胃癌患者内皮细胞微颗粒的检测及其意义

目的比较胃癌患者与正常对照组血浆中内皮细胞微颗粒（EMPs）、血管性血友病因子（vWF）水平的变化,探讨EMPs检测在胃癌中的意义。方法采用流式细胞术及酶联免疫吸附试验（ELISA）检测68例胃癌患者和20例正常对照者血浆中EMPs、vWF的水平,分析这些指标与临床特征的关系。结果胃癌患者血浆vWF及EMPs水平显著高于正常对照组（P〈0.01）,血浆vWF及EMPs水平Ⅲ、Ⅳ期患者高于Ⅰ、Ⅱ患者（P〈0.01）,有淋巴结及肝转移者高于无淋巴结及肝转移者（P〈0.01）,有静脉浸润者高于无静脉浸润者（P〈0.01）,浸润至肌层、浆膜层者高于浸润至黏膜或黏膜下层者（P〈0.01）。结论血浆EMPs可准确反映内皮功能,内皮功能紊乱参与胃癌的转移过程,EMPs可作为判断胃癌的病情、预后、疗效的指标之一。     

Spray drying from organic solvents to prepare nanoporous/nanoparticulate microparticles of protein: excipient composites designed for oral inhalation

Objectives The aim of this study was to determine if spray‐drying could successfully produce microparticles containing the model protein trypsin in a form suitable for inhalation. Methods Trypsin was spray‐dried with raffinose from a methanol : n‐butyl acetate solvent system (MeOH : BA). The solvent system was then adjusted to include water, and trypsin was co‐spray‐dried with raffinose, trehalose or hydroxpropyl‐β‐cyclodextrin. The spray‐dried products were characterised by SEM, XRD, DSC, TGA and FTIR. Protein biological activity and in‐vitro deposition of trypsin : excipient nanoporous/nanoparticulate microparticles (NPMPs) was also assessed. Key findings The inclusion of water in a MeOH : BA solvent system allowed for the successful production of NPMPs of trypsin : excipient by spray‐drying. Trypsin formulated as trypsin : excipient NPMPs retained biological activity on processing and showed no deterioration in activity or morphological characteristics when stored with desiccant at either 4 or 25°C. Hydroxpropyl‐β‐cyclodextrin showed advantages over the sugars in terms of producing powders with appropriate density and with greater physical stability under high‐humidity conditions. Fine particle fractions of between 41 and 45% were determined for trypsin : excipient NPMPs. Conclusions NPMPs of trypsin : excipient systems can be produced by spray‐drying by adjustment of the solvent system to allow for adequate solubility of trypsin.     

幽门螺杆菌根除前后对CagA~+冠心病患者血管内皮功能的影响

目的:探讨幽门螺杆菌(Hp)根除前后细胞毒素相关基因A阳性(CagA+)对冠状动脉粥样硬化性心脏病(冠心病)患者血管内皮功能的影响。方法:选择Hp+CagA+冠心病患者57例为根除治疗组,同期住院的57例Hp-CagA-冠心病患者为对照组。在常规治疗的基础上,根除治疗组接受根除Hp治疗,对照组接受安慰剂治疗,研究期为6个月。两组研究对象在实验前后接受血脂、外周血循环内皮微颗粒(cEMP)水平及肱动脉对反应性充血的内皮依赖血管扩张反应(FMD)检测。结果:治疗后根除治疗组患者的血浆总胆固醇、cEMP水平比治疗前明显降低,FMD明显升高(P均<0.05)。治疗前根除治疗组总胆固醇高于对照组(P<0.01),治疗后两组总胆固醇比较差异无统计学意义(P>0.05)。结论:根除Hp+CagA+,可改善合并Hp+CagA+感染冠心病患者的血管内皮功能。     

腰硬联合麻醉下输入不同种类液体对剖宫产产妇血栓弹力图和血小板微颗粒影响

目的用血栓弹力图(TEG)和血小板微颗粒(PMP)检测观察腰硬联合麻醉下快速输入不同种类液体对剖宫产产妇凝血功能的影响。方法选择在我院分娩的孕36～40周选择性剖宫产的产妇120例,随机分组3组,分别快速输入相应的液体:①平衡液;②琥珀酰明胶(血定安);③羟基淀粉。3组病人均选择腰硬联合麻醉,分别于麻醉前、手术切皮之前和术毕后30 min内,进行TEG和PMP检测。结果平衡液组R值和K值术后比术前显著减小(P<0.05),Angle值术后比术前显著增加(P<0.01),CI指数明显增加(P<0.05);血定安组MA(mm)术中和术后分别明显减少(P<0.05);羟基淀粉组K值比平衡液组增加(P<0.05);血定安组(mm)Angle(°)比平衡液组减小(P<0.05);羟基淀粉组Angle(°)值比平衡液组明显减小(P<0.01);血定安组(mm)CI值比平衡液组减小(P<0.05);羟基淀粉组CI值比平衡液组明显减小(P<0.01)。血小板计数和血小板微颗粒检验结果显示3组血小板微颗粒均无显著变化(P>0.05),3组血小板均比术前减少(P<0.05)。结论术前不同种类液体快速扩容,两组胶体液对产妇的高凝状态有一定稀释作用,对预防术后静脉血栓形成可能有一定的作用,而平衡液无此作用。3组剖宫产产妇凝血功能指标仍在正常范围。     

Phosphatidylserine surface expression and integrin αIIbβ3 activity on thrombin/convulxin stimulated platelets/particles of different sizes

