外排体促进骨再生的研究进展

外排体是来源于内吞作用的一种小囊泡,在骨再生治疗领域成为一种新的选择。这种小囊泡在细胞间传递物质(如蛋白质、RNA等),从而调节靶细胞的分化、增殖功能等。外排体通过直接调节间充质干细胞的成骨分化、刺激成骨细胞的增殖和活性、调节破骨细胞的成熟和活性以及增加血管生成能力来促进骨再生。同时还能避免生物治疗所带来的细胞毒性和免疫抑制问题。此外外排体疗法的并发症(如肿瘤、血栓形成等)相对于生物治疗来说更少。     

外排体在脑胶质瘤中的研究进展

外排体是细胞分泌到细胞外基质中的一种功能性囊泡,胶质瘤外排体在胶质瘤的发生发展、辅助诊断和治疗中的作用日益受到重视。本文对常见胶质瘤外排体标志物、介导免疫治疗、抗肿瘤治疗载体、干预肿瘤细胞信号通路等方面进行总结。     

外排体是肿瘤优势生存的开拓者

外排体(exosomes)是活细胞分泌的具有脂质双分子层和大量内含物的微囊泡结构。肿瘤细胞来源的外排体不仅在肿瘤细胞之间的通信中充当信使的功能,也能作为载体影响远处的正常机体细胞,为肿瘤细胞的增殖、分化、转移及免疫抵抗起到保驾护航的作用。     

结核和非小细胞肺癌患者胸腔积液的蛋白质组学研究

目的通过比较结核和非小细胞肺癌患者的胸腔积液蛋白质组成差异,寻找潜在鉴别诊断标志蛋白质。方法收集结核性胸腔积液样本和非小细胞肺癌胸腔积液样本,使用聚丙烯酰胺凝胶电泳—纳升液相色谱—超高分辨飞行时间质谱进行蛋白质鉴定。结果结核性胸腔积液中共检测出人类蛋白质517种和233种结核菌蛋白质,肺癌胸腔积液共检测出人类蛋白质375种。蛋白功能分析显示结核性胸腔积液中细胞周期和细胞运动的蛋白质富集明显,而肺癌胸腔积液中Ras信号转导和DNA甲基化的蛋白质富集明显。其中白介素Iα(IL1A)仅存在于肺癌胸腔积液中,结核菌大膜蛋白1(Mmpl1)和结核菌蛋白质Rv2567仅存在于结核性胸腔积液样本中。结论 IL1A、Mmpl1、Rv2567可能成为鉴别结核性胸腔积液和肺癌胸腔积液的潜在标志蛋白。     

肺癌组织中ICAM-1测定的临床意义

目的研究肺癌组织中细胞间粘附分子-1的表达及其临床意义。方法应用流式细胞仪检测技术检测了60例人肺癌组织中细胞间粘附分子-1的表达,并与正常肺组织中细胞间粘附分子-1的表达比较,进一步探讨肺癌细胞产生的1型细胞间粘附分子在肺癌侵袭和转移过程中的作用。结果60例肺癌组织中有54例ICAM-1表达明显增强,其含量明显高于正常组织(P<0.01),Ⅲ-Ⅳ期肺癌组织中ICAM-1的表达明显高于Ⅰ-Ⅱ期组。结论ICAM-1与肺癌的侵袭和转移有关,有可能作为预测肺癌转移、复发及预后的指标。     

人脂肪间充质干细胞来源的外排体促进大鼠创伤性脑损伤后神经功能恢复

目的研究人脂肪间充质干细胞(h AMSCs)来源的外排体对创伤性脑损伤(TBI)的治疗作用及其可能的机制。方法分离健康成人脂肪MSCs,通过超滤法提取外排体。将大鼠分成:假手术组,PBS对照组,MSC治疗组,exosomes治疗组。于TBI建模24 h后,治疗组分别沿损伤边缘区局部注射,PBS 30μL,MSC 2×10~5个细胞/只,exosomes 25μg总蛋白量/只,总体积30μL。在建模前和TBI后1、3、7、10、13、16和30 d测试所有大鼠的m NSS评分和前肢踩空试验。3和7d处死大鼠,提取大鼠脑组织总RNA,实时定量PCR检测大鼠炎性因子TNF-α和IL-1β的表达,30 d处死大鼠,tunel-neun双标免疫荧光检测TBI后神经元凋亡。结果外排体的治疗显著促进TBI后的神经功能的恢复,治疗效果与MSC治疗效果相当,其机制可能是通过抑制大鼠TBI后急性炎性反应,减少神经元凋亡。结论人脂肪间充质干细胞来源的外排体促进脑外伤后神经功能的恢复,这将为临床提供一种新的更安全的TBI治疗手段。     

