脑梗死患者血浆炎症因子、纤溶活性的变化及对神经功能的影响

目的通过观察急性脑梗死患者血浆中的Ⅷ因子相关抗原(vWF)、P-选择素(P-Selectin)、白细胞介素-6(IL-6)、组织型纤溶酶原激活物(t-PA)及快速抑制物(PAI-1)的变化规律,探讨炎症因子及纤溶活性在脑梗死发生、发展、转归过程中的作用。方法采用酶联免疫吸附(ELISA)及发色底物法对40例急性脑梗死患者发病后6,12,24及72h血浆vWF,P-selectin,IL-6,t-PA及PAI-1活性进行动态测查。并与32例自愿者健康对照组进行比较。结果在急性脑梗死患者发病24h内,vWF(147.4±26.2)μg/ml、P-selectin(21±4)ng/ml、IL-6(114.5±1.5)μg/ml、PAI-1(0.79±0.24)au/ml活性明显增高,t-PA活性(0.24±0.15)iu/ml明显降低,与对照组比较差异有显著性意义(P<0.05);随时间推移vWF(118.6±24.4)μg/ml、P-selectin(14±3)ng/ml、IL-6(108.7±4.4)μg/ml、PAI-1(0.56±0.20)au/ml活性有所降低,而t-PA活性(0.43±0.11)iu/ml升高,但与正常对照组相比仍有差异(P<0.05)。结论(1)急性脑梗死患者早期存在内皮细胞损伤;(2)炎症因子参与脑部缺血性损害;(3)纤溶活性下降,说明t-PA及PAI-1参与了血栓形成的病理过程。     

小檗碱对HL-60细胞增殖、凋亡及血管内皮生长因子受体2表达的影响

本研究旨在观察小檗碱对人早幼粒白血病HL-60细胞的增殖、凋亡及血管内皮生长因子受体-2(VEGFR2)表达的影响。选用HL-60细胞体外培养,在不同浓度的小檗碱(6-96μg/ml)作用下,应用CCK-8法检测小檗碱对HL-60细胞的增殖抑制作用;应用流式细胞仪检测HL-60细胞的凋亡水平及细胞周期的分布;用RT-PCR及Western blot分别检测VEGFR2 mRNA及VEGFR2蛋白表达的变化。结果显示,小檗碱能抑制HL-60细胞的增殖,促进其凋亡,呈浓度和时间依赖关系。随着小檗碱浓度增加,G1期细胞增多,S期细胞减少,同时VEGFR2 mRNA和VEGFR2蛋白表达减少。结论:小檗碱可抑制HL-60细胞增殖,促进细胞凋亡,改变HL-60细胞周期,下调VEGFR2 mRNA及VEGFR2蛋白的表达。     

The effects of sampling from a peripheral venous catheter compared to repeated venepunctures on markers of coagulation,inflammation, and endothelial function

Abstract

Peripheral venous (PV) catheters are often used for serial blood sampling, but studies suggest that PV catheters increase markers of coagulation activation and inflammation. Whether the increase is caused by irritation of the vessel wall or diurnal variation is unknown. We therefore compared the effects of a PV catheter and repeated venepunctures on markers of coagulation, inflammation, and endothelial function.

A PV catheter was inserted at 07:45 in a hand vein in 10 healthy subjects, and blood samples were collected at 8:00, 10:00, 12:00, and 14:00. In the contralateral arm, blood was simultaneously obtained by venepunctures. Measures of coagulation, i.e., activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, prothrombin fragment 1?+?2 (F1?+?2) and thrombin-antithrombin (TAT), inflammation, i.e., interleukin 6 (IL-6) and C-reactive protein (CRP), and endothelial function, i.e., plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), von Willebrand factor (vWF), and tissue factor (TF) were measured in plasma.

The concentrations of TAT and F1?+?2 were significantly increased (10:00; p?<?.01, 12:00; p?<?.05, and 14:00; p?<?.01) in PV catheter samples compared with venepuncture samples. There was a minor increase in PT and INR and no increase in APTT, fibrinogen, CRP, PAI-1, tPA, vWF, and TF, with no differences between sampling methods. IL-6 concentrations increased in many PV catheter samples and venepuncture samples, but the response varied between the subjects.

Blood collection through a PV catheter induces coagulation activation, whereas endothelial function is not affected. More studies are needed to disclose the effect of blood sampling on IL-6.     

