Effects of cimetidine on platelet function and thrombosis

AIM:To observe the effects of cimetidine(Cim) on platelet function and thrombosis. METHODS:After incubated with Cimin vitro, rat platelets were activated with ADP or thrombin. The platelet aggregation, platelet malondialdehyde(MDA) formation, platelet intracellular free calcium( [Ca²⁺]_i), and thromboxane B₂ (TXB₂) were measured. The effects of Cim on electric-induced thrombosis in rat carotid artery were examined. RESULTS:Cim potentiated ADP induced platelet aggregation, increased the thrombin induced [Ca²⁺]_i and MDA formation, decreased TXB₂. Also, Cim shortened the duration of electric-stimulated occlution time in rat carotid artery. CONCLUSION:Cim increased platelet function and accelerated thrombosis.     

Fibrinolytic activity and its mechanisms in various leukemic cell lines

AIM:To study the relation between expression of uPAR and annexinⅡ and fibrinolytic activity in various leukemic cell lines. METHODS:The plasma activity was measured under the reaction between cells of NB4, SHI-1, K562, Jurkat, Raji and plaminogen by chromogenic assay. The protein expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by flow cytometry method. The mRNA expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by RT-PCR. RESULTS:The plasma activity in SHI-1 cells and NB4 cells were higher obviously than that in Raji, K562 and Jurkat cells. The protein expression ratio of uPAR and annexinⅡ in NB4 cells were (13.15±1.61)% and (95.97±1.19)%， respectively, they were (99.00±0.26)%, (90.35±2.15)% respectively in SHI-1 cells, and they were lower in K562, Jurkat, Raji cells. The expression of annexinⅡ mRNA in NB4 cells was higher than that in SHI-1 cells, and they were undectectable in K562 and Jurkat cells. The expression of uPAR mRNA in NB4 and SHI-1 cells were higher than that in Jurkat and K562 cells. The expression of uPAR mRNA in Raji cells was undectectable. CONCLUSION:The primary hyperfibrinolysis in leucocythemia cells was observed, and relation was closely with the expression of annexinⅡ. It might be the main reason for bleeding and disseminated intravascular coagulation in patients with acute promyelocytic leukemia and acute monocytic leukemia.     

Effects of Tongxinluo on activation of platelet in rabbit model of atherosclerosis

AIM: To study the effects of Tongxinluo on the activation of platelets in a rabbit model of atherosclerosis. METHODS: New Zealand rabbits were randomly divided into 7 groups: normal group, model group, the groups treated with high, medium and low doses of Tongxinluo micropowder (0.15, 0.3 and 0.6 g·kg^-1·d^-1), atorvastatin group (2.5 mg·kg^-1·d^-1), and aspirin group (12.5 mg·kg^-1·d^-1). The rabbits in normal group was fed with common diet for 12 weeks, and the rabbits in model group were fed with high-fat diet for 12 weeks to establish atherosclerosis model. The rabbits in the rest groups were treated with the corresponding drugs, at the same time to give high-fat diet. Fasting for 12 h after the last treatment, whole blood was collected to perform the blood routine test, and to measure serum and plasma levels of lipids, platelet factor 4 (PF4) and soluble CD62P (sCD62P). Flow cytometry was used to analyze platelet calcium ion concentration. Electron microscopy was used for platelet superfine observations, and light microscopy for observing the pathological changes. RESULTS: Compared with normal group, the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), platelet counts, and mean platelet volume in model group were significantly elevated, and the levels of PF4, sCD62P and calcium were also significantly increased (P < 0.05). Compared with model group, except aspirin group, the levels of TG, TC and LDL-C in high, medium and low doses of Tongxinluo groups and atorvastatin group were effectively decreased. The platelet counts and mean platelet volume in all treatment groups were markedly decreased, and the serum levels of PF4, sCD62P and Ca²⁺ in platelet (P < 0.05) were reduced. In electron microscopic observation, the shape of platelet was regular and organelles distributed uniform in normal group. However, in model group, the shape of platelet was irregular, pseudopodia forming was obviously observed, and α particles and dense granules decreased, indicating that the platelet was activated. To a different extent, the platelet shape, increase in the number of α particles and dense granules were improved in treatment groups and the damage of the cytoplasm was attenuated. Through histopathological observation, the intimal was smooth and complete in normal group. In the model group, the intimal thickness markedly increased, foam cell aggregated, and plaque was formed. Compared with model group, the intimal thickening and the number of foam cells were significantly decreased, and plaque formation was not obvious in atorvastatin group and high dose of Tongxinluo group. The pathological damages in the other treatment groups were alleviated in different degrees. CONCLUSION: Tongxinluo significantly inhibits the activation of platelets in the process of atherosclerosis, and has important clinical value to delay the atherosclerotic thrombosis.     

