小胶质细胞通过外泌体释放烟酰胺磷酸核糖转移酶

目的探索炎症条件下小胶质细胞释放烟酰胺磷酸核糖转移酶(NAMPT)的机制。方法采用原代培养的小胶质细胞和小胶质细胞株BV2细胞,以缺糖缺氧再灌注(OGD/R)、TNF-α和ATP诱导细胞炎症,以经典的蛋白释放途径抑制剂和P2X7受体阻断剂、钙离子载体、钙离子螯合剂、磷脂酶D(PLD)途径抑制剂为干预,并采用普通透射电镜、免疫电镜、超速离心法分离和Western蛋白印迹法等确定NAMPT的定位和表达。结果 OGD/R和TNF-α诱导细胞的炎症中,(1)小胶质细胞可主动释放NAMPT;(2) NAMPT不经过经典的内质网/高尔基体途径释放;(3)胞外ATP可增强炎症条件下的NAMPT释放,且NAMPT释放由P2X7受体和细胞内Ca2+介导;(4) PLD抑制剂正丁醇,PLD si RNA和PI3K抑制剂wortmannin显著降低OGD/R和ATP诱导的小胶质细胞NAMPT释放;(5)在排除释放性自噬、内体和释放性溶酶体的机制后,以普通扫描电镜和免疫电镜鉴定具有外泌体的大小和形态特征的细胞外囊泡含有大量NAMPT;(6)以超速离心收集经OGD/R处理的小胶质细胞释放的外泌体,Western蛋白印迹实验进一步证实NAMPT和外泌体标记蛋白有共同表达;(7) NAMPT相对于外泌体蛋白标记的量保持不变,表明小胶质细胞外泌体中的NAMPT负荷量是一定的。结论神经炎症过程中,NAMPT由小胶质细胞通过外泌体主动释放。     

外泌体在糖尿病肾病早期诊断和治疗中的研究进展

随着糖尿病发病率的升高,目前糖尿病肾病(DKD)已成为慢性肾脏病的首要病因之一,除肾脏病理活检外,通过血清、尿液特异标志物等获得糖尿病肾病早期诊断对改善患者预后有重要意义。外泌体是一种细胞外囊泡,可参与细胞间信息交流、免疫调节等病理生理过程。由于外泌体具有易获取、稳定性高、诊断特异性高等优点,目前已成为糖尿病肾病病理生理机制的探索及早期诊断的研究热点。间充质干细胞外泌体可降低糖尿病肾病肾组织内白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)等炎症因子表达,抑制炎症反应、改善肾脏纤维化,已有临床试验证实干细胞治疗对糖尿病患者有保护肾脏的作用。近年来研究发现,细胞自噬作用减弱是糖尿病肾病发生及发展的重要病理生理机制之一,外泌体可通过雷帕霉素靶蛋白(mTOR)途径调节细胞自噬,减轻糖尿病肾病足细胞损害,因此自噬相关基因或蛋白可成为糖尿病肾病潜在的治疗靶点。     

外泌体在心肌疾病中的研究进展

外泌体是具有脂双层膜的细胞外囊泡,其通过携带丰富的微小RNA(miRNA)等生物分子传递信息,是细胞间通讯的关键介质。外泌体通过参与炎症反应、内皮细胞损伤、心肌纤维化等过程来影响各种心肌疾病的发生发展,在心肌的损伤和修复中起着非常重要的作用。本文主要就近几年发现的外泌体在各种心肌疾病中的机制、诊断及治疗等方面的作用展开综述。     

外泌体微小RNA作为肺癌标志物的研究进展

肺癌是当今致死率较高的癌症之一,其早期诊断对于提高治疗效果至关重要.外泌体是肿瘤细胞信号传导过程中的一种重要方式,其内容物包括蛋白质、脂类物质、mRNAs、微小RNA(microRNAs)等.肿瘤细胞和微环境中其他细胞均可以向细胞外基质中释放外泌体,进而参与调控肿瘤的发生和发展.外泌体可体现其来源细胞的部分重要生物学特...     

外泌体在2型糖尿病发病机制及诊疗标志物开发中的研究进展

外泌体是一种纳米级大小的细胞外分泌囊泡,具有脂质双分子层结构,可以携带多种DNA,RNA以及蛋白质等生物分子,是细胞间信号传递的重要载体.大量研究结果表明,外泌体及其内含物可通过多种途径影响胰岛素的分泌和组织敏感性,参与糖脂代谢和胰岛素抵抗的调控,在包括糖尿病在内的内分泌及代谢性疾病发生发展过程中发挥重要作用.本文就外...     

免疫细胞来源的细胞外囊泡在肿瘤治疗中的研究进展

细胞外囊泡是一种由细胞主动向外释放的膜性小囊泡,可装载DNA、RNA、脂质和蛋白质,是细胞间通信的重要调节者.细胞外囊泡由外泌体、微囊泡和凋亡小体组成,其中研究最全面的是外泌体.免疫细胞来源的细胞外囊泡通过调节免疫系统来增强或抑制免疫活动,在抗肿瘤过程中发挥重要作用.本文综述了细胞外囊泡的生物发生、提取与纯化和载药方法...     