调节性T细胞外泌体对肿瘤调节作用的研究进展

外泌体是由细胞分泌的异质性纳米级胞外囊泡,富含多种RNA、DNA片段、脂质和蛋白质等生物活性物质,能参与细胞间的物质交换与信息交流转送。作为细胞间交流和传递信息的一种新媒介,其在免疫应答、血管生成、炎症反应、细胞凋亡及肿瘤发生发展等过程中均有重要的调节作用。调节性T细胞分泌外泌体参与调节免疫应答、器官移植、自身免疫病以及肿瘤的发生发展与转移等过程。本文主要综述调节性T细胞来源外泌体的研究进展和生物学特征,与肿瘤细胞来源外泌体的异同点及其在肿瘤微环境的形成维持、肿瘤诊断与治疗方面的潜在应用价值。     

外泌体非编码RNA在肺癌肿瘤微环境中的作用

正肺癌是全球发病率和死亡率第一的恶性肿瘤。2018年约有210万例新增肺癌患者及180万死亡病例,严重威胁着各地人民的健康[1]。根据细胞的病理特征和分化程度,肺癌主要被分为非小细胞肺癌(non-small-cell lung cancer,NSCLC)和小细胞肺癌。前者占所有肺癌类型的85%左右[2-3],其主要亚型包括腺癌、鳞状细胞癌和大细胞癌。近年来,随着科学技术的飞速发展以及对细胞外囊泡,尤其是外泌体探索的日趋深入,外泌体检测逐渐成为液体活检技术的重要组成部分,为肺癌研究提供了新思路。     

外泌体在缺血性脑卒中的研究进展

正1外泌体概述外泌体是直径在30~100 nm的胞外囊泡,通过细胞被释放到细胞外液中~([1])。它们存在于生物体液中,诸如血液和脑脊液。外泌体携带有DNA、RNA、蛋白质和脂质等。由于外泌体的微泡结构为其内在的小分子提供了一个安全稳定的环境,同时这些信号小分子利用循环系统在胞间信号交换发挥重要作用,这让外泌体表现出一个成熟、稳定的信号传输系统~([2])。研究发现,外泌体中的m RNA和micro RNA     

PKC参与化学修饰的LDL对ABCA1表达和功能的调节

目的 探讨PKC信号途径对oxLDL和acLDL诱导的小鼠巨噬细胞ABCAl表达的影响。方法 分别以100μg/ml acLDL和oxLDL温育小鼠巨噬细胞系RAW264.7细胞24 h,同时加入PKC信号途径的激活剂(PMA)或抑制剂(GF109203X),应用胆固醇外排实验、半定量RT-PCR和Western印迹,检测小鼠巨噬细胞内ABCAl功能、mRNA及蛋白质表达的水平。结果 1.0 μmol/L GF109203X可分别使aeLDL孵育组的胆固醇外排率下降至对照组的56.0％,ABCA1 mRNA水平下降至(54.0±8.2)％,蛋白质水平下降至(68.1±2.0)％;oxLDL孵育组的胆固醇外排率下降至对照组的47.0％,ABCA1 mRNA水平下降至(43.0±5.0)％,蛋白质水平下降至(73.0±10.0)％。160 nmol/L PMA作用24 h可分别使acLDL孵育组的胆固醇外排率升高至134.0％,oxLDL孵育组升高至125.1％;ABCA1 mRNA水平升高至(211.0±17.0)％,蛋白质水平升高至(305.0±21.0)％。结论 PKC信号途径在oxLDL和acLDL对小鼠巨噬细胞内ABCA1表达调控中发挥作用,该信号途径的激活可上调ABCA1的表达,促进ABCA1介导的胆固醇外排。     

Dipeptidyl peptidase I cleaves matrix-associated proteins and is expressed mainly by mast cells in normal dog airways

Dipeptidyl peptidase I (DPPI) is a cysteine protease found in many tissues, including the lung. Major cell types expressing DPPI in vitro include myelomonocytic cells, cytotoxic T cells, and mast cells. After activation and degranulation, cytotoxic T cells and mast cells secrete DPPI. With a goal of clarifying possible roles for DPPI in lung diseases, we sought to identify cells expressing DPPI in lung tissue, hypothesizing that lung mast cells are major producers of DPPI and that secreted DPPI cleaves extracellular matrix proteins. To address these hypotheses, we used immunohistochemical techniques to localize DPPI in normal dog airways, lung, and cultured mast cells, and we used purified DPPI to examine cleavage of matrix-associated proteins in vitro. We found that mast cells are the major identifiable source of DPPI in airways and that macrophages are the major source in alveoli. Within mast cells, DPPI localizes to cytoplasmic granules. We also found that DPPI endoproteolytically cleaves the extracellular matrix proteins fibronectin and collagen types I, III, and IV. The finding of DPPI in airway mast cells and its cleavage of matrix proteins suggest the possibility that DPPI plays a role in mast cell-mediated turnover of matrix proteins and in airway remodeling of chronic airway diseases such as asthma.     