Expression of adhesion molecules in hepatocellurlar carcinoma and its clinical significance

AIM: To investigate the expression of adhesion molecules in hepatocellular carcinoma (HCC), and analyze its clinical significance. METHODS: The expressions of adhesion molecules of tumor tissues of 64 cases and adjacent tissues of 12 cases of HCC were detected with RT-PCR. RESULTS: ①The expression rates of E-cadherin, ICAM-1, CD44, CD44V, α₅, β₁ were 90.62%, 93.75%, 50.00%, 96.88%, 100%, 100%, respectively, and there was a significant difference between CD44 and other adhesion molecules. ②The expression level of E-cadherin, ICAM-1, CD44, CD_44V, α₅,β₁ in liver cancer tissues were 1.24±0.54, 0.96±0.37, 0.62±0.73, 0.86±0.33, 0.97±0.49, 1.41±0.24, respectively, and there was a significant difference between CD44 and E-cadherin, β₁. ③The expression level of E-cadherin and CD44 mRNA declined as HCC stage become higher, and there was a statistical difference in the expression level of CD44 mRNA between Ⅰ-Ⅱ stage and Ⅳ stage. The expression level of ICAM-1, α₅, β₁ had a trend to rise as HCC stage become higher, and there was a statistical difference in the expression level of ICAM-1 between Ⅰ-Ⅱ stage and Ⅳ stage. ④The expression level of ICAM-1,CD_44V, α₅, β₁ had positive correlation with tumor volume, tumor nodules, tumor metastasis, and had negative correlation with tumor encapsulation. E-cadherin and CD44 had negative correlation with tumor volume, tumor nodules, tumor metastasis, and had positive correlation with tumor encapsulation. All showed no significant correlation with the level of AFP , the degree of cirrhosis and the function of liver. CONCLUSION: There was a significant difference in the expression level of adhesion molecule mRNA in HCC, and their expression had Spearman correlation with each other. The expression level of adhesion molecule mRNA is associated with tumor volume, tumor nodules and tumor metastasis.     

Effect of thrombopoietin on platelet activation invitro

AIM:To study the effects of thrombopoietin(TPO) and other hematopoietic cytokines on platelet activation. METHODS: Using fluorescent-labeled monoclonal antibodies and flow cytometry. RESULTS:The percentage range of platelets activated by 120 ng/mL TPO was 8.89%~39.92%,mean value was 17.43%; However,there were no effects of TPO 40 ng/mL and GM-CSF 100 ng/mL and IL-3 100 ng/mL on platelet activation. CONCLUSION:Higher concentration of TPO directly stimulated platelet activation.     

Effect of polymorphonuclear leukocytes on washed platelet aggregation

AIM:To investigate the effect of non-activated or activated polymorphonuclear leukocytes(PMN) on washed platelet aggregation. METHODS:Born's method was used to determine platelet aggregation.RESULTS:non-activated PMN (5×10⁹ cells/L) significantly suppressed washed platelet aggregation induced by ADP or arachidonic acid. Aspirin enhanced this inhibition. N-formyl-methiongl-leucy-phenylalanine (fMLP)-or platelet-activating factor (PAF)-stimulated PMN strongly induced platelet aggregation, and the induction effect of PMN suspension was more active than that of PMN supernatant. Aspirin had no significant inhibitory effect on platelet aggregation induced by fMLP-or PAF-activated PMN. CONCLUSIONS:Different conditions of PMN (activated or non-activated) had the nearly opposite action on normal platelet reactivity. Briefly, non-activated-PMN inhibited platelet reactivity, whereas activated PMN stimulated it.     

