OBJECTIVETo study the mechanism of restenosis after angioplasty and to clarify the effect of thrombin and its receptor on restenosis development.

METHODSBalloon catheter-induced injury was adopted to induce intimal hyperplasia of the carotid arteries in rats. Antisense thrombin receptor (ATR) cDNA was transfected by perfusing recombinant LXSN ATR plasmid/nanoparticle complex into the segment of the injured carotid artery.

RESULTSPCR result showed integration of the recombined gene. Dot blot showed the expression of antisense TR mediated by recombinant LXSN ATR plasmid/nanoparticle complex in the wall of common carotid arteries of the experimental group rats, which enabled to inhibit TR gene expression and intimal hyperplasia of the injured arteries.

CONCLUSIONSThrombin and its receptor play an important role in the formation of neointima after the injury, which provides a potential clue in developing a new approach for prevention and treatment of restenosis after angioplasty.

Animals ; Carotid Arteries ; Hyperplasia ; Rats ; Receptors, Thrombin ; metabolism ; Thrombin ; pharmacology ; Tunica Intima ; metabolism

OBJECTIVEThis study explores the effects of different fibrin glue combination modes on the survival, proliferation, and apoptosis of dental follicle cells (DFCs), as well as to evaluate the feasibility and effectiveness of fibrin glue as transplantation material.

METHODSThe membranes of surviving DFCs were marked using 3,3'-dioctadecyloxa carbocyanine perchlorate (DIO), and the cell number was counted by using ImageJ2x software. The apoptotic cells were marked with prodium iodide (PI).

RESULTSCompared with that of the 3D-2 and 2D-1 groups, the degradation speed of the 3D-1 group was the slowest. DFCs could survive and grow well in fibrinogen with a concentration of 15 mg · mL⁻¹ supplemented with thrombin with a concentration of 2 U · mL⁻¹. In particular, the 3D-1 combination mode was significantly conducive to cell proliferation and stretching.

CONCLUSIONFibrin glue can be used as an effective cell transplantation material. The different combination modes have certain effects on cell proliferation. The 3D-1 combination mode is more conducive to the survival and proliferation of DFCs than other modes.

Apoptosis ; Cell Proliferation ; Cell Survival ; Dental Sac ; cytology ; Fibrin Tissue Adhesive ; pharmacology ; Fibrinogen ; Humans ; Thrombin

This study was aimed to investigate the growth-promoting activity of thrombin on mesenchymal stem cells (MSC) and its mechanisms. Human bone marrow MSC were cultured in serum-free medium supplemented with graded concentrations of thrombin, and the proliferation status of MSC was detected by MTT test. The expression levels of protease-activated receptors (PAR) and c-MYC gene were detected by PCR. Activated Akt signaling pathway was revealed by Western blot, and specific inhibitors of the signaling pathways were used to confirm the effects. The results showed that thrombin stimulated MSC proliferation in a dose-dependent manner; the minimal concentration of thrombin for stimulating MSC growth was 0.5 U/ml, and the promoting effect reached its maximum when thrombin at a dose of 8 U/ml was employed. PCR results showed that MSC expressed the two types of PAR1 and PAR2. After PAR1 was blocked with a specific inhibitor SCH79797, the growth-promoting effect of thrombin was inhibited, while this phenomenon was not observed when MSC were exposed to FSLLRY-NH2, a specific inhibitor for PAR2. Further experiments showed that after exposure to thrombin, the AKT signaling pathway in MSC was promptly activated, and c-MYC expression was greatly up-regulated. Meanwhile, when LY294002, a specific AKT inhibitor, was added into the culture medium, the up-regulation of c-MYC expression was reduced, accompanied by the low rate of MSC growth. It is concluded that thrombin can stimulate MSC proliferation by eliciting PAR1-mediated AKT activation and subsequent up-regulation of c-MYC expression.
Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Receptors, Thrombin ; metabolism ; Signal Transduction ; drug effects ; Thrombin ; pharmacology