Anthrax toxin: structures,functions and tumour targeting

Anthrax toxin, the major virulence factor of Bacillus anthracis, consists of three polypeptides: protective antigen (PrAg), lethal factor (LF) and oedema factor (EF). To intoxicate mammalian cells, PrAg binds to its cellular receptors and is subsequently activated via proteolysis, yielding a carboxyl-terminal fragment which coordinately assembles to form heptamers that bind and translocate LF and EF into the cytosol to exert their cytotoxic effects. Substantial progress has been made in recent years towards the characterisation of the structure and function of anthrax toxin, and this has greatly facilitated rational drug design of antianthrax agents. There is also emerging evidence that toxins can be manipulated for cancer therapy. LF can efficiently inactivate several mitogen-activated protein kinase kinases (MAPKKs) via cleavage of their amino-terminal sequences. Consequently, antitumour effects of wild type lethal toxin were observed after treatment of mitogen-activated protein kinase (MAPK)-dependent tumours such as human melanomas. Modification of the toxin’s proteolytic activation site limits its cytotoxicity to certain cell types and creates a versatile method of treatment. One approach that has successfully achieved specific tumour targeting is the alteration of the furin cleavage of PrAg so that it is not activated by furin, but, alternatively, by proteases that are highly expressed by tumour tissues, including matrix metalloproteases and urokinase.     

人附睾蛋白4通过上调MMP9及VEGF降低顺铂对卵巢癌细胞增殖和迁移的影响

目的 探讨人附睾蛋白4(HE4)对顺铂作用后卵巢癌细胞增殖和迁移的影响及潜在机制。方法 构建HE4质粒,脂质体Lipofectamine 2000转染人卵巢癌细胞系HO8910PM,通过Western blot验证稳定转染细胞株构建成功。通过CCK8实验检测HE4过表达对顺铂作用后细胞增殖的影响;通过Transwell实验检测HE4过表达对顺铂作用后细胞迁移的影响;同时通过Western blot实验检测促迁移相关蛋白基质金属蛋白酶9(MMP9)和血管内皮生长因子(VEGF)的表达。结果 过表达HE4人卵巢癌细胞系HO8910PM构建成功;相比于转染空载体的HO8910PM细胞,HE4可明显降低顺铂对细胞增殖的抑制作用,促进HO8910细胞的增殖;同时,HE4过表达细胞中MMP9及VEGF蛋白表达水平也相应提高。结论 HE4可能通过上调MMP9及VEGF表达降低顺铂对卵巢癌细胞增殖和迁移的影响。     

子痫前期患者组织因子、组织因子途径抑制物与血清可溶性血管内皮生长因子受体1的相关性

目的探讨子痫前期患者外周血组织因子(TF)、组织因子途径抑制物(TFPI)与及可溶性血管内皮生长因子受体1(s Flt-1)水平的相关性。方法酶联免疫吸附试验(ELISA)检测健康孕妇、非重度子痫前期孕妇、重度子痫前期孕妇血浆TF、TFPI水平以及血清s Flt-1水平;免疫组化检测各组胎盘TF抗原的表达。结果 3组患者血浆及胎盘TF、血清s Flt-1水平均有显著差异,且随病变程度的增加逐渐升高。3组TF/TFPI比值有显著差异,随病变程度的增加亦逐渐升高。与非重度PE组相比,重度PE组血清s Flt-1水平显著升高(P0.05)。在子痫前期患者中,血浆TF、TF/TFPI比值与血清s Flt-1水平呈正相关。3组中胎盘TF表达与血清s Flt-1水平无相关性。在TF165.9 pg/m L组中,血浆、胎盘TF、血浆TFPI与血清s Flt-1水平呈正相关。结论子痫前期患者血浆TF、TF/TFPI比值与血清s Flt-1水平存在正相关关系。     

Effect of tyrosine kinase inhibitors,imatinib and nilotinib,in murine lipopolysaccharide-induced acute lung injury during neutropenia recovery

Introduction

Neutrophil recovery has been implicated in deterioration of oxygenation and exacerbation of preexisting acute lung injury (ALI). The aim of this study was to investigate whether imatinib or nilotinib was effective on lipopolysaccharide (LPS)-induced ALI during neutropenia recovery in mice.

Methods

Mice were rendered neutropenic with cyclophosphamide prior to the intratracheal instillation of LPS. Imatinib or nilotinib was administrated by oral gavage during neutropenia recovery. In order to study the effects of drugs, mice were killed on day 5 and blood, bronchoalveolar lavage (BAL) fluid and lung tissue samples were obtained. The lung wet/dry weight ratio and protein levels in the BAL fluid or lung tissue were determined.