Detection of monoclonal immunoglobulin heavy chain gene rearrangements and its application in diagnosis of B-cell non-Hodgkin lymphoma

AIM: To establish a reliable and feasible protocol for detection of monoclonal immunoglobulin heavy chain (IgH) gene rearrangements for routine diagnosis of B-cell non-Hodgkin lymphoma (B-NHL). METHODS: Using the primer combinations of FR2, FR3, LJH and VLJH, mode tube A+ tube B, and semi-nested PCR, the monoclonal IgH gene rearrangements in 121 cases of B-NHL, 58 cases of T-cell non-Hodgkin lymphoma (T-NHL) and 19 cases of reactive lymphoid hyperplasia were detected. The differences of clonality detection rate between B-NHL group and T-NHL group, B-NHL group and reactive lymphoid hyperplasia group, and between the use of FR2, FR3 and FR2+FR3 primers were analyzed. RESULTS: The clonality detection rates of B-NHL, T-NHL and reactive lymphoid hyperplasia were 81% (96/118), 4% (2/54) and 0% (0/19). There were remarkable differences between B-NHL group and T-NHL group, B-NHL group and reactive lymphoid hyperplasia group in monoclonal IgH gene rearrangements (P<0.05). In B-NHL group, monoclonality was found in 58% of the cases using primer FR2, 55% using FR3, and 81% using the combination of both primers, with significant differences (P<0.05). CONCLUSION: Using the primer combinations of FR2, FR3, LJH and VLJH, detection of paraffin-embedded tissues, the method of tube A+tube B mode and semi-nested PCR for determining monoclonal IgH gene rearrangements is feasible and reliable, and the clonality detection rate is high enough for clinical diagnosis of B-NHL.     

Effects of different group B streptococci strains on platelet activation

AIM: To explore the ability of different group B streptococci (GBS) strains on inducing platelet activation. METHODS: Six strains of GBS, separated from the septic patients with thrombocytopenia, were used as the inducers. Light transmission aggregometry was used to measure platelet aggregation. Scanning electron microscopy (SEM) was performed to investigate the interaction of platelets with bacteria. The expression of platelet CD62P, Toll-like receptor 2 (TLR2) and TLR4 was determined by flow cytometry and Western blotting. Furthermore, the activity of platelet TLR2 (or TLR4) was blocked by anti-TLR2 (or anti-TLR4) monoclonal antibody, and the platelet aggregation induced by GBS was detected. RESULTS: Only 3 of 6 GBS strains isolated from the septic patients induced platelet aggregation and up-regulated the expression of CD62P and TLR2 in the platelets (P < 0.05), but not TLR4. Incubation with anti-TLR2 antibody, but not anti-TLR4 antibody, significantly blocked platelet aggregation induced by GBS.CONCLUSION: Some GBS strains from the patients are able to trigger platelet activation in vitro, and platelet TLR2 may play an important role in the interaction between GBS and platelets.     

Inhibitory effects of propolis on platelet adhesion in flowing blood in vitro

AIM:To investigate effects of propolis-ethanol extract on platelet activity in flowing blood. METHODS:Intima-injury model was made by in vitro perfusion. Coverslips was coated by using fibrinogen and human Ⅲ type collagen to detect platelet adhesive rate when blood went through fibrinogen and collagen surface in 1 000/s shear rate. Three experimental groups were set up: negative control group (24% ethanol), experimental group (0.1 g/L, 24% propolis-ethanol extract) and positive control group (0.1 g/L, ferulic acid). RESULTS:Intima-injury model was made. On fibrinogen and collagen, platelet adhesion on area of experimental group was lower than that in negative control group (P<0.01). There was no significant difference between experimental group and positive control group (P>0.05). Propolis-ethanol extract decreased more obviously the platelet adhesion area on fibrinogen surface than that in collagen surface (P<0.01). CONCLUSION:Propolis-ethanol extract inhibits platelet activation and reduces its adhesion on fibrinogen and collagen surface, especially in fibrinogen surface.     

