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。     

肿瘤细胞外泌体中融合基因的检测

目的:探究肿瘤细胞分泌的外泌体中是否可以检测到来源于源细胞的融合基因m RNA。方法:培养人非小细胞肺癌NCI-H3122细胞,采用外泌体试剂盒提取细胞上清中的外泌体,并用Western Blot实验验证外泌体是否提取成功。分别提取外泌体以及H3122细胞中的总RNA,将RNA反转录为c DNA,通过PCR反应扩增v1型EML4-ALK融合基因片段,经琼脂糖凝胶电泳确定目的条带后,将两种PCR产物送公司进行基因测序,最后对测序结果进行比对分析。结果:从H3122细胞上清中成功提取了外泌体,并且在外泌体中检测到来源于H3122细胞的m RNA;从H3122细胞外泌体中检测到EML4-ALK融合基因,并发现外泌体中的EML4-ALK融合基因的融合形式为V1,与在H3122细胞中检测到的EML4-ALK融合基因的融合形式完全相同。结论:肿瘤细胞分泌的外泌体中可以检测到肿瘤细胞来源的m RNA,并且外泌体中的m RNA可以反映肿瘤细胞m RNA的基因融合情况等生物学特性。     

间充质干细胞来源外泌体对脑内小胶质细胞极化和炎症因子释放的影响及机制研究

摘要 目的：探究间充质干细胞外泌体对脑内小胶质细胞极化和炎症因子释放的影响及其机制。方法：收集体外培养的间充质干细胞上清,超高速冷冻离心获取外泌体。采用纳米颗粒系统和透射电子显微镜分别检测外泌体粒径大小、形态结构和功能完整性。通过免疫荧光、ELISA和细胞流式等方式检测LPS刺激下,外泌体对BV2细胞的表型极化和炎症因子释放的影响。采用Western Blot法检测间充质干细胞外泌体对BV2细胞JAK1/STAT3通路活化的影响。结果：（1）间充质干细胞分泌的外泌体粒径大小主要介于40-100 nm,透射电镜显示外泌体形态呈典型膜性\"杯盘\"状结构;（2）流式结果表明,相比于对照组,LPS组能显著激活M1型小胶质细胞表面标志物CD11b和M2型小胶质细胞表面标志物CD206的表达,而经外泌体处理,CD11b的表达显著被抑制,CD206显著升高。同时ELISA结果证实,相比于LPS组,外泌体组分泌的促炎症因子（IL-1β、IL-6）和NO水平显著降低（P<0.05）,抗炎因子（IL-10）显著升高 (P<0.05);（3）间充质干细胞外泌体显著提高了BV2细胞JAK1/STAT3通路的磷酸化水平。结论：间充质干细胞外泌体通过激活JAK1/STAT3通路有效促进脑内小胶质细胞M1型向M2型极化。     

肝细胞癌患者血清外泌体microRNA表达鉴定

目的:探讨肝癌细胞外泌体中差异表达的microRNAs(miRNAs)在肝细胞癌(HCC)诊断中的应用价值。方法:通过高通量测序筛选肝癌细胞外泌体中差异表达的miRNAs。实时定量PCR验证差异表达分子;检测差异表达的miRNAs在健康人(Health)、慢性乙型肝炎患者(CHB)、肝硬化患者(LC)及乙型肝炎病毒阳性的肝细胞癌患者(HCC)血清外泌体中的表达。结果:高通量测序筛选到肝癌细胞外泌体中差异表达的miRNA共88种,其中58种表达上调,30种表达下调。选择其中8种差异表达的miRNAs进行q RT-PCR验证,结果显示,此8种miRNAs在细胞上清外泌体、细胞内、癌与癌旁组织中的表达趋势与测序结果一致。miR-221-3p和miR-224-5p在HCC组外泌体中的表达水平显著高于Health组、CHB组和LC组(P0.01),miR-124-3p和let-7a-5p在HCC组外泌体中的表达水平显著低于其他各组(P0.05)。四个组中,miR-21-5p、miR-191-5p、miR-34a-5p和miR-122-5p的表达水平不存在显著性差异(P0.05)。结论:血清外泌体中的miR-221-3p、miR-224-5p、miR-124-3p和let-7a-5p可能成为肝细胞癌的候选标志物。     