Diallyl trisulfide selectively causes Bax- and Bak-mediated apoptosis in human lung cancer cells

Garlic-derived organosulfur compounds (OSCs) are highly effective in affording protection against chemically induced pulmonary carcinogenesis in animal models. We now demonstrate that garlic constituent diallyl trisulfide (DATS) suppresses viability of cultured human lung cancer cell lines H358 (anon-small cell lung cancer cell line) and H460 (a large cell lung cancer cell line) by causing G2-M phase cell cycle arrest and apoptotic cell death. On the other hand, a normal human bronchial epithelial cell line BEAS-2B was significantly more resistant to growth inhibition and apoptosis induction by DATS compared with lung cancer cells. We also found that even a subtle change in the OSC structure could have a significant impact on its biological activity. For example, DATS was significantly more effective than either diallyl sulfide or diallyl disulfide against proliferation of lung cancer cells. The DATS-mediated G2-M phase cell cycle arrest was explained by down-regulation of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C protein expression leading to accumulation of Tyr15 phosphorylated (inactive) Cdk1. The DATS-induced apoptosis correlated with induction of pro-apoptotic proteins Bax, Bak and BID, and a decrease in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL in lung cancer cells but not in BEAS-2B. Knockdown of Bax and Bak proteins conferred significant protection against DATS-induced apoptotic cytoplasmic histone-associated DNA fragmentation. On the other hand, BID protein was dispensable for DATS-induced apoptosis. In conclusion, the present study indicates that Bax and Bak proteins are critical targets of DATS-induced apoptosis in human lung cancer cells.     

Differential accumulation of surfactant protein A, B, and C mRNAs in two epithelial cell types of hyperoxic lung

The physiologic response of the lung to oxygen toxicity is complex, and similar among all mammals studied. Acute exposure to 100% O2 results in severe decreases in respiratory function and is accompanied by alterations in pulmonary surfactant metabolism, including the regulation of surfactant proteins A, B, and C (SP-A, SP-B, SP-C). Because surfactant proteins and their mRNAs can be expressed in alveolar epithelial type II cells, and nonciliated bronchial epithelial (Clara) cells, we were interested in determining if alterations in the abundance of SP-A, SP-B, and SP-C mRNAs occurred differentially in these two cell types during hyperoxic lung injury. Using quantitative in situ hybridization, we found that hyperoxic lung injury resulted in nearly 20-fold increases in SP-A and SP-B mRNAs in Clara cells, with relatively small (2-fold or less) increases in type II cells. Immunohistochemical analysis suggested a commensurate increase in SP-A protein in Clara cells. SP-C mRNA was only detected in type II cells, and changed little in hyperoxic lung. Because Clara cells are not known to produce surfactant, and appear to lack SP-C mRNA, these observations suggest that increased SP-A and SP-B may serve nonsurfactant functions in hyperoxic lung.     

Type I and III collagen protein precursors and mRNA in the developing human lung

Extracellular matrix proteins have a prominent role in both ontogenesis and fibrogenesis in the human lung. The aim of this study was to analyse the expression of newly formed precursor proteins and mRNA of collagen types I and III in developing human lung tissues from 12 to 40 weeks of gestation, and also in neonatal disorders such as respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Lung tissues were obtained at autopsy from 60 non-malformed cases. All tissues were analysed by immunohistochemistry and 24 were also investigated by mRNA in situ hybridization. The precursor proteins and mRNA of both collagens were expressed in abundance in pulmonary arteries and veins during all developmental periods. In RDS and BPD, precursor proteins and mRNAs of both collagen types were increased within alveolar walls. The cells in these locations showed alpha-smooth muscle actin, vimentin, and variable desmin immunoreactivity. Collagen I and III precursor proteins and mRNA were also observed in pleura, bronchi, bronchioles, and around chondrocytes during all developmental periods and in diseased lung. In conclusion, collagens I and III were expressed in a similar way in and around various cell types in the developing lung and their expression was increased within alveolar walls in RDS and BPD. Myofibroblast-type cells appeared to produce mRNA for both types of collagen in alveoli.     

Growth hormone (GH) action in the developing lung: changes in lung proteins after adenoviral GH overexpression.