Nucleostemin gene expression in human breast tumor tissues and its relation with clinical pathology

AIM: To study the expression of nucleostemin (NS) gene in human breast tumor tissues and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor.METHODS: Total RNA was isolated from human breast tumor tissue.The methods of electrophoresis and RT-PCR were used in measuring NS gene expression level,and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor were analyzed.RESULTS: The results indicated that there was no NS gene expression detected in normal breast tissues,and NS gene expression in malignant breast tumor tissues (P<0.01) was higher than that in the benign breast tumor tissues.The higher histological grades of the breast cancer showed the stronger NS gene expression (P<0.01),the higher TNM stages of the breast cancer showed the stronger NS gene expression (P<0.01),and the level of NS gene expression had not correlation with the histological types (P>0.05).CONCLUSION: It is suggested that there is no relation of NS gene expression level with histological types of the breast cancer,but there is a marked correlation of NS gene expression level with the histological grades and TNM stages.     

Expression of NK4 gene in human pancreatic cancer cells and its effect on growth of human vascular endothelial cells

AIM:The current study was designed to construct eukaryotic expression vector containing NK4 gene and transfect it into human pancreatic cancer cell lines.METHODS:The recombinant of pcDNA3/hNK4 was digested by restriction enzyme,the NK4 gene was cloned into a high effective eukaryotic expressing plasmid which contains CMV2 immediate early gene promoter and then transiently introduced into the pancreatic cancer cell line SW1990 by lipo fectamine and clonal cel lines that secrete high levels of NK4 protein were isolated.The expression of NK4 was observed by RT-PCR and Western blot,in vitro the vascular endothelial cell proliferation inhibiting activity of NK4 was examined by 3-[4,5-dimethylthiazolzyl]-2,5-diphenyl tetrazolium bromide(MTT)method.RESULTS:A specific expression of NK4 gene mRNA by lipofectamine-mediated transfer exhibited only in SW1990/NK4 cells,Western blot analysis demonstrated that there was positive expression of NK4 protein(50 kD).The NK4 inhibited proliferation of the vascular endothelial cellsin vitro.CONCLUSION:The recombinant of pRC/CMV2-hNK4 is a high effective expressing eukaryotic vector.The bio-engineering product of the NK4 is an angiogenesis inhibitor and may play an important role in the gene therapy for tumor.     

Construction of human HSV-1 UL40 gene recombinant and its application in siRNA screening

AIM: To construct the recombinant of human herpesvirus Ⅰ(HSV-1) UL40 gene and to screen siRNAs of silencing efficiently human HSV-1 UL40 gene expression.METHODS: The recombinant UL40-EGFP plasmid (pEGFP-N1-UL40) was constructed by cloning the UL40 gene into pEGFP-N1.siRNA target UL40 gene and pEGFP-N1-UL40 were cotransfected into Vero cells.The effects of RNAi were detected by green fluorescence signals.RESULTS: siRNA3,siRNA4 reduced prominently the expression of UL40 gene.The silence efficiency was 76.99% and 84.00% respectively.CONCLUSION: We succeed in construction of the pEGFP-N1-UL40 recombinant,and screen out siRNA3 and siRNA4 of silencing efficiently human HSV-1 UL40 gene expression.     