外泌体在淋巴瘤中的研究进展

摘要：病毒感染和环境污染等使得淋巴瘤的发病率逐年升高,早期诊断和精准治疗具有十分重要的临床意义。外泌体是一种脂质双层膜结构的微小囊泡,介导了细胞间交流和信息交换。近几年许多研究证实外泌体是淋巴瘤的发生、进展和耐药的重要机制。外泌体内核酸和小分子可用于淋巴瘤的早期诊断和预测患者预后。材料学修饰可显著增强外泌体治疗的靶向性和治疗效能。本文总结了外泌体生物学特性、分离和鉴定方法、与肿瘤相关性、及其在淋巴瘤中的研究进展,为淋巴瘤的预警和治疗提供参考。     

人脐带干细胞来源的外泌体促进体外培养雪旺细胞迁移的研究

目的:外泌体是活细胞分泌的来源于多囊泡体的膜性囊泡,其主要作用包括携带与运输。雪旺细胞是周围神经再生中非常优秀的种子细胞,但其迁移能力较差,影响修复效果。本文旨在探讨外泌体和雪旺细胞共培养是否可以促进雪旺细胞迁移。方法:本实验通过分离纯化人脐带干细胞外泌体和大鼠坐骨神经雪旺细胞并鉴定,随后将其共培养于Transwell小室观察雪旺细胞迁移率。结果:通过人脐带干细胞超高速离心法得到的外泌体高表达干细胞标志物CD44(92.2±3.6%)、CD73(99.1±0.6%),并且低表达单核细胞表面抗原CD14(0.5±0.06%)以及造血干细胞表面抗原CD34(0.4±0.07%),外泌体鉴定高表达CD81和CD9;雪旺细胞培养鉴定纯度达(92.3±2.7)%;均符合实验要求。通过Transwell小室实验发现外泌体可以明显促进雪旺细胞的迁移,并且具有一定剂量关系。结论:外泌体可以提高雪旺细胞的迁移能力,从而使雪旺细胞在组织工程领域中的应用产生巨大突破。     

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。     

膀胱癌细胞来源外泌体的提取与鉴定

目的:提取并鉴定膀胱癌5637细胞来源外泌体。方法:收集膀胱癌5637细胞培养上清液,采用多步骤离心法提取膀胱癌5637细胞外泌体。透射电子显微镜观察外泌体形态及颗粒直径。Bradford法定量外泌体蛋白含量。蛋白质免疫印迹鉴定外泌体标志蛋白。结果:20 m L 5637细胞培养基可收集约50-80μg外泌体。膀胱癌5637细胞来源外泌体呈典型的茶杯样形态,外泌体颗粒直径大约在30-150 nm。膀胱癌5637细胞外泌体提取物中可检测到标志蛋白CD63、TSG101、Hsp70和Hsp90表达。结论:多步骤离心法可以用于提取膀胱癌5637细胞外泌体,为后续开展膀胱癌5637细胞外泌体作用与机制的研究奠定了基础。     

