结肠直肠癌中PISD的表达及其与临床病理的关系

目的:检测Wnt通路靶基因PISD在结肠直肠细胞及组织标本中的表达情况及其与临床病理的关系。方法:应用Western印迹法测定结肠直肠癌细胞株中PISD的蛋白表达水平,应用免疫组化法测定PISD在组织标本中的表达情况及其与临床病理的关系。结果:Western印迹法结果显示PISD在7种结肠直肠癌细胞株中均呈阳性表达,免疫组化结果显示PISD在结肠直肠癌组织中表达阳性,灵敏度为62.9%。结论:PISD在结肠直肠癌细胞及组织中表达上调,但与肿瘤的转移无明显相关。     

Effect of bacterial peptidoglycan on erythrocyte death and adhesion to endothelial cells

Peptidoglycans, bacterial wall components, have previously been shown to trigger eryptosis, the suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine exposing erythrocytes adhere to the vascular wall at least partially by interaction of erythrocytic phosphatidylserine with endothelial CXC chemokine ligand 16 (CXCL16). The present study explored whether peptidoglycan exposure fosters the adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC). To this end, HUVEC were treated for 48 h with peptidoglycan (10 μg/ml) and CXCL16 abundance determined by confocal microscopy and FACS analysis. Moreover, human erythrocytes were exposed for 48 h to peptidoglycan (10 μg/ml) and phosphatidylserine exposure estimated from binding of fluorescent annexin-V, cell volume from forward scatter in FACS analysis and erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber. As a result, bacterial peptidoglycan exposure was followed by increased CXCL16 expression in HUVEC as well as erythrocyte shrinkage, phosphatidylserine exposure and adhesion to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly attenuated but not abrogated in the presence of either, erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml) or CXCL16 neutralizing antibody directed against endothelial CXCL16 (4 μg/ml). In conclusion, exposure to peptidoglycan increases endothelial CXCL16 expression and leads to eryptosis followed by phosphatidylserine- and CXCL16-mediated adhesion of eryptotic erythrocytes to vascular endothelial cells.     

微粒与心血管疾病

微粒是血管内皮细胞、血细胞等不同类型的细胞活化及凋亡时释放的具有很多生物学活性的小囊泡,微粒表面表达磷脂酰丝氨酸(PS),可通过检测微粒表面的PS发现微粒的来源,细胞的活化及凋亡。很多心血管疾病与细胞的活化和凋亡相关,有相应微粒水平的升高,因此微粒的检测可能为临床心血管病的早期检测提供帮助,为心血管疾病的诊断治疗提供新的思路。     

Safety of Soy-derived Phosphatidylserine in Elderly People

Abstract

Phosphatidylserine (PS) is a phospholipid which has been claimed to enhance neuronal membrane function, and can be derived from several sources. Earlier studies used brain cortex derived PS, of which the human tolerability of 300 mg daily in 130 patients has been shown. The human tolerability of PS derived from soy-bean has not been reported, although it is widely sold as a nutritional supplement which may improve cognitive function in the elderly. We report the results of a study of the safety of two dosages of soy-phosphatidylserine (S-PS) in elderly.

Subjects were 120 elderly of both sexes who fulfilled the more stringent criteria for age-associated memory impairment; some also fulfilled the criteria for age-associated cognitive decline. Subjects were allocated at random to one of the three treatment groups: placebo, 300 or 600 mg S-PS daily. Standard biochemical and hematological safety parameters, blood pressure, heart rate and adverse events were assessed at baseline, after 6 and 12 weeks of treatment.

No significant differences were found in any of the outcome variables between the treatment groups after Bonferonni-Holme correction.

In conclusion, soy derived PS is a safe nutritional supplement for older persons if taken up to a dosage of 200 mg three times daily.     