Study of platelet aggregations in patients with coronary heart disease and the effect of ticlopidine treatment

AIM: To investigate the platelet aggregations in patients with coronary heart disease(CHD) and the effect of ticlopidine treatment. METHODS: Platelet aggregations induced by adenosine diphosphate(ADP), epinephrine(EPI), collagen(Coll), arachidonic acid(ACA) in CHD group before and after ticlopidine treatment were measured by turbidity assay. RESULTS: Maximum ratios of platelet aggregations (max%) induced by ADP, EPI in CHD group (0.78±0.23, 0.86±0.25) were significantly higher than that of control group (0.65±0.19, 0.73±0.21, P＜0.05). After the treatment with ticlopidine, they were lowered obviously (0.68±0.18, P＜0.05;0.75±0.20, P＜0.05). There were no difference in max% induced by Coll and ACA between two groups and there were no significantly changes of max% induced by Coll and ACA by ticlopidine in CHD group. CONCLUSION: The platelet aggregations were increased in patients with coronary heart disease and could be inhibited by ticlopidine.     

Effect of platelet activating factor on the action potential and potassium currents in guinea-pig ventricular myocytes

AIM: To investigate the effects of platelet activating factor (PAF) on the action potential and potassium currents in guinea-pig ventricular myocytes. METHODS: By using whole-cell patch clamp technique, the effects of PAF on APD₉₀, I_K1 and I_K were investigated in enzymatically dispersed single guinea-pig ventricular myocytes. RESULTS: With 5 mmol/L ATP in the pipette electrode, 1 μmol/L PAF increased APD₉₀ from (225.8±23.3) ms to (352.8±29.8) ms (n=5, P<0.05), decreased I_K1 and I_K tail currents from (-6.1±1.3) nA to (-5.6±1.1) nA (n=5, P<0.05) at -120 mV and from (173.5±16.7) pA to (152.1±11.5) pA (P<0.05, n=4) at +30 mV, respectively. In contract, without ATP in the pipette electrode, 1 μmol/L PAF shortened APD₉₀ from (153.0±24.6) ms to (88.2±19.4) ms (n=5, P<0.01). Incubation of myocytes with 1 μmol/L glibenclamide, a blocker of I_KATP restored prolongation of APD induced by PAF. CONCLUSION: In guinea-pig ventricular myocytes, with 5 mmol/L ATP in the pipette, PAF prolonged APD partly due to the inhibition of I_K and I_K1, while with 0 mmol/L ATP in the pipette, PAF induced an activation of I_KATP, hence a decrease in APD was observed. Therefore, PAF might amplify the heterogeneity between ischemia and normal cardiac myocytes during ischemic reperfusion, which might play a vital role in the pathogenesis of the arrhythmias induced by ischemia/reperfusion.     

In vitro perfusion method during cardiac graft preservation and its application in adenovirus mediated gene transfection

AIM: To establish a method of in vitro donor heart perfusion in murine cardiac transplantation during preservation and apply it in adenovirus mediated gene transfection for donor heart. METHODS: Donor heart was transfected with recombinant adenovirus and stored for 2 hours after harvest, then it was transplanted heterotopically in abdomen. The grafts were appraisal by palpitation. Marked gene products were determined by X-Gal staining, and T cell infiltration was determined by immunohistochemistry. The activation markers of recipients' lymphocytes were examed by cytometry. RESULTS: The grafts survival rate is 100% after perfusion and cold storage. The LacZ staining became strong 1 week after transplantation. The grafts remained an intact structure and no apparent T cell infiltration. The activation status of recipients' lymphocytes were not enhanced by transfected cardiac graft. CONCLUSION: In vitro perfusion during graft cold preservation is feasible for adenovirus mediated gene transfection.     

