Venous thromboembolism ( VTE) remains the third most common cardiovascular disease with a vague pathogenesis.Conventional biomarkers exhibit poor performance in the diagnosis, surveillance and prognosis of VTE.MicroRNAs ( miRNAs ) are a class of evolutionarily conserved small non-coding RNAs that are involved in the regulation of gene expression and protein translation An array of experimental studies has shown the importance of miRNAs for disease initiation/progression.Circulating miRNAs are found in plasma, serum and other body fluids in an apparently stable form.Recent evidence revealed that circulating miRNAs, a novel family of regulatory molecules, emerge as a promising class of biomarkers in many cardiovascular diseases, malignancies as well as VTE.This review describes current understanding of miRNA biogenesis and the origins and types of circulating miRNAs and gives an outline of recent work on circulating miRNAs as well as its challenges and perspectives of the clinical utility of circulating miRNA in VTE.

Objective:To express the recombinant single chain Fv(scFv) in E.coli and reduce immunogenicity and molecular weight of a monoclonal antibody specific for human platelet.Methods:The variable regions of the heavy and light chains of platelet specific antibody SZ 2 were amplified by reverse transcription and polymerase chain reaction.VH and VL gene segments were cloned into pUC Tm and joined together with a (gly 4ser) 3 linker.The resulting scFv was expressed in PET expression system.The expressed recombinant protein was characterized by its size on SDS PAGE,by Western blot,by flow cytometry and its functions.Results:The VH and VL genes were homologous with the published gene sequences of mouse antibody variable region.The recombinant scFv was expressed mostly in the form of inclusion bodies,and the yield was up to 25% of the total cell proteins.Functional studies showed that SZ 2 scFv could bind to platelet and could suppress platelet aggregation induced by ristocatin and thrombin.Conclusion:A recombinant SZ 2 scFv specific against platelet was developed and characterized.

AIM To study the action of danshengsuan (Dan) on expression of CD11b, P selectin, ICAM 1, VCAM 1 and E slectin in vitro. METHODS Adheshion molecule expression on the cell surface was measured by flow cytometry. RESULTS Treatment of neutrophils with N fomyl met leu phe(fMLP), resulted in a marked increase of CD11b expression. Dan inhibited the effects of fMLP in a concentration dependent manner. Dan did not inhibit the increase of P selectin expression of thrombin actived human blood platelets. Treatment of cultured human umbilical vein endothelial cells (HUVEC) with TNF ? resulted in an increase of ICAM 1, VCAM 1 and E slectin. Dan inhibited the increase of VCAM 1 and E slectin expression, not ICAM 1. CONCLUSION Dan inhibited expression of adhesion molecules CD11b, ICAM 1, VCAM 1 in human neutrophils and HUVEC.

Aim To study the effects of ferulic acid (FA) on E-selectin expression in human umbilical vein endothelial cells (HUVECs) activated by lipopolysaccharide and leukocyte-endothelial cell adhesion. Methods The effects of FA on E-selectin and E-selectin mRNA expression were determined by flow cytometry and reverse transcription polymerase chain reaction. The effect of FA on HL60-HUVEC adhesion was evaluated with the method of staining the cells by Rose Bengal. Results The expression of tively). Conclusion FA can inhibit the expression of E-selectin and E-selectin mRNA and HL60-HUVEC adhesion. This may contribute to its protective effect against ischemia-reperfusion injury.

Purpose　The aim is to isolate and purify Protein C(PC) and Protein S(PS) from no-albumin human plasma by rivanol precipitation. Methods　The isolated and purified steps included adsorption onto and elution from barium, salting-out, ion-exchange chromatography, affinity chromatography,preparative isoelectric focusing and so on. Results　The molecular weights of the obtained PC and PS were (61±0.9)kD and (83±0.8)kD, respectively, the isoelectric point, 4.70±0.03 and 5.20±0.03,and the yield, 28.3% and 12.6%. The purified PC and PS were shown to be highly homogeneous by capillary zone-electrophoresis(CZE), and rich in Glu, Leu and Gly or Asp, Glu and Leu respectively. Conclusion　 The methods could be used for large-scale isolation and purification of PC and PS from no-albumin human plasma.

AIM: To investigate the clinical significance in determination of the P- selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P- selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently - labeled SZ - 51 by direct FCM comparing with indirect FCM and enzyme- linked immunosorbent assay (ELISA). RESULTS: The level of P- selectin on platelet membrane is higher in DM (23.92 % + 15.83 % ), in hyperlipidemia ( 18.34 % + 9.46 % ) and in cerebral infarction ( 19.32 % + 10.38 % ) than normal subjects (3.38 % + 1.11% ) ( P < 0.01 ). In addition, similar results on P - selectin were obtained by indirect FCM and ELISA in patients with DM and cerebral infarction. CONCLUSION: FITC - labeled SZ - 51 - IgG can be used in FCM, and it would be a new and sensitive method in detecting platelet activation.