A lipophilic free radical initiator, 2,2′-azobis (2,4-dimethylvaleronitrile) (AMVN) enhances caspase-dependent apoptosis induced by hyperthermia

Purpose: A free radical initiator, 2,2'-azobis (2-amidinopropane) dehydrochloride (AAPH), was previously found to enhance apoptosis by hyperthermia. Here, but more lipophilic free radical initiator, 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) was investigated for its effects as a possible heat sensitizer. Materials and methods: Human myelogenous monocytic leukaemia U937 cells were treated with hyperthermia combined with a various concentration of AMVN for investigating its ability to induce apoptosis and various parameters to identify the pathway. Results: Combined treatment of hyperthermia and AMVN induced DNA fragmentation markedly, while hyperthermia or AMVN alone induced marginal DNA fragmentation. Fractions of cells showed low mitochondrial membrane potential and increased superoxide production after the combined treatment. Experiments using various caspase inhibitors and a fluorogenic monitor of caspase 3 activities indicated that caspase acts both up- and down-stream of mitochondria. Conclusions: AMVN is suggested to be a potential heat sensitizer effective at a lower concentration than AAPH. The possible mechanism is discussed.     

Changes in erythrocyte membrane phospholipid composition induced by physical training and physical exercise

Summary The effects were investigated of physical training and exercise on lipids of the erythrocyte membrane of healthy students. Membrane cholesterol and phospholipids were analysed simultaneously by thin-layer chromatography with a flame ionization detector and the fatty acid composition was determined by gas chromatography. Physically trained students had similar physical characteristics to control students but a significantly higher aerobic capacity, estimated as the maximal oxygen uptake and anaerobic threshold. Of the phospholipids examined, only the content of membrane phosphatidylserine was significantly lower in the trained group. Fatty acid analysis showed that the amount of docosahexaenoic acid in membrane phosphatidylserine was lower in the trained group. There was no significant difference between the fatty acid compositions of membrane phosphatidylcholine in the two groups. Maximal exercise decreased membrane phosphatidylserine in the control group but not in the trained group. It also significantly decreased the relative amounts of unsaturated fatty acids in both phosphatidylcholine and phosphatidylserine in the untrained group. Maximal oxygen uptake was negatively correlated with the amount of erythrocyte membrane phosphatidylserine. These results would indicate that both physical training and acute exercise decrease phosphatidylserine and polyunsaturated fatty acids in erythrocyte membranes, possibly due to lipid peroxidation, suggesting limited enhancement of erythrocyte defense mechanisms in adaptation to chronic oxidative stress.     

The inhibition of protein C anticoagulant activity by anti-β2-glycoprotein I (β2GPI) antibodies isolated from patients with antiphospholipid syndrome by chromatography methods

Antiphospholipid antibodies (aPL) are associated with an increased risk of thrombosis; however, the mechanism remains unknown. Recent studies have focused on the impediment of protein C anticoagulant activity by anti-β₂-glycoprotein I (β₂GPI) antibodies (aβ₂GPI Ab). We purified IgG fractions containing a high concentration of aβ₂GPI Ab from patients with antiphospholipid syndrome (APS) and then investigated the effect of purified aβ₂GPI Ab on the activity of activated protein C (APC). Using a three-step chromatography method (DEAE-sepharose column, phosphatidylserine polyacrylamide gel column dependent on the presence of β₂GPI, and protein G column chromatography), we successfully isolated anti-β₂GPI IgG from nine patients with APS. Seven of nine samples inhibited APC activity in a concentration-dependent manner only in the presence of β₂GPI, as observed by a chromogenic assay that was able to determine thrombin activity even in the presence of APC. The extent of APC inhibition by these fractions appeared to be related to aβ₂GPI Ab titers of the purified IgG. However, the inhibitory effect of IgG from patients was not detected in the absence of β₂GPI. IgG purified from three normal subjects did not affect APC activity. Herein, we show a useful method for the isolation of IgG containing a high concentration of aβ₂GPI Ab. Moreover, the present findings indicate that inhibition by aβ₂GPI Ab on APC anticoagulant activity could explain one of the mechanisms for the thrombotic state in APS. Received: April 9, 2001 / Accepted: July 10, 2001     

A role of phosphatidylserine externalization in clearance of erythrocytes exposed to stress but not in eliminating aging populations of erythrocyte in mice