Results

Treatment with imatinib or nilotinib significantly attenuated the LPS-induced pulmonary edema, and this result was supported by the histopathological examination. The concentrations of tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and myeloperoxidase in BAL fluid were significantly inhibited by imatinib or nilotinib in mice of ALI during neutropenia recovery. The mRNA expressions of platelet-derived growth factor receptor-β and c-KIT in imatinib or nilotinib group were significantly lower than LPS group.

Conclusions

Our data indicated that imatinib or nilotinib effectively attenuated LPS-induced ALI during neutropenia recovery. These results provide evidence for the therapeutic potential of imatinib and nilotinib in ALI during neutropenia recovery.     

Prognostic value of interleukin-6, plasma viscosity, fibrinogen, von Willebrand factor, tissue factor and vascular endothelial growth factor levels in congestive heart failure

BACKGROUND: Congestive heart failure (CHF) carries a poor prognosis with a high mortality rate, frequent hospitalizations and increased risk of thrombotic complications such as stroke. Cytokines may contribute to the progression and prothrombotic state of CHF, including the pro-inflammatory interleukin-6 (IL-6) and the pro-angiogenic vascular endothelial growth factor (VEGF), both of which are raised in CHF. The procoagulant properties of both cytokines may be mediated via tissue factor (TF), a potent clotting activator. We hypothesized that plasma levels of these markers, as well as levels of plasma viscosity, fibrinogen, soluble P-selectin and von Willebrand factor (markers of abnormal rheology, clotting, platelet activation, and endothelial damage, respectively) will be useful in predicting morbidity and mortality in chronic stable CHF. METHODS AND RESULTS: One hundred and twenty consecutive out-patients with chronic stable CHF (92 males; mean [SD] age 64 [11] years, mean [SD] left ventricular ejection fraction of 29 [6]%) were recruited and followed for 2 years during which 42 patients reached a clinical end-point of all-cause mortality and cardiovascular hospitalizations, including stroke and myocardial infarction. Plasma IL-6 (P=0.003) and TF (P=0.013) levels, but not other research indices, were higher in those who suffered events compared with those without events. Predictors of end-points were high (> or =median) TF (P=0.011), and IL-6 (P=0.023) levels, as well as the lowest quartile of a left ventricular ejection fraction (P=0.007). A strong correlation was present between TF and IL-6 levels (r=0.59; P<0.0001) and with VEGF levels (r=0.43; P<0.0001). CONCLUSION: IL-6 and TF are predictors of poor prognosis in chronic CHF, raising the hypothesis that IL-6 may contribute to the progression and thrombotic complications of CHF via its actions on TF expression. Although VEGF did not independently predict outcome in chronic CHF, the possibility arises that it may act with IL-6 to induce TF expression.     

The relationship of TFPI, Lp(a), and oxidized LDL antibody levels in patients with coronary artery disease

OBJECTIVES: The aim of the present study was to determine and correlate tissue factor pathway inhibitor (TFPI), lipoprotein (a) (Lp(a)), oxidized low-density lipoprotein (LDL) antibody (oLAB), and thiobarbituric acid reactive substances (TBARS; as a marker of lipid peroxidation) levels in patients with coronary artery disease (CAD) and in a control group. DESIGN AND METHODS: Peripheral blood samples from patients with coronary heart disease were provided by the Department of Cardiology. Serum oLAB, Lp(a), plasma total TFPI, and plasma-free TFPI levels were determined by ELISA. Serum TBARS levels were determined by a spectrophotometric method using thiobarbituric acid. RESULTS: The CAD and the control group were matched for age and sex. Serum Lp(a), oLAB, and plasma total TFPI levels in patients with coronary heart disease were found to be significantly higher than in the control group (P < 0.001). But there was no difference in plasma-free TFPI levels between patients with CAD and the control group (P > 0.05). In patients with single (P < 0.05), double, and triple vessel (P < 0.01) disease, the mean serum Lp(a) levels were significantly higher than in the control group. On the other hand, in patients with single vessel disease (P < 0.05), double vessel disease (P < 0.05), and triple vessel disease (P < 0.001), plasma total TFPI levels were found to be significantly higher than in the control group. We also found a significant positive correlation (r = 0.28, P < 0.05) between serum Lp(a) and plasma total TFPI levels in CAD. In the patient group, TBARS, total cholesterol, triglyceride (TRG), and LDL cholesterol levels were found to be significantly higher than those in the control group. In addition, high-density lipoprotein (HDL) cholesterol levels were found to be significantly lower than the control group. CONCLUSIONS: These results suggest that elevated plasma levels of total TFPI, Lp(a), and oLAB may be useful diagnostic and monitoring markers in patients with CAD.     

