Objeetive To explore change of platelet glycoproteins Ⅵ(GPⅥ)in patients with type 2 diabetes mellitus and its clinical significance.Methods The surface expression of platelet glycoprotein Ⅵ was determined by flow cytometry in 56 patients with type 2 diabetes mellitus and 61 normal individuals.Plasma GP Ⅵ concentration was measured by ELISA in 30 patients with type 2 diabetes mellitus.Platelet surface CD62P was analyzed by flow cytometry and HbA1c was determined in Datients with type 2 diabetes mellitus.Results The geometric mean fluorescence of platelet surface GPⅥ in Datients with type 2 diabetes mellitus was 93.66±35.24,which was significantly enhanced compared with normal subjects (62.83±24.2)(t=-4.927,P＜0.05).Nine patients were positive in plasma GPⅥ among 30 Datients with type 2 diabetes mellitus.Plasma GP Ⅵ concentrations in 9 positive patients were conversely correlated with platelet surface GPⅥ expression(r=-0.633,P＜0.05).However,there was no correlation between platelet Surface CD62P and plasma HbA1c concentrations, and the correlation coemcient is -0.333 and -0.417,respectively(P＞0.05).There was no correlation between platelet surface GP Ⅵ expression with C062P and HbA1c in patients with type 2 diabetes mellitus and the conrrelation coefficient is -0.009 and -0.217,respectively(P＞0.05).Conclusions GPⅥ expression on platelet surface is elevated in Datients with type 2 diabetes mellitus and the determination of platelet surface GPⅥ and plasma GPⅥ concennlation may helP to prognosticate the risk of thrombotic events and may play an important role in evaluating platelet activation in patients with type 2 diabetes mellitus.

Objective To establish an UPLC method for the determination of sinomenine in Sinomenine External Applied Powder. Methods The UPLC method was carried out on a C18 column by using acetonitrile-water-ethylene diamine (50:50:0.25) as mobile phase. The flow rate was 0.2 mL/min; the sample quantity was 2 μL; the detection wavelength was 283 nm. Results The peak time was within 1 min or so. The calibration curve of sinomenine was in the linear range of 34.2–2188.0 ng. Conclusion The method is simple, rapid, stable and reliable, which can be used for the determination of sinomenine in Sinomenine External Applied Powder.

Manual microscopic differential of white blood cell has been challenged by multiparameter flow cytometry,using monoclonal antibodies to define the different leukocyte types.Compared with manual differential,flow cytometry method is more sensitive,specific,objective and has good repeatability.Recent studies demonstrated flow cytometric differential correlates well with manual microscopic method and has good clinical performance for blast and immature granulocyte.Meanwhile more leukocyte populations can be identified with flow cytometric method,such as lymphocyte subset and CD 16 + monocyte,thus helping in monitoring blast in acute leukemia,B lymphocyte proliferative disorder differential diagnosis and minimal residual disease.With the development and improvement of flow cytometric differential,it might be a candidate reference method of leukocyte differential,gradually applied in the routine work.

Objective To express and purify recombinant human platelet glycoprotein Ⅵ extracellular domain and prepare the polyclonal antibody against it.Methods Human platelet glycoprotein Ⅵ extracellular domain fragment (123~268 residues) was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-3x.The recombinant plasmid was constructed and expressed in E.coli after induction with isopropyl-?-D-thiogalactopyranoside (IPTG).The fusion protein was identified by Western blot analysis after purification by affinity chromatography.Rabbits were immunized with the purified fusion protein, and the collected rabbit antiserum was evaluated by sandwich ELISA, Western blot and flow cytometry.Results The coding sequence of GPVI extracellular domain was successfully inserted into pGEX-3x.Sequencing result showed that the cloned gene was identical as reported.After induction, a Mr 42kd fusion protein was expressed and confirmed by Western blot, which was identical to that expected.The titers of the antisera were up to 1∶[KG-*2]128. Sandwich ELISA result demonstrated that the prepared antibody recognized GPVI in human platelet. Western blot and flow cytometry revealed that the prepared antibody reacted with GPVI of platelet lysate and the native GPVI on human platelet surface.Conclusions Using purified prokaryotic expressed the fusion protein as antigen, the specific antibody was elicited in the immunized animals. The prepared polyclonal antibodies react specially with GPVI on human platelet surface and can be used for further studies of GPVI.

Direct oral anticoagulants,include direct thrombin inhibitor and direct factor Xa inhibitor.As the pharmacokinetics and pharmacodynamics of these drugs are known,their plasma concentration is not food-effective,and theoretically it is not necessary to monitor routinely.However, clinical practice in recent years has shown that anticoagulant effect of DOACs is required to evaluate in patients with thrombosis, major bleeding, emergency surgery, hepatic/renal dysfunction and other special situations.Recent studies have shown that routine coagulation assays such as activated partial thromboplastin time(APTT) and thrombin time(TT) can be used as laboratory screening tests for direct thrombin inhibitor dabigatran and prothrombin time(PT) can be used as laboratory screening test for direct factor Xa inhibitor rivaroxaban.DOAC′s quantitative measurements include dilute thrombin time(dTT),hemoclot thrombin inhibitor (HTI),ecarin clotting time (ECT) and ecarin chromogenic assay (ECA) for direct thrombin inhibitor and anti-FXa assay(rivaroxaban calibration) for rivaroxaban.Laboratories should establish their own monitoring range when performing these assays.