间充质干细胞外泌体对小鼠急性肝衰竭的保护作用

目的:研究小鼠骨髓间充质干细胞(Mouse bone marrow mesenchymal stem cells, m BMMSC)来源的外泌体(Exosome, Exo)对四氯化碳(Carbon tetrachloride, CCl4)诱导的急性肝衰竭(Acute liver failure, ALF)的保护作用。方法:采用全骨髓培养法提取Balb/c小鼠原代骨髓间充质干细胞并利用流式细胞仪进行鉴定,用试剂盒混合细胞上清后提取干细胞分泌的外泌体,透射电镜(Transmission electron microscope, TEM)鉴定外泌体形态,纳米粒径追踪分析仪(Nanoparticle tracking analysis, NTA)鉴定外泌体的粒径,Western Blot鉴定外泌体表面的标志蛋白。激光共聚焦显微镜(Confocal laser scanning microscope, CLSM)下观察肝细胞内吞外泌体。小鼠肝脏的免疫荧光切片观察外泌体的肝脏内分布。Balb/c小鼠随机分为健康对照组(Healthy)、CCl4损伤组(CCl4)、CCl4损伤外泌体治疗组(Exo)。观察小鼠7 d内的生存时间。检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)、尿酸(UA)的含量,HE染色组织切片观察肝脏病理变化,流式细胞仪和磁珠细胞试剂盒(Cytometric bead array, CBA)检测血清中白介素6(IL-6)、白介素12(IL-12)和γ干扰素的含量(IFN-γ)。结果:成功提取并鉴定了小鼠骨髓间充质干细胞与其外泌体,NTA测得干细胞外泌体的粒径在100 nm左右,电镜下外泌体呈现囊泡状,Western Blot测得外泌体表达CD9和CD63蛋白。CLSM下观察到L02肝细胞内吞外泌体且随时间递增,免疫荧光切片观察到外泌体靶向肝脏且在肝脏组织中均匀分布。动物实验表明相比于CCl4组,Exo组小鼠的生存时间显著延长,血清中ALT、AST、LDH和UA的含量显著降低,肝脏组织切片显示肝脏损伤明显减少,血清中IL-6, IL-12和IFN-γ的含量显著降低。结论:小鼠骨髓间充质干细胞分泌的外泌体可能通过抑制炎症减轻四氯化碳诱导的急性肝衰竭。     

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。     

Intercellular lipid in oilseeds

Summary Electron microscopic observations of three oilseeds, zucchini, yucca, and okra, have revealed a substance occurring in the intercellular spaces in the embryos of these seeds. Extraction methods and histochemical tests for light microscopy have characterized the material as lipid. Disappearance of the intercellular lipid during seed germination suggests that it may be utilized by the germinating seed.     

For uptake of yolk precursors,epithelial cell-oocyte gap junctional communication is required by insects representing six different orders

For uptake of vitellogenin protein into nascent yolk spheres, communication through open gap junction channels between the follicle epithelium and oocyte is required by six different insects representing six different orders. It was recently shown in the hemipteran, Oncopeltus fasciatus, that endocytic uptake of yolk protein resulting in the formation of nascent yolk spheres depended upon an intact epithelium communicating with the oocyte through patent gap junctions. Following treatment with octanol, which down-regulated gap junctions below the level of dye coupling, vitellogenin uptake was terminated. Yet, for another hemipteran, Dysdercus intermedius, it has been shown that yolk spheres can form even when all epithelial cells have been stripped from the oocyte. To determine if the mechanism seen in Oncopeltus is present in other insects, we utilized the same techniques to study nascent yolk sphere production in a dipteran, Drosophila melanogaster, a lepidopteran, Actias luna, a hymenopteran, Xylocopa virginica, a coleopteran, Tenebrio molitor and an orthopteran, Acheta domesticus. In each of these, when gap junctions were down-regulated yolk uptake quickly stopped. That six different insects from six different orders all required a gap junctionally transmitted chemical signal of epithelial cell origin suggests that this mechanism is widespread throughout the insects.     

Two cholesterol pools in Acholeplasma laidlawii membranes

Cholesterol exchange kinetics between [14C]cholesterol-labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine-cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.     