Platelets stimulated by a combination of thrombin/convulxin have been shown to develop two to three populations characterized by different phosphatidylserine (PS) surface expression and integrin αIIbβ3 activity. To determine how these markers are distributed on the surface of platelets/particles, we studied Annexin V and PAC-1 binding to platelets/particles of different sizes by flow cytometry analysis and evaluated influences of calpain and caspase inhibitors on thrombin/convulxin-activated platelets. Analysed platelets/particles were divided by their sizes, according to the standard size beads, into seven populations from 0·37 to 4·8 μm. PAC-1 binding/μm² was almost equal in platelets/particles ranging from 1·2 to 4·8 μm and was significantly lower on smaller-sized particles sizes (0·37–0·7 μm). PS surface exposure/μm² was high in the particles of 0·37–1·2 μm and very low in platelets (2·6–4·8 μm). Upon thrombin/convulxin stimulation caspase inhibitors prevented microparticle (MP) formation, while a calpain inhibitor stimulated MP formation. It was also shown that stimulated platelets are heterogeneous not only in their ability to activate αIIbβ3 integrin complex and expose PS on their surface, but also in the distribution of activation markers, which strongly depends on platelet/particle size and that platelets/particles of different sizes provide different responses to the same stimulus.     

Development of a time-resolved immunofluorometric assay for quantifying platelet-derived microparticles in human plasma

Platelet-derived microparticles (PMPs) are considered a marker of platelet activation. They vary considerably in size, and flow cytometry, the predominant method used to assay PMPs, is only detecting larger PMPs (> 0.1 μm).

We describe here a method that quantifies the amount of PMP-located GPIIb antigen in detergent-treated platelet-free plasma (PPP) by means of a one-step time-resolved immunofluorometric assay (TR-IFMA). This assay uses a streptavidin-coated microwell plate and two different monoclonal antibodies to GPIIb (CD41), one conjugated to biotin and the other labeled with europium ion. A wide linear range standard curve with low background and a high sensitivity was obtained. Pre-assay ultracentrifugation or filtration of PPP extensively reduced the fluorometric signal, indicating that the GPIIb antigen is mainly particle-located. A strong correlation between the amount of GPIIb and PMP as detected by flow cytometry was found. Consequently, the assay can be used to study PMP-related phenomena and, in contrast to flow cytometry, can be used on frozen samples and is independent of PMP size.     

内皮细胞微粒对人脐静脉内皮细胞VCAM-1和ICAM-1表达的影响

目的:探讨内皮细胞微粒(Endothelial microparticles,EMPs)对人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)血管细胞粘附分子-1(Vascular cellular adhesion molecule-1,VCAM-1)和细胞间粘附分子-1 (Intercellular adhesion molecule 1,ICAM-1)表达的影响.方法:取生长良好的第4、5代HUVECs,将细胞以1×105个/ml密度接种于5 cm2的培养皿中,待细胞生长近80％融合时进行干预.按0、1×102、1×103、1×104、1×105个ml浓度的EMPs进行分组及刺激,每组12个样本.在共同培养24h后收集细胞.分别采用实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测VCAM-1和ICAM-1 mRNA和蛋白的表达.结果:HUVECs受EMPs刺激,VCAM-1和ICAM-1 mRNA及蛋白表达显著增加,且EMPs的这种作用呈浓度依赖性增强(均P＜0.05).结论:EMPs能上调HUVECs VCAM-1和ICAM-1的表达,EMPs不仅是内皮功能障碍的标记物,还能加重内皮功能损害.     

Increased levels of microparticles originating from endothelial cells, platelets and erythrocytes in subjects with metabolic syndrome: Relationship with oxidative stress

Background and aims

The Metabolic Syndrome (MetS) is associated with increased cardiovascular risk. Circulating microparticles (MP) are involved in the pathogenesis of atherothrombotic disorders and are raised in individual with CVD. We measured their level and cellular origin in subjects with MetS and analyzed their associations with 1/anthropometric and biological parameters of MetS, 2/inflammation and oxidative stress markers.

Methods and results

Eighty-eight subjects with the MetS according to the NCEP-ATPIII definition were enrolled in a bicentric study and compared to 27 healthy controls. AnnexinV-positive MP (TMP), MP derived from platelets (PMP), erythrocytes (ErMP), endothelial cells (EMP), leukocytes (LMP) and granulocytes (PNMP) were determined by flow cytometry. MetS subjects had significantly higher counts/μl of TMP (730.6 ± 49.7 vs 352.8 ± 35.6), PMP (416.0 ± 43.8 vs 250.5 ± 23.5), ErMP (243.8 ± 22.1 vs 73.6 ± 19.6) and EMP (7.8 ± 0.8 vs 4.0 ± 1.0) compared with controls. LMP and PNMP were not statistically different between groups. Multivariate analysis demonstrated that each criterion for the MetS influenced the number of TMP. Waist girth was a significant determinant of PMP and EMP level and blood pressure was correlated with EMP level. Glycemia positively correlated with PMP level whereas dyslipidemia influenced EMP and ErMP levels. Interestingly, the oxidative stress markers, plasma glutathione peroxydase and urinary 8-iso-prostaglandin F₂ α, independently influenced TMP and PMP levels whereas inflammatory markers did not, irrespective of MP type.

Conclusion

Increased levels of TMP, PMP, ErMP and EMP are associated with individual metabolic abnormalities of MetS and oxidative stress. Whether MP assessment may represent a marker for risk stratification or a target for pharmacological intervention deserves further investigation.     

Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay

Introduction

Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification.

Materials and Methods

Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated.

Results

Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV + MPs (P = 0.002) and platelet-derived MPs (PMPs) (P = 0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV + MPs (P = 0.0004) and PMPs (P = 0.0004). A single freeze thaw cycle of samples led to an increase in AnnV + MPs (P = 0.0020) and PMPs (P = 0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels.

Conclusions

This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.