This dissertation is to determine the biopotency of hemostat which processed in different places by establishing a bioassay method of Bletillae Rhizoma based on the thrombin time. Contrast test is the main methodology. Specifically, the reference substance of Bletillae Rhizoma is determined by comparing with the control substance of vitamin K1 using thrombin time, which is calibrated the Bletillae Rhizoma. The hemostatic biopotency is calculated by using the method of "parallel line assay method based on quantitative responses" (3.3) from different processed products. It indicates that there is a strong linear correlation between Bletillae Rhizoma and control drugs (Y = 66.332-23.913X, R2 = 0.995 3). The hemostatic biopotency of Bletillae Rhizoma from different processed products ranged between 821.93-1 187.53 U x g(-1) shown in the paper, and all of them can meet the requirements of the test. The methodology has an appropriate instrument precision (RSD 3.8%), intermediate precision (RSD 4.6%), repeatability (RSD 3.2%) and stability (RSD 3.7%). Therefore, it can be turned out that the methodology which established in the dissertation is good at determinating the hemostatic biopotency of Bletillae Rhizoma and it is reliable, simple and repeatable.
Animals ; Drugs, Chinese Herbal ; pharmacology ; standards ; Hemostatics ; pharmacology ; standards ; Orchidaceae ; chemistry ; Rats ; Rats, Sprague-Dawley ; Rhizome ; chemistry ; Thrombin Time

OBJECTIVETo investigate the expression of mDia1 (mammalian diaphanous 1)in platelet and the role of mDia1 or phosphatidylinositol 3-kinase (PI3K) in the process of thrombin-induced platelet aggregation.

METHODSThe extent of platelet aggregation was measured by a platelet aggregation system and the expression of mDia1 and its relation with F-actin in quiescent, spreading or aggregated platelets by Western blot.

RESULTSThere was no significant difference in mDia1 expression level between quiescent and activated platelets. mDia1 moved from a Triton-X100-soluble cytosolic fraction to insoluble cytoskeleton fraction after thrombin induced platelets aggregation. Anti-mDia1 antibody could inhibit this aggregation. PI3K inhibitor Wortmannin or Ly294002 inhibited the thrombin induced platelet aggregation and the above mentioned mDia1 translocation.

CONCLUSIONPI3-kinase mediates the thrombin-induced platelet aggregation through mDia1 pathway.

Actins ; Animals ; Blood Platelets ; metabolism ; Humans ; Phosphatidylinositol 3-Kinases ; Platelet Aggregation ; Platelet Aggregation Inhibitors ; pharmacology ; Thrombin ; pharmacology

OBJECTIVETo investigate the effect of benzo(a)pyrene (BaP) on platelet aggregation and expression of P-selectin.

METHODSBlood samples were collected from healthy volunteers and the platelets was washed. Platelet aggregation was monitored by aggregometer and the expression of P-seletin was detected by whole blood flow cytometry.

RESULTBaP (10 μmol/L, 1 μmol/L and 0.1 μmol/L) did not induce platelet aggregation; however, preincubation with BaP (10 μmol/L) significantly enhanced ADP-induced platelet aggregation (P < 0.01) and platelet aggregation was (80 ± 10)%, while BaP-preincubation failed to enhance platelet aggregation under collagen and thrombin stimulation. Flow cytometry showed that preincubation with BaP increased ADP-induced, but not thrombin-induced P-selectin expression (P < 0.01).

CONCLUSIONBaP can stimulate ADP-induced platelet aggregation and P-selectin expression, probably through the interaction with ADP-mediated signal pathway.

