淫羊藿来源外泌体的提取和鉴定分析

〔摘 要〕 植物源外泌体样纳米颗粒是从植物组织中分离的直径约 40 ~ 300 nm 的双层膜性囊泡,由活体细胞分泌产生, 是细胞间传递生物信息的重要载体,具有沟通细胞间通讯的作用,该膜性囊泡在生理调节和病理发展中都发挥重要作用, 是近年来研究的热点,为更深入研究外泌体生物学作用机制,提取高质量的外泌体是首要步骤,本研究通过超速离心法提 取并用蔗糖重水垫法纯化淫羊藿外泌体,经电镜鉴定符合外泌体显微鉴别特征。希望本研究能为研究者们开拓中草药外泌 体提取研究提供一些参考。     

MiRNA in melanoma-derived exosomes

Proteins, RNAs and viruses can be spread through exosomes, therefore transport utilizing these nanovesicles is of the great interest. MiRNAs are common exosomal constituents capable of influencing expression of a variety of target genes. MiRNA signatures of exosomes are unique in cancer patients and differ from those in normal controls. The knowledge about miRNA profiles of tumor-derived exosomes may contribute to better diagnosis, determination of tumor progression and response to treatment, as well as to the development of targeted therapies. We summarize the current knowledge with regard to miRNAs that are found in exosomes derived from tumors, particularly from melanoma.     

未成熟树突状细胞-胃癌细胞融合细胞来源的exosome诱导特异性抗肿瘤免疫

目的观察未成熟树突状细胞（DC）-胃癌细胞融合细胞来源的exosome诱导特异性抗肿瘤免疫的效果。方法以细胞培养技术将BALB／c小鼠未成熟Dc与人胃癌细胞SGC-7901融合,并提取融合细胞分泌的exosome作为瘤苗。将人胃癌细胞株接种于BALB／c小鼠以建立4组荷瘤小鼠模型,A、B、C组小鼠接种SGC-7901细胞。D蛆小鼠接种BGC．823细胞。各组2周后开始在腹腔接种相应瘤苗。A组瘤苗为对照用PBS;B组溜苗为BALB／c小鼠来源的未成熟Dc,c组及D组瘤苗为未成熟DC-SGC7901融合细胞来源的exosome。每组取6只小鼠收集脾细胞悬液,进行体外细胞毒实验。剩余每组6只小鼠留待观察生存期。结果A、B、C、D组小鼠脾细胞悬液的细胞毒性分别为（27．22±7．31）％、（58．28±7．80）％、（72．36±6．28）％、（43．15±5。60）％,4组小鼠的存活时间分别为（14．2±1．3）d、（29．2±1．8）d、（37．6±2．2）d、（22．1±2．6）d。方差分析提示组间差异有统计学意义（P〈0．05）;S-N-K检验提示各组之间的差异均有统计学意义（P〈0．05）。结论未成熟DC-胃癌细胞融合细胞来源的exosome作为新型、高效肿瘤疫苗,可诱导抗胃癌细胞的特异性免疫反应。     

Critical role of exosomes in sperm–egg fusion and virus-induced cell–cell fusion

In mammals, two integral membrane proteins, sperm IZUMO1 and egg CD9, regulate sperm–egg fusion, and their roles are critical, but yet unclear. Recent studies, however, indicate interesting connections between the sperm–egg fusion and virus-induced cell–cell fusion. First, CD9-containing exosome-like vesicles, which are released from wild-type eggs, can induce the fusion between sperm and CD9-deficient egg, even though CD9-deficient eggs are highly refractory to the fusion with sperm. This finding provides strong evidence for the involvement of CD9-containing, fusion-facilitating vesicles in the sperm–egg fusion. Secondly, there are similarities between the generation of retroviruses in the host cells and the formation of small cellular vesicles, termed exosomes, in mammalian cells. The exosomes are involved in intercellular communication through transfer of proteins and ribonucleic acids (RNAs) including mRNAs and microRNAs. These collective studies provide an insight into the molecular mechanism of membrane fusion events.     

成纤维细胞系3T3细胞来源exosome对小鼠乳腺癌细胞增殖能力的影响

目的 观察小鼠成纤维细胞系3T3来源的外泌小体（exosome）对小鼠乳腺癌细胞4T1增殖能力的影响,并探索其中可能的机制。方法 PureExo Exosome提取试剂盒提取3T3细胞上清液中的exosome,按照不同浓度及时间作用于4T1细胞,CCK8法检测4T1细胞的增殖能力,BrdU/PI双掺入法测定细胞DNA合成及细胞周期;免疫印迹法（Western blot）及荧光定量实时PCR（qPCR）检测人表皮生长因子受体2（epidermal growth factor receptor-2, EGFR2,也称HER2）及下游PI3K/AKT信号转导通路相关蛋白的变化。利用HER2单克隆抗体靶向药物赫赛汀（Herceptin）,观察exosome是否影响4T1细胞对于Herceptin敏感度。结果 exosome处理组OD450吸光度值显著高于对照组（P<0.05）,细胞增殖及细胞周期进程加快。Western blot及qPCR实验提示随着exosome浓度的增加,HER2表达逐渐升高, AKT磷酸化水平增加。而同时给予exosome可明显增加4T1细胞对Herceptin的敏感度。结论 小鼠成纤维细胞系3T3来源exosome可促进小鼠乳腺癌细胞4T1增殖及周期进程,并且可能通过HER2激活其下游PI3K/AKT信号通路发挥上述作用。     

