细粒棘球蚴原头节miR-71的分泌表达特征分析

目的分析在胰岛素、阿苯达唑和人工胃液刺激下,细粒棘球蚴原头节miR-71的分泌表达特征。方法从新疆屠宰场采集绵羊肝、肺细粒棘球蚴包囊,经固定、包埋及切片后,用miR-71探针进行杂交。与异硫氰酸荧光素(FITC)标记的抗Digoxin抗体(Anti-Digoxin-FITC)作用,经4’,6-二脒基-2-苯基吲哚(DAPI)核酸染料染色后,在荧光显微镜下观察miR-71在包囊壁和原头节中的定位,分析miR-71在原头节中的分布,确定miR-71在原头节中的表达情况。在人工胃液(人工胃液组)、胰岛素(胰岛素组)或阿苯达唑(阿苯达唑组)等不同条件下培养原头节,收集上清并用差速离心法分离外泌体,采用透射电镜观察外泌体形态结构,利用纳米粒径分析仪测定外泌体的粒径分布;利用外泌体生物标志分子烯醇化酶和14-3-3蛋白的抗体蛋白质免疫印迹(Western blotting)鉴定外泌体;采用实时荧光定量PCR(qPCR)检测外泌体中miR-71的丰度。结果原位杂交结果显示,miR-71在囊壁生发层和原头节中均有表达。人工胃液组、胰岛素组和阿苯达唑组原头节分泌的外泌体粒径分别为48.9、49.7和65.4 nm,均在40~120 nm内;外泌体形态呈由膜包裹的球形。Western blotting从外泌体中检出烯醇化酶和14-3-3蛋白,表明外泌体提取成功。qPCR结果显示,人工胃液组、胰岛素组和阿苯达唑组原头节分泌的外泌体中,miR-71的表达量分别是其对照组的1.84、1.87和2.38倍(t=12.8、26.7、29.3,均P<0.01)。结论miR-71在细粒棘球蚴囊壁和原头节中广泛表达,且可以通过外泌体进行分泌;经胰岛素、人工胃液和阿苯达唑刺激后其分泌表达水平升高。     

再生障碍性贫血患者外周血T细胞FasL的表达分析

目的 比较分析再生障碍性贫血(AA)患者T细胞上Fas配体(FasL)的表达及其在胞内外的分布情况，观察AAT细胞胞浆FasL的释放特性及细胞毒作用。方法 以正常人“静息”T细胞和人为诱导活化的T淋巴母细胞为参照，采用免疫荧光标记和流式细胞技术，检测FasL在AAT细胞表面及胞浆中的分布；用体外大剂量PHA-过性刺激的方式诱导T细胞胞浆FasL的释放；以Jurkat细胞为靶向，同时辅以单抗阻断及超速离心的方法，观察AAT细胞FasL的释放特性和杀伤效应。结果 AA T细胞表面及胞浆中均有FasL的异常表达，其中胞浆FasL的异常表达尤为显著；高浓度胞浆FasL可在大剂量PHA-过性刺激时迅速向胞外释放；PHA刺激后的AAT细胞培养上清有明显的Jutkat细胞杀伤活性，该效应可被FasL McAb阻断，或经超速离心加以去除。结论 AAT细胞是一种处于预活化状态的细胞，具有与T淋巴母细胞相似的活化特征，含有高浓度、能以细胞外体形式诱导释放、具备完整活性的胞浆FasL是其异常活化的一大特点。     

卵巢功能减退患者卵泡液外泌体miRNA表达谱分析研究

目的利用高通量测序技术筛选卵巢储备功能减退(DOR)与卵巢功能正常卵泡液外泌体中差异表达的miRNAs,探讨其对DOR的发病影响。方法收集DOR患者和卵巢功能正常者(对照组)的卵泡液各15例,各组每5例卵泡液混合为均一样本提取外泌体,共进行3次生物学重复。使用Illumina HiSeq4000测序平台对DOR组和对照组卵泡液外泌体中的miRNAs进行测序分析,使用miRanda和TargetScanS软件、GO以及KEGG数据库对差异显著的miRNAs进行靶基因预测、生物学功能富集以及信号通路预测。结果与对照组相比,DOR卵泡液外泌体有80个显著差异表达的miRNAs,其中31个表达上调(包括hsa-miR-4792、hsa-let-7c-3p和hsa-miR-184等),49个表达下调(包括hsa-miR-136-3p、hsa-miR-296-3p和hsa-miR-337-5p等)(P<0.05);通过miRanda和TargetScanS的算法预测所有差异表达miRNA的靶基因共17159个,经GO和KEGG分析发现,这些靶基因主要富集的生物学功能有电离型谷氨酸受体信号通路、氨基酸转运调节、脂多糖介导信号通路及发育相关的凋亡过程等,主要参与调节的信号通路有趋化因子信号通路、环磷酸腺苷信号通路、肿瘤坏死因子信号通路及丝裂原活化蛋白激酶信号通路等。结论来源于DOR和卵巢功能正常者的卵泡液外泌体的miRNAs表达谱存在显著差异,差异表达的miRNAs可能在DOR的发生中起到重要的调控作用,这些miRNAs可能经过靶基因参与调节多种生物学功能以及信号通路进而影响DOR的病理生理过程。     

