断和治疗带来新的契机。     

用聚乙二醇分离富集microRNA的方法探讨

目的探讨用聚乙二醇（polyethylene glycol,PEG）溶液分离富集miRNA的操作方法和分离富集效果,并与Ambion公司的miRNA分离试剂盒分离效果进行比较。方法用PEG溶液和Ambion公司的miRNA分离试剂盒从肝脏组织总RNA中分离富集miRNA,用变性琼脂糖和变性聚丙烯酰胺凝胶电泳鉴定分离效果,并在富集的miRNA中用RT—PCR扩增miR-122以鉴定是否有效地回收了miRNA。结果PEG和Ambion公司的miRNA分离试剂盒都能有效地富集miRNA,PEG富集的RNA片段比Ambion公司的试剂盒的大。结论PEG溶液能有效地分离富集miRNA,和Ambion公司的miRNA分离试剂盒分离效果相当,并具有操作简便、快捷及成本低廉的优点。     

Modification of Lipase by Polyethylene Glycol

Lipase was modified using polyethylene glycol activated by p-nitrochloroformate. The hydrolytic activity of the polyethylene glycol-derivatised lipase (PEG-lipase) was relatively low compared with that of the unmodified enzyme in aqueous system. The esterification activity, however, was enhanced following the modification. The rate of esterification of butyric acid was higher than that of oleic acid. Benzene was the best solvent for the esterification reaction.     

一种用聚乙二醇制备微粒体的方法

介绍一种用聚乙二醇(PEG)制备微粒体的方法.大鼠肝匀浆经聚乙二醇-6000凝聚,及两次高速离心即可得到微粒体组分,与超速离心方法比较,可省去超速离心步骤,又缩短了分离制备的时间,是一种比较简单易行的方法．     

聚乙二醇对菠萝蛋白酶的化学修饰

方法:用琥珀酸酐法活化的聚乙二醇对菠萝蛋白酶进行化学修饰,得到菠萝蛋白酶的修饰酶,对比研究三种菠萝蛋白酶:修饰酶、混合酶、天然酶的热稳定性及酸碱稳定性,考察金属离子对三种菠萝蛋白酶的影响。结果:当在55℃水浴保温100min后天然酶活力只保留20%,混合酶活力保留37%,修饰酶活力保留58%;在pH3.0-4.5及pH6.0-7.0的条件下,修饰酶活力高于天然酶活力。当Ca2 的浓度达到0.05mg/mL时,修饰酶的活力高达257.66%;当Mg2 的浓度达到0.035mg/mL时,修饰酶的活力高达147.25%。一价离子Na 对三种菠萝蛋白酶无明显影响。结论:修饰的菠萝蛋白酶对温度和pH值的稳定性均比天然酶有很大程度的提高。混合酶的活力介于天然酶和修饰酶之间说明聚乙二醇对菠萝蛋白酶有一定的保护作用。二价离子Ca2 、Mg2 对三种菠萝蛋白酶活力均有不同程度的激活作用。     

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e. low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.     

Precipitation of collagens by polyethylene glycols

Types I, II, and III collagens are readily precipitated at neutral pH by polyethylene glycols (PEG). As the molecular weight fraction of the polyethylene glycols increases, they become more effective as precipitants on a weight basis. The amount of PEG required for precipitation depends on the pH, the ionic strength, and the nature of the buffer or salts present. In tissue culture media, low concentrations of collagens and procollagens may be quantitatively precipitated and readily collected by low-speed centrifugation. Polyethylene glycol precipitation can be used to obtain collagens and procollagens from tissue culture media at either analytical or preparative scale, and since the polyethylene glycols do not bind to collagens, the precipitates may be further analyzed directly by chromatographic or electrophoretic methods.     

黄蝉离体生殖细胞的融合

近期研究者用不同的方法成功地从花粉和花粉管中分离出生殖细胞(Zhou等1986,1988;Zhou 1988,Tanaka 1988,Tanaka等1989,周嫦和吴新莉1990,徐是雄1991a)。这些生殖细胞在离体培养的条件下,不但能够保持生活力,而且还能     

A Phase-Transfer Glucosamination of Phenols Catalyzed by Polyethylene Glycol

Glycosylation of phenols with α-D-glucosaminyl chloride peracetate catalyzed by polyethylene glycol (PEG) was carried out in a solid-liquid phase transfer system at room temperature. The results were compared with those previously obtained for the catalysis with various crown ethers. The catalytic activity of PEG in this reaction was found to be comparable with those of 15-crown-5 and aromatic crown ethers.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 335–336.Original Russian Text Copyright © 2005 by Kur’yanov, Priskoka, Chupakhina, Chirva.     