干细胞来源外泌体的生物学特性研究进展

目前,干细胞来源外泌体可通过不同的方法对其进行鉴定及提取,从而为干细胞来源外泌体的研究奠定了基础。研究发现干细胞来源外泌体在非肿瘤环境下可通过免疫调节、抗炎、抗氧化、调节蛋白及促进组织功能恢复等方式参与机体的调节功能,在缺氧、脂多糖及转染等外源诱导作用下可干预干细胞分泌外泌体的功能及外泌体的分泌作用。而在肿瘤环境下,多数研究报道干细胞外泌体具有促肿瘤的作用,而通过基因转染干细胞衍生的外泌体对肿瘤具有抑制作用。     

优化脂肪间充质干细胞外泌体提取方法的研究

目的 本研究旨在探讨用外泌体提取试剂盒提取脂肪间充质干细胞源性外泌体的优化方法。方法 采用组织块贴壁法分离提取和培养原代脂肪间充质干细胞。收集1000mL 3～6代脂肪间充质干细胞的细胞上清，用100KD超滤离心管进行浓缩，外泌体提取试剂盒提取外泌体，测定外泌体悬液蛋白浓度后进行鉴定。结果 脂肪间充质干细胞的形态主要以长梭形为主；外泌体悬液的蛋白浓度，经BCA法测定为7.66 mg/mL;透射电镜、NTA、蛋白质印迹法结果均符合外泌体的典型特征。结论 在使用外泌体提取试剂盒(invitrogen)之前，用100KD的超滤管对1000mL细胞上清进行浓缩的方法可以成功提取到外泌体，而且是一种有效节省外泌体提取试剂盒剂量的方法。     

治疗性外泌体的研究进展

外泌体是一种活细胞分泌的囊性小泡,携带大量具有组织或细胞特异性的蛋白质、脂质及遗传物质,可调控不同的生理活动,因此作为一类新兴的治疗药物被广泛研究。间充质干细胞(mesenchymal stem cells, MSCs)和树突状细胞(dendritic cells, DCs)衍生的外泌体是研究较为广泛的两类外泌体,已有许多临床前及临床研究表明其在肺部疾病、肝脏疾病、神经系统疾病及肿瘤等疾病中展现出良好的治疗效果。另外,巨噬细胞、肿瘤细胞和植物细胞等众多其他细胞衍生的外泌体也因其治疗潜力受到越来越多的关注。除了天然来源的外泌体,工程化外泌体的研究也取得许多进展。已报道的外泌体工程化手段种类繁多,如外泌体靶向修饰、外泌体包载活性成分等。本文总结了不同来源的治疗性外泌体的研究进展,并讨论了外泌体的应用前景与未来可能遇到的挑战。     

外泌体与胆道闭锁肝纤维化研究进展

胆道闭锁（BA）是一种严重的婴幼儿肝胆系统疾病,很快会进展为不可逆的肝纤维化,最终导致肝衰竭,威 胁患儿生命。及时有效地遏制 BA肝纤维化是延长或实现患儿自体肝生存的关键。外泌体作为一种包含特殊的脂 质、蛋白质、核酸的纳米级囊泡,具有一定的生理和病理功能,被认为是一种新型的细胞间通讯方式。近年来,随着对 外泌体的研究升温,其参与调节BA肝纤维化的功能也逐渐被关注,并作为细胞间信号传递的载体影响纤维化进程： 能传递或影响纤维结缔组织生长因子、转化生长因子 β1、间接促进白细胞介素（IL）-17分泌、参与肝纤维化相关的 Notch通路与Hedgehog（Hh）通路以及调节肝星状细胞迁移。尤其,已有体外实验证实了来自脂肪组织来源间充质干 细胞及人脐带间充质干细胞的外泌体能发挥抑制纤维化作用。本文拟对外泌体与BA肝纤维化的关系进行综述,期 望能够为临床治疗BA肝纤维化提供新的方向。     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg.kg) or i.p. (50 mg.kg) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) l.h. kg in the male rat and 10.6 (95% CI: 7.5, 15.0) l.h. kg in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p 0.001) in plasma obtained from the male (8.8 2.0%) compared with the female rat (11.7 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg x kg(-1)) or i.p. (50 mg x kg(-1)) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) 1 x h(-1) x kg(-1) in the male rat and 10.6 (95% CI: 7.5, 15.0) 1 x h(-1) x kg(-1) in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was approximately 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p < 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p < 0.001) in plasma obtained from the male (8.8 +/- 2.0%) compared with the female rat (11.7 +/- 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Interindividual variability in metabolic activation in humans in vivo

In assessing interindividual variability in metabolic activation, the toxic metabolite is often too unstable for conventional analysis. Possible alternatives include a stable product of the reactive metabolite e.g. cysteinyl derivatives of N-acetyl-4-benzoquinoneimine, the toxic metabolite of paracetamol, adducts with DNA or protein, and indirect measurement of the activity of the enzyme(s) producing the active metabolite. An example of the last approach is the use of furafylline, a highly specific inhibitor of human CYP1A2, to determine the extent of the metabolic activation of the cooked food mutagens PhIP and MeIQx. The extent of inhibition, determined from levels of unchanged amine in urine, is an indirect measure of the activity of the activation pathway. Further refinement of this approach, allied to improved measures of the biological process of interest should prove of value in evaluating interindividual variability and its role in the risk assessment process.     