细胞分泌的囊状小体exosomes的研究进展

有多种细胞都能分泌一种被称为exosomes的小囊泡，这些囊泡由细胞内的内吞小体出泡产生，它包裹着特殊的蛋白质，在信息传递中起着很重要的作用。特别是在免疫系统中，exosomes能够将外来抗原传递给T细胞，并且在免疫调节中发挥作用。exosomes作为一种免疫治疗的新手段，可以应用在肿瘤治疗和免疫耐受等方面。     

CPC derivedexosome protects cardiomyocytes from oxidative stress北大核心CSCD

Aim To explore the anti-apoptotic function of cardiac progenitor cells (CPCs) -derived exosome in vitro. Method CPCs were isolated from mouse heart using Magnetic Cell Sorting(MACS) system. Flow Cytometry (FC) determine the purity of stem cell surface antigen-1 positive (Sca-1+) CPCs. Exosome was purified from conditional medium, and confirmed by Western blot using CD63 as a marker, Nanoparticle Trafficking Analysis (NTA) was used to detect the diameters and concentration of exosome. Then the cells were divided into control groups and CPC-exosome pre-protec-tion groups. H2O2 was added into H9c2 cells to induce oxidative stress. Western blot was adopted to determine the expression of cleaved caspase-3. Results (1) Immunofluorescence showed that CPCs isolated by MACS were positively expressing Sca-1 protein; FC analysis showed that typical purity of Sca-1+ CPCs from the first preparations was more than 95 % . (2) WB demonstrated that CD63 of exosome isolated from CCM was positively expressed, and NTA results showed that the diameters of exosome were (82. 33 ± 3. 06) nm (n = 3). Microscope detected PKH-26 labeled exosome appeared in the cytoplasma of H9c2 cells. (3) Western blot showed the CPC-exosome pre-protection groups significantly down-regulated the levels of cleaved caspase-3 compared to the control groups (P <0.05). Conclusion CPC can secrete exosome which carries many important cargos, which can effectively gather in H9c2 cells. CPC-exosome can protect H9c2 cells from the oxidative stress induced by H2O2. Our results highlight a new perspective strategy for cardiac disease.     

外泌体在肿瘤发展中的研究进展

外泌体是细胞经过"内吞-融合-外排"等一系列调控过程而形成的细胞外纳米级小囊泡。外泌体可以携带蛋白,运送RNA,在细胞间物质和信息转导中起重要作用。外泌体可能通过调控免疫功能,促进肿瘤血管新生和肿瘤转移,以及直接作用于肿瘤细胞等途径,影响肿瘤的进展。外泌体可应用于肿瘤的诊断。本文总结了近年来有关外泌体在肿瘤发展中作用的研究进展。     

Molecular cloning and characterization of ecto-5′-nucleotidase from the venoms of Gloydius blomhoffi

A Gloydius blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences from known ecto-5′-nucleotidases (ecto-5′-NTs). Molecular cloning of ecto-5′-NT from G. blomhoffi brevicaudus venom predicted that it was a glycosyl phosphatidylinositol-anchored membrane protein containing 588 amino acid residues with 7 potential N-linked glycosylation sites. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian ecto-5′-NT sequences. This is the first report of the primary structure of ecto-5′-NT from a reptile. Gel-filtration chromatography of fresh venom from Gloydius blomhoffi blomhoffi, a subspecies of G. blomhoffi, revealed that at least a part of ecto-5′-NT is bound to exosome-like vesicles.     

Exosome swarms eliminate airway pathogens and provide passive epithelial immunoprotection through nitric oxide

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The suppression of cervical cancer ferroptosis by macrophages: The attenuation of ALOX15 in cancer cells by macrophages-derived exosomes

Induction of cancer cell ferroptosis has been proposed as a potential treatment in several cancer types. Tumor-associated macrophages(TAMs) play a key role in promoting tumor malignant progression and therapy resistance. However, the roles and mechanisms of TAMs in regulating tumor ferroptosis is still unexplored and remains enigmatic. This study shows ferroptosis inducers has shown therapeutic outcomes in cervical cancer in vitro and in vivo. TAMs have been found to suppress cervical cancer cel...