Translocation in the staminal hairs ofSetcreasea purpurea

Summary Investigations into plant intercellular communication were initiated through an examination of plasmodesmata and cell-to-cell passage of molecular probes in the staminal hairs ofSetcreasea purpurea. Plasmodesmata connecting staminal hair cells of small buds are filled with an electron-opaque homogenous material. To examine the permeation selectivity of plasmodesmata, molecular probes made up of fluorescein isothiocyanate (FITC) complexed with amino acids and peptides were injected into the staminal hair cells and the spread of these fluorescent molecules through the symplast, was monitored. Molecules composed of FITC complexed to single amino acids with polar and aliphatic R groups travel rapidly, while those which include peptides travel slowly. Dye molecules composed of an amino acid with an aromatic side group do not pass from cell to cell at all. It is hypothesized that the material occluding the plasmodesmata constitutes the diffusion barrier, by presenting a hydrophilic environment which allows passage of molecules with maximum molecular weights of 700–800 daltons, but which retains those with aromatic side groups.     

Closing and opening of gap junction pores between two- and threedimensionally cultured tumor cells

Intercellular signal transfer via gap junction pores in cultured multicell spheroids of BICR/M1R-K cells decreases with increasing spheroid age. In two days old spheroids the pores allow passage of Lucifer yellow molecules. Two days later, this fluorescent dye is retained in the injected cell even though the cells are still electrically coupled. Gap junction plaques of considerable size are still found in 9 days old spheroids, when the cells are completely uncoupled. The same cells growing as monolayer cultures do not exhibit such a gradual closing of their gap junction pores: Their coupling is established at first cell contact, probably by a gradual opening of the pores, which remain open even up to 9 days in culture.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

转录因子的胞间移动在植物生长发育中的作用

多细胞生物体的生长发育依赖于细胞和细胞之间物质的交流和信号的传递。细胞命运的特化受到来自旁临细胞信息的调控。作为细胞-细胞通讯方式之一的蛋白质胞间运输广泛的存在于植物各种发育过程中。本文总结了近年来有关植物重要发育调控转录因子在细胞间移动的研究进展,综述了这些因子移动的细胞学基础和分子调控模型,并对今后蛋白胞间移动研究面临的挑战和需引入的新技术手段进行了展望。     

Intercellular communication and cell cooperation in growth control of T-lymphocytes

T-lymphocyte stimulation is strictly dependent on cell cooperation. Cell contact dependent cell cooperation has been described as well as cooperation by soluble mediators, the lymphokines. We here present a unifying hypothesis proposing that both types of cell cooperation are performed by the same mediators.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

神经组织缝隙连接

近年来神经组织中缝隙连接的分布和功能研究取得了一些显著进展。分子生物学方法的应用促进了ＧＪ结构,亚型及生物物理特性的揭示,染料偶联实验和Ｃａ＾２＋成像技术为ＧＪ的功能研究提供了直观有效的手段。ＧＪ的调控涉及ＧＪ的表达,导通性的改变等环节。ＧＪ胞间通讯的基本形式是交换第二信使和电偶联。     

Cell signaling during development of Dictyostelium

Continuous communication between cells is necessary for development of any multicellular organism and depends on the recognition of secreted signals. A wide range of molecules including proteins, peptides, amino acids, nucleic acids, steroids and polylketides are used as intercellular signals in plants and animals. They are also used for communication in the social ameba Dictyostelium discoideum when the solitary cells aggregate to form multicellular structures. Many of the signals are recognized by surface receptors that are seven-transmembrane proteins coupled to trimeric G proteins, which pass the signal on to components within the cytoplasm. Dictyostelium cells have to judge when sufficient cell density has been reached to warrant transition from growth to differentiation. They have to recognize when exogenous nutrients become limiting, and then synchronously initiate development. A few hours later they signal each other with pulses of cAMP that regulate gene expression as well as direct chemotactic aggregation. They then have to recognize kinship and only continue developing when they are surrounded by close kin. Thereafter, the cells diverge into two specialized cell types, prespore and prestalk cells, that continue to signal each other in complex ways to form well proportioned fruiting bodies. In this way they can proceed through the stages of a dependent sequence in an orderly manner without cells being left out or directed down the wrong path.