Preparation of Long-Acting Superoxide Dismutase Using High Molecular Weight Polyethylene Glycol (41,000-72,000 Daltons)

Superoxide dismutase (SOD) has demonstrated therapeutic potential for treating a variety of conditions including radiation injury, oxygen toxicity, reperfusion injury, and inflammation, especially arthritis. However, the native enzyme's short half-life in plasma (6 minutes in mice. 25 minutes in man) limits the enzyme's effectiveness in many applications, or requires infusion of large doses. High doses of SOD derived from either natural or rDNA sources may increase the potential for immunologic sensitization. One effective use of native SOD is intra particular administration for treatment of arthritis, where injection of SOD into joints retards elimination (15 hour terminal half-life), allowing the effective use of lower doses.

To overcome the limitations resulting from rapid clearance. various researchers have increased the persistence of SOD by cross-linking SOD or by attaching polymeric substances, including dextrans, albumin, Ficoll, polyvinyl alcohol or polyethylene glycol (PEG). PEG is relatively safe; however. the amount of modification by PEG. is the MW range 1.900-5.000 daltons, which is necessary to optimally increase serum persistence and reduce immunogenicity, results in the loss of much of the enzymatic activity.

In this report we describe the preparation of SOD adducts containing I to 4 strands of high MW PEG (41,000-72,000 daltons). The MW range of these adducts, measured by steric exclusion HPLC based on protein standards, is 200,000 to over 1,100,000 daltons. The number of PEG strands attached per SOD dimer (32,000 daltons) was measured by HPLC. Because of the low degree of protein modification required to produce very high MW products, these PEG-SODS retain 90%-100% of the SOD activity of the native enzyme. Additionally, these very large adducts demonstrate longer persistence and lower immunogenicity and antigenicity compared to the more highly modified PEG-SODS containing low MW PEG (i.e., 7-16 strands of 5.000 dalton methoxy-PEG).     

Preparation of Long-Acting Superoxide Dismutase Using High Molecular Weight Polyethylene Glycol (41,000-72,000 Daltons)

Superoxide dismutase (SOD) has demonstrated therapeutic potential for treating a variety of conditions including radiation injury, oxygen toxicity, reperfusion injury, and inflammation, especially arthritis. However, the native enzyme's short half-life in plasma (6 minutes in mice. 25 minutes in man) limits the enzyme's effectiveness in many applications, or requires infusion of large doses. High doses of SOD derived from either natural or rDNA sources may increase the potential for immunologic sensitization. One effective use of native SOD is intra particular administration for treatment of arthritis, where injection of SOD into joints retards elimination (15 hour terminal half-life), allowing the effective use of lower doses.

To overcome the limitations resulting from rapid clearance. various researchers have increased the persistence of SOD by cross-linking SOD or by attaching polymeric substances, including dextrans, albumin, Ficoll, polyvinyl alcohol or polyethylene glycol (PEG). PEG is relatively safe; however. the amount of modification by PEG. is the MW range 1.900–5.000 daltons, which is necessary to optimally increase serum persistence and reduce immunogenicity, results in the loss of much of the enzymatic activity.

In this report we describe the preparation of SOD adducts containing I to 4 strands of high MW PEG (41,000–72,000 daltons). The MW range of these adducts, measured by steric exclusion HPLC based on protein standards, is 200,000 to over 1,100,000 daltons. The number of PEG strands attached per SOD dimer (32,000 daltons) was measured by HPLC. Because of the low degree of protein modification required to produce very high MW products, these PEG-SODS retain 90%-100% of the SOD activity of the native enzyme. Additionally, these very large adducts demonstrate longer persistence and lower immunogenicity and antigenicity compared to the more highly modified PEG-SODS containing low MW PEG (i.e., 7–16 strands of 5.000 dalton methoxy-PEG).