Theophylline kinetics in peripheral tissues in vivo in humans

Several biochemical and cellular effects have been described for methylxanthines under in vitro conditions. However, it is unknown, whether threshold concentrations required to exert these effects are attained in target tissues in vivo. We therefore employed the microdialysis technique for measuring theophylline concentrations in peripheral tissues under in vivo conditions.Following in vitro and in vivo calibration, microdialysis probes were inserted into the medial vastus muscle and into the periumbilical subcutaneous adipose layer of healthy volunteers. Following single oral dose administration of 300 mg or i.v. infusion of 240 mg theophylline, in vivo time courses of theophylline concentrations were monitored in tissues and plasma. Major pharmacokinetic parameters (c_max, t_max, AUC) were calculated for plasma and tissue time courses. The mean AUC_tissue /AUC_plasma-ratio was 0.56 (p.o.) and 0.55 (i.v.) for muscle and 0.55 (p.o.) and 0.72 (i.v.) for subcutaneous adipose tissue.We conclude that microdialysis provides important information on the distribution and the tissue pharmacokinetics of theophylline.Abbreviations FPIA Fluorescence polarisation immuno assay - AUC Area under the curve - t_max Time to peak concentration - c_max Peak concentration     

休克时血中氧自由基的变化

本实验测定10名休克患者血浆和红细胞的丙二醛(MDA)、血浆总抗的氧化活性(AOA)的含量。结果表明:休克病人红细胞膜和血浆 MDA 含量(4.298±0.722;5.348±0.834)与对照组(3.235±0.682;4.356±1.081)比较明显增高(P<0.05);血浆 AOA(39.65±7.858)与对照组(48.21±10.81)比较明显降低(P<0.01)。提示:休克时,患者机体内自由基反应增强是引起组织细胞损伤的原因之一。     

Changes in angiopoietin expression in glomeruli involved in glomerulosclerosis in rats with daunorubicin-induced nephrosis

AIM: To study the potential pathological role of endogenous angiopoietins in daunorubicin-induced progressive glomerulosclerosis in rats. METHODS: Seventy male Wistar rats were allocated randomly into a daunorubicin group (DRB; n=40) or a control group (n=30). The rats in the DRB group were injected with DRB (15 mg/kg), in their tails. Subsequently, at intervals of 1, 2, 4, 6, 8, and 12 weeks, 5 male Wistar rats in each group were chosen randomly for 24 h urinary protein quantitative measurements (24 h UPQM), and determination of plasma tumor necrosis factor alpha (TNF-alpha), angiopoietin-1 (Ang1), and angiopoietin-2 (Ang2) levels. Kidney sections were examined by electron microscopy, Periodic Acid Schiff (PAS) staining, immunohistochemical staining and in situ hybridization histochemistry. RESULTS: As glomerulosclerosis progressed in the DRB group, expression of Ang1 mRNA and protein in glomeruli decreased and expression of TNF-alpha protein, Ang2 mRNA and protein in glomeruli increased. Expression of Ang1 mRNA and protein in glomeruli were negatively correlated with 24 h UPQM, Fn protein expression, and mean area of extracellular matrix (MAECM). In comparison, expression of Ang2 mRNA and protein in glomeruli were positively correlated with 24 h UPQM, Fn protein expression and MAECM; furthermore, there was a positive correlation between plasma Ang2 and 24 h UPQM. Plasma TNF-alpha and expression of TNF-alpha in glomeruli were positively correlated with expression of Ang2 mRNA and protein in glomeruli. There was a negative correlation between Ang1 protein expression and Ang2 protein expression in glomeruli. CONCLUSION: During DRB-induced glomerulosclerosis, podocyte injury led to a shift in the balance of Ang1 and Ang2 in glomeruli. Increased TNF-alpha in plasma and glomeruli may upregulate Ang2 expression in glomeruli. Elevated Ang2 in both plasma and glomeruli may mediate protein permeability through the glomerular filtration barrier. Moreover, local expression of Ang2 may facilitate the progress of glomerulosclerosis by upregulating a component expression of extracellular matrix.     

Trichinellosis in immigrants in Switzerland

We describe a case of trichinellosis diagnosed at the Division of Infectious Diseases, Hospital of Lugano, in January 2009. This case was associated with a cluster of cases and was traced to the consumption of contaminated meat after a wild boar hunt in Bosnia.