Objective To establish a novel method to detect autoantibodies against platelatespecific receptors by flow cytometric bead assay and study its clinical application. Methods The beads were coated with monoclonal antibodies SZ2, SZ22, SZ21 and 7E3 against platelet GP Ⅰ b, GP Ⅱ b, GP Ⅲa and GP Ⅱ b/Ⅲ a, respectively. Captured platelet glycoprotein and beads complex was detected by FITC labeled polyclonal goat antihuman immunoglobulin using flow cytometer. The platelet samples that reacted with antibodies (SZ2, SZ22, SZ21 and 7E3) negatively and positively were tested, respectively. Each sample was repeated 20 times to generate intra-day CV for the MFI and once a day for 8 days to generate inter-day CV values. The 85 ITP patients, 17 NITP patients and 50 controls from the First Affiliated Hospital of Soochow University during March 2006 to December 2008 were included in the studies. The sensitivity and specificity of these four platelet antibodies to diagnose ITP were analyzed using ROC curve. The results were compared with MAIPA. Results The CV of the intra-day-assay for samples negative to antibody SZ2, SZ22,SZ21 and 7E3 were 3.26%, 2. 86%, 1.65% and 4. 94%, respectively; While the CV of the intra-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 6. 16%, 4. 88%, 5.20% and 5. 85%,respectively. The CV of the inter-day-assay for samples negative to antibody SZ2, SZ22, SZ21 and 7E3 were 5. 86%, 4. 74%, 5.69% and 7.56%, respectively; While the CV of the inter-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 7.53%, 5.49%, 7.11% and 6.25%,respectively. The MFI for SZ2 in ITP group, NITP group and healthy control group were 1.49(0. 88-16. 24),1.12(1.00-1.33), 1.01 (0. 83-1.37), respectively, which showed significant differences (H = 36.89,P＜0.01). The MFI for SZ22 in the three groups were 1.55 (0.84-11.30), 1.13(1.03-1.29), 0.98(0. 85-1.24), respectively (H=28.41, P ＜0.01). The MFI of SZ21 were 1.50 (0.87-11.04), 1.13(0.97-1.32), 1.05 (0.85-1.48), respectively (H=54.42, P＜0. 01). The MFI for7E3 were 1.51(0. 84-9.81), 1.05(0.86-1.13), 1.03 (0.74-1.28), respectively (H =31.97, P ＜0.01). Based on ROC analysis, with cut-off values of 1.37, 1. 24, 1.48 and 1.28 for SZ2, SZ22, SZ21 and 7E3,respectively, the AUC were 0. 86, 0.90, 0. 87 and 0. 84, respectively. The sensitivities of the assays were 58. 82% (50/85), 52. 94% (45/85), 52.94% (45/85) and 51.76% (44/85), respectively. When all four antibodies were used, the sensitivity was increased to 74. 12% (63/85), which was higher than that of MAIPA [ 50. 59% (43/85) ,χ2 = 6. 78, P ＜ 0. 05) ]. Conclusion Flow cytometric bead assay can be used to detect four platelet-specific autoantibodies simultaneously, and may be a useful method to aid in the diagnosis of ITP.

AIM: To investigate the clinical significance in determination of the P-selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P-selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently-labeled SZ-51 by direct FCM comparing with indirect FCM and enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of P-selectin on platelet membrane is higher in DM (23.92%?15.83%), in hyperlipidemia (18.34%?9.46%) and in cerebral infarction (19.32%?10.38%) than normal subjects (3.38%?1.11%) (P

AIM: To clone and express the metalloproteinase domain of human von Willebrand factor-cleaving protease (vWF-cp). METHODS: The metalloproteinase domain of human vWF-cp, amplified from the plasmid containing the vWF-cp cDNA gene by using polymerase chain reaction, was cloned into pUC18, and its accuracy was verified by sequencing. Then the domain was inserted into the multiclone site of pET28a(+) and included a 6?His Tag at its amino terminal. After induced by IPTG, the recombinant protein was purified by using a Ni-NTA column and confirmed by Western blot. RESULTS: Comparison of the nucleotide sequence of our cloned domain with the GenBank sequence revealed no difference. High-level expression of the recombinant protein was yielded after 5-hour induction, which amounted to 28% of total bacteria protein in inclusion body. Western blot demonstrated that it possessed high specificity. CONCLUSION: The metalloproteinase domain of vWF-cp was high efficiently expressed in Escherichia coli. This might contribute to the further study of the relationship between its structure and function. [

AIM: To obtain a McAb that can inhibit the function of matrix metalloproteinase-2 (MMP-2), we expressed the fibronectin-like domain of MMP-2 (MFD) in vitro and prepared a McAb against MMP-2. METHODS: The purified MFD protein was used to immunize BALB/C mouse three times. Then the spleen of mouse was taken out and hybridized with hybridoma cells SP2/0. The positive cell clones were screened with ELISA method. The subtype and tissue specificity of the McAb were identified and its effect on endothelial cell migration and tube-formation was analyzed. RESULTS: After the spleen cells of the mouse and hybridoma cells SP2/0 were hybridized, a piece of cells that continuously secreted McAb against MMP-2 was obtained and named SZ-117. The titers of this McAb in culture supernatants and ascites were 2?10~-3 and 2?10~-5 , respectively. The heavy chain of the McAb belongs to IgG1 subclass. The McAb identified native MMP-2. MMP-2 existed in the stromal tissue of stomach, cholecystis, spleen, ovarian, prostate, salping and lymph node. It inhibited the invasion behavior of endothelial cells Eahy926 and pancreatic carcinoma cells 1990 and inhibited the tube-formation of Eahy926 cells. CONCLUSION: A useful tool for testing MMP-2 is obtained and it will be helpful to look for a kind of new anti-tumor material.