Growth hormone (GH) recently has been shown to be expressed in the neonatal rat lung during alveolarization. The possible functional importance of lung GH in lung function, therefore, has been assessed by determining changes in GH-responsive proteins in the developing rat lung after the overexpression of the GH gene in this tissue. GH overexpression was achieved using an adenovirus that expressed the mouse GH gene. This adenovirus was effective in inducing mouse GH expression in cultured rat lung L2 epithelial cells. It was also shown to be strongly expressed in the alveoli of 14-day-old rat pup lungs 10 days after it was administered by intratracheal injection, during a period of rapid lung development. Expression of the transgene in these pups was accompanied by changes in lung protein concentrations determined by two-dimensional gel electrophoresis and mass spectrometry. The lung concentrations of specific enzymes (nucleotide diphosphate kinase B, Cu/Zn superoxide dismutase, glutathione-S-transferase, and aldehyde reductase-1) were increased by the adenoviral expression of mouse GH, as were the concentrations of beta subunit G-protein calponin 2, beta-5 tubulin, retinoblastoma binding protein 4, and fetuin A. In contrast, the lung concentrations of haptoglobin and major acute phase alpha-1 protein were reduced by adenoviral expression of mouse GH. Although most of these proteins have not previously been identified as GH-responsive proteins, these results demonstrate actions of GH in the rat lung and support the possibility that GH acts as an autocrine/paracrine during early lung development.     

Type I epithelial cells are the main target of whole-body hypoxic preconditioning in the lung

Whole-body hypoxic preconditioning (WHPC) prolongs survival of mice exposed to severe hypoxia by attenuating pulmonary edema and preserving gas exchange. However, the cellular and molecular mechanism(s) of this protection remains unclear. The objective of this study was to identify the cellular target(s) of WHPC in the lung. Conscious mice were exposed to hypoxia (7% O(2)) for 6 hours with or without pretreatment of WHPC ([8% O(2)] x 10 min/[21% O(2)] x 10 min; 6 cycles). Hypoxia caused severe lung injury, as shown by the development of high-permeability-type pulmonary edema and the release of lactate dehydrogenase and creatine kinase into the airspace and the circulation. All these signs of hypoxic lung injury were significantly attenuated by WHPC. Hypoxia also caused a remarkable release of type I cell markers (caveolin-2 and receptor for advanced glycation end products) in lung lavage that was almost completely abolished by WHPC. Conversely, hypoxia-induced release of type II cell markers (surfactant-associated proteins A and D) was only marginal, and was unaffected by WHPC. Electron microscopic analysis demonstrated considerable hypoxic damage in alveolar type I cells and vascular endothelial cells. Notably, WHPC completely eliminated hypoxic damage in the former and alleviated it in the latter. Type II cells appeared normal. Furthermore, WHPC up-regulated protein expression of cytoprotective genes in the lung, such as heat shock proteins and manganese superoxide dismutase. Thus, WHPC attenuates hypoxic lung injury through protection of cells constituting the respiratory membrane, especially hypoxia-vulnerable type I epithelial cells. This beneficial effect may involve up-regulation of cytoprotective genes.     

Rat monoclonal antibodies to mouse lung components for analysis of fibrosis

Six rat monoclonal antibodies to mouse lung membrane fraction have been characterized. Each has unique binding properties and can be used to stain particular lung components in paraffin sections. One antibody, 133-13A, recognizes a 100-kDa glycoprotein on lung tumor cells, but stains only macrophage-like cells in normal or fibrotic lung sections as determined by immunogold electron microscopy. The monoclonal antibody 273-34A binds to a 112-kDa protein on the surface of normal mouse lung fibroblasts. Immunogold electron microscopy demonstrates antibody binding to capillary endothelial cells, but not to fibroblasts. Type I, or Type II cells. Staining of fibrotic sections with MoAb 273-34A is dramatically enhanced over staining of normal lung. A third antibody, 370-8A, gives a general staining pattern throughout normal lung that is intensified in fibrotic lung. Another MoAb 328-41A mediates intense nuclear fluorescence of lung tumor cells and cells in lung sections. It binds to 14- and 17-kDa proteins that may be high mobility group (HMG) nuclear proteins. Enzyme inhibition studies and immunofluorescence staining patterns on normal lung indicate that elastin may be the target of MoAb 328-35B. MoAb 327-5B binds to normal mouse lung fibroblasts and red blood cell membranes. These last three MoAbs stain macrophages in fibrotic lung, but give a general pattern of light epithelial cell stain in normal lung sections indicating macrophage engulfment of the normal cell antigen during fibrosis. These antibodies should be useful in identifying cell types and molecular mechanisms involved in early stages of fibrosis induced by different chemical insults.