Role of ERK5 in platelet activation in vitro and arterial thrombosis in vivo

AIM: To investigate the role of extracellular signal-regulated kinase 5 (ERK5) in platelet aggregation in vitro and arterial thrombosis in vivo. METHODS: The expression and phosphorylation levels of ERK5 in human platelet were detected by Western blot. The effects of ERK5 selective inhibitor XMD8-92 on platelet aggregation and dense granule secretion were detected by Chrono-Log aggregometer. The effect of ERK5 on in vivo thrombosis was analyzed using an FeCl₃ artery thrombosis model. The effects of XMD8-92 on protein kinase B (PKB/Akt) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) phosphorylation levels were determined by Western blot. RESULTS: ERK5 was stably expressed in human platelets and its phosphorylation level increased significantly after platelet activation (P<0.05). XMD8-92, a selective inhibitor of ERK5, inhibited platelet aggregation and dense granule secretion in response to several platelet stimulators (P<0.05). The results of Western blot showed that XMD8-92 inhibited Akt phosphorylation level by down-regulating PTEN Ser370 phosphorylation and enhancing PTEN activity. The pathway was further confirmed using platelet specific PTEN deficiency mice. The first occlusion time was obviously extended in the mice intravenously given XMD8-92 in the FeCl₃-induced carotid artery injury model. CONCLUSION: ERK5 plays a role in platelet activation and arterial thrombosis by influencing PTEN and Akt phosphorylation.     

The study of cytosine deaminase gene/5-fluorocytosine in tumor gene therapy

Cytosine deaminase (CD) gene is a kind of suicide gene, cytosine deaminase can catalyze the conversion from 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), this combination therapy (CDgene/5-FC) were widely used in cancer therapy. The results of many researches in different cancer cells have demonstrated that it can inhibit tumor growth significantly. The mechanism includes 5-FU’s direct killing tumor cells and its bystander effect. It can also be used as a potential purging method for autologous hematopoietic cells transplantion after chemotherapy in breast cancer patients. CD/5-FC combined with other therapies would be an important future research approach.     

植物叶片衰老及调控

综述了对植物叶片衰老过程中的结构和代谢变化,衰老细胞程序性死亡(PCD)与过敏性反应PCD的关系,重点介绍了植物激素在叶片衰老过程中的调控作用,以及叶片衰老的其它调控因素。     

Correlation analysis between visualization of hemorrheologic changes and platelet function in patients with acute coronary syndrome after percutaneous coronary intervention

AIM:To intuitionally observe the characteristics of blood rheology in the patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI) for 1 year to 3 years by micro-channel array flow analyzer (MC-FAN) combined with other platelet function indexes, and to explore the correlations between the test results of MC-FAN and platelet function. METHODS:This study brought 74 patients with ACS after PCI for 1 year to 3 years into test group, and 21 healthy subjects were enrolled as normal group. The levels of platelet aggregation test (PAgT), platelet adhesiveness test (PAdT), P-selectin, platelet-derived growth factor BB (PDGF-BB) and von Willebrand factor (vWF) were detected. MC-FAN HR300 was used to detect the transiting time (MC-FAN TT) of the blood passing through the model body capillaries. The differences of the test results between the 2 groups were compared, and the correlations between the results of MC-FAN and platelet function in the patients with ACS after PCI were also explored. RESULTS:Compared with normal group, the MC-FAN TT in test group was prolonged (P<0.01), the ability of erythrocyte deformation was weakened, and the leukocyte attaching the vascular wall and platelet adhesion and aggregation relatively increased. The levels of PAgT, PAdT, P-selectin and PDGF-BB in test group were all higher than those in normal group (P<0.01). No difference of vWF between the 2 groups was observed. The intergroup correlation analysis showed that there were correlations between MC-FAN TT and platelet function, in which 10 μL MC-FAN TT and 30 μL MC-FAN TT had the most significant correlation with P-selectin (r=0601, P<0.01; r=0334, P<0.01), 60 μL MC-FAN TT had the most significant correlation with PAgT (r=0527, P<0.01), and 100 μL MC-FAN TT had the most significant correlation with PAdT (r=0. 815, P<0.05). CONCLUSION:The visualization of hemorrheologic changes and platelet function in the patients with ACS after PCI are abnormal.There are correlations between MC-FAN TT and platelet function.The results of MC-FAN can objectively evaluate the blood rheology of the patients, and provide the reference for clinical treatment.