动脉粥样硬化人群中血液外泌体的蛋白质谱分析

目的预测血液中外泌体在动脉粥样硬化中的作用及机制。方法招募56个受试对象,分成两个比较组:健康人群与动脉粥样硬化人群、高血压人群与高血压合并动脉粥样硬化人群。用试剂盒提取受试者血液中外泌体,利用蛋白质谱分析技术检测外泌体中蛋白表达,将蛋白表达谱的结果进行基因功能(GO)分析。结果GO分析显示,在生物学过程中,两个比较组中都出现显著差异的有5个条目,分别是"固有免疫反应的正向调节"、"免疫反应激活信号转导"、"固有免疫反应激活"、"固有免疫反应激活信号转导"和"固有免疫反应激活细胞表面受体信号通路";在分子功能类别中,两个比较组中有2个条目是重复出现的,分别是"抗原结合"和"酶结合";在两个组别比较中都出现差异蛋白有3个,分别是蛋白酶亚基α6(PSMA6)、蛋白酶亚基α7(PSMA7)和膜联蛋白A2。结论固有免疫系统可能在动脉粥样硬化疾病发病中起重要作用。PSMA6、PSMA7和膜联蛋白A2有可能成为动脉粥样硬化疾病防治的新的靶蛋白。     

Tumor-derived exosomes: Nanovesicles made by cancer cells to promote cancer metastasis

Nanomedicine usually refers to nanoparticles that deliver the functional drugs and siRNAs to treat cancer. Recent research has suggested that cancer cells can also make nanoparticles that also deliver functional molecules in promoting cancer metastasis, which is the leading cause of various cancer mortalities. This nanoparticle is called tumor-derived vesicles, or better-known as tumor-derived exosomes (TEXs). TEXs are nanoscale membrane vesicles (30–140 nm) that are released continuously by various types of cancer cells and contain tumor-derived functional biomolecules, including lipids, proteins, and genetic molecules. These endogenous TEXs can interact with host immune cells and epithelial cells locally and systemically. More importantly, they can reprogram the recipient cells in favor of promoting metastasis through facilitating tumor cell local invasion, intravasation, immune evasion, extravasation, and survival and growth in distant organs. Growing evidence suggests that TEXs play a key role in cancer metastasis. Here, we will review the most recent findings of how cancer cells harness TEXs to promote cancer metastasis through modulating vascular permeability, suppressing systemic immune surveillance, and creating metastatic niches. We will also summarize recent research in targeting TEXs to treat cancer metastasis.     

星形胶质细胞外泌体对缺氧缺血神经元的保护作用

目的 探索星形胶质细胞外泌体对缺氧缺血神经元的影响。方法 体外培养大鼠星形胶质细胞,通过差速离心法获取细胞上清液中的外泌体。采用透射电镜、Nanosight和Western blot鉴定外泌体;采用BCA法检测外泌体蛋白浓度。体外培养大鼠神经元,分为对照组、外泌体组、氧糖剥夺（OGD）组和OGD+外泌体组（n=3）。OGD组和OGD+外泌体组给予无糖培养基和缺氧处理;外泌体组和OGD+外泌体组分别给予终浓度为22 μg/mL的外泌体处理,对照组和OGD组给予等体积PBS处理。采用ELISA法检测神经元乳酸脱氢酶（LDH）水平;TUNEL法检测神经元凋亡指数。结果 外泌体鉴定结果显示差速离心法提取的外泌体符合外泌体特点;与对照组和外泌体组相比,OGD组LDH值和凋亡指数显著增加（P < 0.05）;与OGD组相比,OGD+外泌体组LDH值和凋亡指数均显著降低（P < 0.05）。结论 星形胶质细胞来源的外泌体对神经元缺氧缺血损伤有保护作用。     

间充质干细胞外泌体在骨关节炎治疗修复中的研究进展

骨关节炎(OA)是种以关节软骨退变为特征的疾病,其缺乏干预性药物,对于晚期OA患者往往通过关节置换术以维持生活质量。外泌体(exosomes)是一种由不同细胞分泌的细胞外囊泡,可以传递DNA、微小RNA(microRNA,miRNA)、mRNA、蛋白质等多种信息,并以此通过多种方式进行细胞间信号传递和功能调节。间充质干...