Age dependent changes in phosphatidylserine (PS) externalization were studied in mouse erythrocytes of different age groups (range 1-55 days) by using a newly developed double in vivo biotinylation (DIB) technique. Around 3-4% of the erythrocytes freshly released in the circulation were PS(+) but this proportion fell rapidly to 1% or less and did not increase at later time points. Blocking erythrocyte clearance from the circulation by in vivo depletion of macrophages (by treatment with clodronate loaded liposomes) for up to 7 days did not result in accumulation of PS(+) erythrocytes in the circulation indicating that the low percentage of PS(+) cells within old erythrocytes (age >40 days) was not related to the clearance of PS(+) erythrocytes by macrophages. In vitro treatment with stress inducing agents like deoxyglucose or Ca(++)/calcium ionophore resulted in a marked induction of PS externalization in mouse erythrocytes and this effect was most prominent in the youngest erythrocyte population (age <10 days). Kinetics of clearance of different age groups of stress exposed erythrocytes after intravenous infusion into recipient mice indicated that the young erythrocytes were cleared at fastest rate from the circulation as compared to erythrocytes of older age groups. Within young erythrocytes exposed to stress, PS(+) erythrocytes were preferentially cleared. Taken together our results suggest that PS externalization is unlikely to have a role in the removal of old erythrocytes from blood circulation but may have a role in the clearance of stressed and damaged young erythrocytes in blood circulation.     

Normal levels of constitutive and death receptor-mediated apoptosis of peripheral blood neutrophils from patients with chronic idiopathic neutropenia

To investigate the role of neutrophil apoptosis in the pathogenesis of chronic neutropenia, we examined constitutive and death receptor-mediated apoptosis ex vivo of peripheral blood neutrophils obtained from six chronic idiopathic neutropenia (CIN) patients and six healthy adult blood donors. Apoptosis was quantified based on phosphatidylserine externalization and caspase-3 activation in freshly isolated neutrophils or after overnight cultivation of neutrophils in the absence or presence of pro- or anti-apoptotic factors, including the pan-caspase inhibitor, zVAD-fmk. Neutrophils from CIN patients receiving treatment with granulocyte colony-stimulating factor appeared to be more prone to constitutive apoptosis than cells from untreated patients; however, further investigations in larger cohorts of patients are needed to validate these pilot studies. Overall, the level of neutrophil apoptosis was similar in patient and control groups, thus supporting the notion that the underlying defect in these neutropenia patients lies elsewhere, such as in the bone marrow microenvironment.     

Enhanced efficacy of curcumin with phosphatidylserine-decorated nanoparticles in the treatment of hepatic fibrosis

Hepatic macrophages have been considered as a therapeutic target for liver fibrosis treatment, and phosphatidylserine (PS)-containing nanoparticles are commonly used to mimic apoptotic cells that can specifically regulate macrophage functions, resulting in anti-inflammatory effects. This study was designed to test the efficacy of PS-modified nanostructured lipid carriers (mNLCs) containing curcumin (Cur) (Cur-mNLCs) in the treatment of liver fibrosis in a rat model. Carbon tetrachloride-induced liver fibrosis in rats was used as an experimental model, and the severity of the disease was examined by both biochemical and histological methods. Here, we showed that mNLCs were spherical nanoparticles with decreased negative zeta potentials due to PS decoration, and significantly increased both mean residence time and area under the curve of Cur. In the rats with liver fibrosis, PS-modification of NLCs enhanced the nanoparticles targeting to the diseased liver, which was evidenced by their highest accumulation in the liver. As compared to all the controls, Cur-mNLCs were significantly more effective at reducing the liver damage and fibrosis, which were indicated by in Cur-mNLCs-treated rats the least increase in liver enzymes and pro-inflammatory cytokines in the circulation, along with the least increase in collagen fibers and alpha smooth muscle actin and the most increased hepatocyte growth factors (HGF) and matrix metalloprotease (MMP) two in the livers. In conclusion, PS-modified NLCs nanoparticles prolonged the retention time of Cur, and enhanced its bioavailability and delivery efficiency to the livers, resulting in reduced liver fibrosis and up-regulating hepatic expression of HGF and MMP-2.