The multi‐functional serpin,protein C inhibitor: beyond thrombosis and hemostasis

Summary. Protein C inhibitor (PCI) is a member of the serine protease inhibitor (serpin) family. PCI was initially found to be an inhibitor of activated protein C, and later shown to be a potent inhibitor of blood coagulation and fibrinolysis such as that mediated by urokinase type‐plasminogen activator. Therefore, the protein came to be known as plasminogen activator inhibitor‐3. It also inhibits proteases involved in fertilization. PCI is broadly conserved, and is found in human, rhesus monkey, cow, rabbit, rat, mouse and chicken. The human PCI gene is located on chromosome 14q32.1 in a cluster of genes encoding related serpins. Sp1‐ and AP2‐binding sites in the 5′‐flanking region act as promoter and enhancer, respectively, for its expression in the liver. PCI mRNA is expressed in many organs in primates, but only in the reproductive organs in rodents. Recent studies using transgenic mice expressing the human gene have suggested that PCI is also involved in regulation of lung remodeling, tissue regeneration, vascular permeability, proteolysis in the kidney and tumor cell invasion. A protease inhibitor‐independent activity of PCI, the prevention of anti‐angiogenesis and metastasis of tumor cells, has also been observed. Thus, PCI is a unique multi‐functional serpin playing diverse roles in the thrombosis and hemostasis in multiple organs and tissues of a variety of species.     

结缔组织生长因子反义寡核苷酸对胰腺癌细胞基质金属蛋白酶-9、血管内皮生长因子表达及细胞增殖与侵袭的影响

目的 通过研究结缔组织生长因子(CTGF)反义寡核苷酸(ASODN)对人胰腺癌细胞基质金属蛋白酶-9(MMP-9)、血管内皮生长因子(VEGF)表达及细胞增殖与侵袭力的影响,深入探讨CTGF在胰腺癌发生、发展中的作用机制,为CTGF ASODN在胰腺癌基因治疗中的运用奠定理论基础.方法 经脂质体介导将CTGF ASODN转染人胰腺癌细胞株SW1990,荧光显微镜及流式细胞仪检测转染效率;逆转录-聚合酶链反应(RT-PCR)及Western blot检测转染细胞CTGF、MMP-9与VEGF mRNA与蛋白表达;噻唑盐(MTT)比色法检测转染细胞的增殖活性;采用Transwell小室检测细胞侵袭能力.结果 (1)荧光显微镜及流式细胞仪检测显示CTGF ASODN可成功转染SW1990细胞.(2)反义组、无义组和单加脂质体组细胞CTGF mRNA表达量分别为0.24±0.09、0.67±0.21、0.63±0.18,反义组较其他组的mRNA表达明显减少(P<0.01);相应的CTGF蛋白表达量分别为0.19±0.07、0.75±0.26、0.71±0.23;相应的MMP-9 mRNA表达量分别为0.21±0.12、0.78±0.35、0.81±0.37;相应的VEGF mRNA表达量分别为0.16±0.06、0.42±0.22、0.43±0.28;相应的MMP-9蛋白表达量分别为0.68±0.22、1.97±0.46、1.92±0.39;相应的VEGF蛋白表达量分别为0.27±0.07、0.52±0.19、0.55±0.18,反义组CTGF、MMP-9和VEGF的表达较其他组显著降低(P<0.01).(3)反义组、无义组、单加脂质体组细胞增殖活性分别为1.67±0.14、2.34±0.17、2.29±0.21,反义组较其他组细胞增殖被显著抑制(P<0.01).(4)反义组、无义组、单加脂质体组微孔滤膜外侧细胞数分别为24.88±6.17、52.37±8.62、55.49±8.83,反义组较其他组微孔滤膜外侧细胞数显著减少(P<0.01).结论 CTGF ASODN转染可有效抑制SW1990细胞CTGF的表达,并降低其MMP-9、VEGF的表达及细胞增殖活性与细胞侵袭力.     