OBJECTIVE:To optimize the preparation technology of Sinomenine topical paste. METHODS:Using“initial vis-cous force”,“holding viscous force”and“peeling strength”as index,heating and stirring time (A,h),heating temperature (B,℃) and the sequence of adding composition (softening agent,blank matrix and sinomenine)(C) as influential factors,the preparation technology of Sinomenine topical paste was optimized by orthogonal test and verified. At the same time,the content of sinomenine was determined by HPLC method. RESULTS:The optimal preparation technology of Sinomenine topical pasta was as follows as adding blank matrix and sinomenine,and then adding softening agent,heating at 80 ℃,stirring for 1 h. In verification test,RSD of comprehensive score for 3 batches of samples were 2.09%(n=3);average contents of samples were 6.7 mg/g, which was in line with the requirement of ≥6.0 mg/g. CONCLUSIONS:The optimal preparation technology of Sinomenine topical pasta is reasonable,stable and feasible. The paste shows good adhesiveness and is qualified in content.

Microcapsule technology is a high and new technology under the focused research and development in 21st century, which has been used in many research fields. However, its researches on preparation field should be paid more attention to, especially the research direction of TCM drug delivery system, which is of great value for development and application. This article reviewed the application progress of microcapsule technology in pharmaceutic preparation field, and discussed the strategic significance of the researches on TCM microcapsule preparation, with a purpose to provide research ideas and paths for the researches on the new formulations of traditional Chinese medicine.

Objective To establish a new flow cytometric immuno-bead array assay (FCIBA)to detect several platelet-specific autoantibodies simultaneously in a single serum sample.Methods A series of beads of different red fluorescent intensity were used for coating with different anti-platelet membrane protein monoclonal antibodies(anti-GP Ⅱb/a,anti-GP Ⅰ a/Ⅱ a,anti-GP Ⅳ,anti-GP Ⅰ b/Ⅸ and anti-HLAABC)to detect five platelet-specific autoantibodies in serum. The beads captured platelet antigenautoantiboay complex.Subsequently,different platelet-specific autoantibodies in a patient serum can be detected simultaneously by the flow cytometry.In addition,we evaluated the new FCIBA,and compared its results with modified antigen capture ELISA method(MACE).The new FCIBA was used to detect five platelet-specific autoantibodies in serums of autoimmune thrombocytopenic purpura(AITP)and nonautoimmune thromboeytopenic purpura patients.Results The new FCIBA can be used to detect five plateletspecific autoantibodies simultaneously(Anti-GP Ⅱ b/Ⅲ a,Anti-GP Ⅰ a/Ⅱ a,Anti-GP Ⅳ,Anti-GP Ⅰ b/Ⅸand Anti-HLA-ABC).The coefficient of variation(CV)of intra-repetition are 4.82%,6.09%,5.04%,5.73%and 5.30%,respectively.The dilution test results are in good logarithm linearity which are 0.997 2,0.996 6,0.998 8,0.996 5 and 0.998 2,respectively. The resuhs of the new FCIBA are highly correlated with those with MACE method,and the coefficient correlation were 0.928 9,0.922 4,0.889 4,0.910 0 and 0.913 4,respectively(P＜0.01).51.69%samples of AITP patients show positive for platelet-specific autoantibodies as detected by the new FCIBA.Among the AITP patients,the positivity of specific autoantibodies for anti-GPⅡb/ Ⅲ a,anti-GP Ⅰ a/Ⅱ a,anti-GP Ⅳ,anti-GP Ⅰb/Ⅸ and anti-HLA-ABC were 40.82%,24.45%,19.39%,32.65%and 17.35%.Among the 40 non-autoimmune thrombocytopenic purpura patients,none of platelet-specific autoantibodies in serum samples can be detected.Conclusion A new FCIBA is established successfully to detect five platelet-specific autoantibodies coefficient correlation.

Platelet surface is rich in glycocalyx. It has been found that platelet glycosylation plays an important role in the physiological hemostasis mechanism, regulating the interaction between platelets and receptor proteins, and dynamically reshaping the surface glycosylation through its own glucose metabolism system. Platelet glycosylation also participates in platelet aging and clearance, and regulates platelet counts. Meanwhile, abnormal platelet glycosylation is closely related to primary immune thrombocytopenia, coronary heart disease and other related diseases, being a potential therapeutic target.

Objective To observe the effect of febuxostat on epithelial-to-mesenchymal transition (EMT) of kidney tubules and the levels of serum IL-6 nad transforming growth factor (TGF) β1 in hyperuricemic rats.Methods Forty male SD rats were divided into 4 groups:normal control group (NC group),oteracil potassium group (OP group),oteracil potassium with febuxostat group (OF group) and oteracil potassium with benzbromarone group (OB group).Each group had 10 rats and balanced in body weights.To induce hyperuricemia,rats were given oteracil potassium by gastric garage once a day for eight weeks.Rats in OF group and OB group were given either febuxostat or benbromarone starting with oteracil potassium,and rats in NC group was given saline only.Blood samples were taken before,and at the end of 4 and 8 weeks of the treatments and serum uric acid,creatinine,blood usea nitrogen (BUN),IL-6 and TGFβ1 contents were measured at each time point.Renal pathological changes were observed via HE and Masson staining,and the expression of α-SMA and E-cadherin were detected by immunohistochemistry.Results Compared with those in NC group,the levels of serum uric acid,creatinine,BUN,IL-6 and TGFβ1 in the another three groups were increased significantly (all P ＜ 0.01).However,the IL-6 and TGFβ1 contents in OF group were much lower than those in OP group (P ＜0.01).HE and Masson staining showed that OF group had less damage and tubulointerstitial fibrosis than OP group and OB group (P ＜0.01).Moreover,the expression of α-SMA was significantly down-regulated (P ＜ 0.01) and that of E-cadherin was significantly up-regulated in OF group compared with those in OP group.Conclusion Febuxostat treatment significantly inhibited EMT and reduced the levels of IL-6 and TGFβ1 in hyperuricemia rats.