宫颈鳞癌组织中PINCH和血管内皮生长因子C蛋白表达的研究

目的 探讨宫颈鳞癌组织中PINCH蛋白和血管内皮生长因子C(VEGF-C)蛋白的表达意义.方法 应用免疫组化SP法检测58例宫颈鳞癌、30名正常宫颈上皮组织中PINCH蛋白和VEGF-C蛋白的表达,并分析PINCH蛋白和VEGF-C蛋白的表达与临床病理特征的关系.结果 58例宫颈鳞癌组织中PINCH和VEGF-C的表达阳性率[分别为(62.1％ (36/58)和67.2％ (39/58)]高于正常宫颈上皮(0),差异有统计学意义(x2值分别为31.512、12.534,P均＜0.001).PINCH蛋白表达与年龄、肿瘤大小、肿瘤组织分化程度无明显相关(P均＞0.05),与有无淋巴结转移及临床分期相关(x2值分别为9.090、8.263,P均＜0.01).VEGF-C的表达与患者年龄、肿瘤大小无相关性(P均＞0.05),与有无淋巴结转移、肿瘤组织分级和临床分期相关(x2值分别为10.775、13.496、5.001,P均＜0.05).PINCH蛋白及VECF-C蛋白表达有关联(C =0.341,P＜0.01).结论 PINCH和VEGF-C可能协同参与了宫颈鳞癌的发生和发展过程,并在宫颈癌的侵袭和转移机制中发挥着重要作用.     

Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate

It remains uncertain how calcium, phosphate and calcitriol regulate parathyroid cell growth. The present study was aimed at examining possible direct effects of these modulators and of the calcimimetic NPS R-467 on parathyroid cell growth in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell cycle antigen Ki 67 expression in a parathyroid cell culture model derived from uraemic patients. The effect of NPS R-467 on parathyroid hormone (PTH) secretion and intracellular [Ca2+]i response was also examined. Increasing the [Ca2+] in the medium from 0.5 to 1.7 mM increased DNA synthesis (P < 0.005) and the number of Ki 67-positive cells (P < 0.005). However, NPS R-467 (0.01-1 microM) inhibited 3[H]thymidine incorporation by 35% in the presence of 0.5 mM [Ca2+]e. Exposure of cells to Ca2+ or NPS R-467 led to a rapid increase of intracellular Ca2+, although the pattern of increase differed. Addition of calcitriol (10-10-10-7 M) to the culture medium suppressed [3H]thymidine incorporation dose-dependently. Finally, high levels of phosphate (3.5 mM) in the medium led to a significant (P < 0.05) increase in [3H]thymidine incorporation. The observed stimulatory effect of Ca2+ in the medium in vitro appears to be at variance with the inhibitory effect of calcimimetic NPS R-467 in vitro. In an attempt to solve these apparent discrepancies, and based on the notion of a reduced calcium-sensing receptor (CaR) expression in parathyroid tissues of uraemic patients, we hypothesize that Ca2+ may regulate parathyroid cell proliferation via two different pathways, with predominant growth inhibition in cases of high CaR expression or activation, but prevailing stimulation of proliferation in cases of low CaR expression.     

A collagen cardiac patch incorporating alginate microparticles permits the controlled release of hepatocyte growth factor and insulin‐like growth factor‐1 to enhance cardiac stem cell migration and proliferation

Cardiac stem cells (CSCs) represent a logical cell type to exploit as a regenerative treatment option for tissue damage accrued as a result of a myocardial infarction. However, the isolation and expansion of CSCs prior to cell transplantation is time consuming, costly and invasive, and the reliability of cell expansion may also prove to be a major obstacle in the clinical application of CSC‐based transplantation therapy after a myocardial infarction. In order to overcome this, we propose the incorporation of growth factor‐eluting alginate microparticles into collagen‐based scaffolds as an implantable biomaterial to promote the recruitment and expansion of CSCs in the myocardium. In order to obtain scaffolds able to enhance the motogenic and proliferative potential of CSCs, the aim of this work was to achieve a sustained delivery of both hepatocyte growth factor and insulin‐like growth factor‐1. Both proteins were initially encapsulated in alginate microparticles by spray drying and subsequently incorporated into a collagen scaffold. Microparticles were seen to homogeneously distribute through the interconnected scaffold pore structure. The resulting scaffolds were capable of extending the release of both proteins up to 15 days, a three‐fold increase over non‐encapsulated proteins embedded in the scaffolds. In vitro assays with isolated CSCs demonstrated that the sustained release of both bioactive proteins resulted in an increased motogenic and proliferative effect. As presently practiced, the isolation and expansion of CSCs for autologous cell transplantation is slow, expensive and difficult to attain. Thus, there is a need for strategies to specifically activate in situ the intrinsic cardiac regenerative potential represented by the CSCs using combinations of growth factors obviating the need for cell transplantation. By favouring the natural regenerative capability of CSCs, it is hypothesized that the cardiac patch presented here will result in positive therapeutic outcomes in MI and heart failure patients in the future. Copyright © 2016 John Wiley & Sons, Ltd.