Adenosine Diphosphate ; pharmacology ; Benzo(a)pyrene ; pharmacology ; Blood Platelets ; drug effects ; metabolism ; Collagen ; pharmacology ; Humans ; P-Selectin ; blood ; drug effects ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology

Differentiation, the stepwise specialization of cells, and transdifferentiation, the apparent switching of one cell type into another, capture much of the stem cell spotlight. But dedifferentiation, the developmental reversal of a cell before it reinvents itself, is an important process too. In multicellular organisms, cellular dedifferentiation is the major process underlying totipotency, regeneration and formation of new stem cell lineages. In humans, dedifferentiation is often associated with carcinogenesis. The study of cellular dedifferentiation in animals, particularly early events related to cell fate-switch and determination, is limited by the lack of a suitable, convenient experimental system. The classic example of dedifferentiation is limb and tail regeneration in urodele amphibians, such as salamanders. Recently, several investigators have shown that certain mammalian cell types can be induced to dedifferentiate to progenitor cells when stimulated with the appropriate signals or materials. These discoveries open the possibility that researchers might enhance the endogenous regenerative capacity of mammals by inducing cellular dedifferentiation in vivo.
Animals ; Cell Differentiation ; Cells, Cultured ; Epidermal Growth Factor ; physiology ; Humans ; Regeneration ; Salamandridae ; physiology ; Serum ; physiology ; Thrombin ; pharmacology

The purpose of this study was to investigate the effect of glutathione (GSH) on blood coagulation. The normal plasma samples and mixed plasma samples were taken randomly, and into which the normal dose and different concentration of GSH were added. The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) and thrombin time (TT) were detected by using coagulation method before and after treatment with GSH. The detection results of normal plasma and mixed plasma containing GSH of different concentration were compared and analyzed with linear regression. The results showed that the APTT and FIB values of the plasma containing 2.5 mg/L glutathione or more, PT values of the plasma containing 10 mg/L glutathione or more, and TT values of the plasma containing 1250 mg/L glutathione or more were significantly different from those results of normal plasma or mixed plasma (P < 0.01) . There was a linear relation between all of the detection results of PT,APTT, FIB, TT and glutathione concentrations. The results of TT, APTT, PT and FIB detection in patient plasma were statistically different (P < 0.01) before and after treatment with normal concentration GSH. It is concluded that glutathione can influence detection results of coagulation function.
Blood Coagulation ; drug effects ; Female ; Fibrinogen ; analysis ; Glutathione ; pharmacology ; Humans ; Male ; Partial Thromboplastin Time ; Plasma ; Prothrombin Time ; Thrombin Time

Rat model of blood stasis syndrome was prepared by subcutaneous injecting of epinephrine hydrochlorid,then the model rats were administrated by Yunnan Baiyao for 15 days. Blood rheology,coagulation function and histopathology were chosen as indicators to evaluate the successful replication of blood stasis syndrome model and the treatment effect of Yunnan Baiyao. UPLC-Q-TOF-MS was used to rapidly analyze the serum samples of blood stasis syndrome rat after 15 days Yunnan Baiyao treatment,Progenesis QI software was employed to identify the alkaloids components. The results showed that Yunnan Baiyao reduced the plasma viscosity and whole blood viscosity of rats with blood stasis syndrome,prolonged thrombin and prothrombin time,reduced fibrinogen content,and effectively improved pathological state such as inflammatory cell infiltration,blood stasis,congestion and edema of various organs in rats with blood stasis syndrome. Seven alkaloids components from Aconitum kusnezoffii,including karacolidine,senbusine B,isotalatizidine,karakoline,denudatine,talatisamine and chasmanine were found in the rat serum after Yunnan Baiyao treatment. Based on the effectiveness of Yunnan Baiyao in the treatment of blood stasis syndrome induced by epinephrine hydrochloride in rats,alkaloids components from the root of A. kusnezoffii absorbed into blood after Yunnan Baiyao treatment were clarified rapidly and accurately with the help of UPLC-Q-TOF-MS. Karacolidine,senbusine B,isotalatizidine,karakoline,denudatine,talatisamine and chasmanine are the pharmacodynamic material basis of the root of A. kusnezoffii for activating blood circulation and removing blood stasis.
Aconitum ; chemistry ; Animals ; Blood Circulation ; drug effects ; Blood Viscosity ; Drugs, Chinese Herbal ; pharmacology ; Prothrombin Time ; Rats ; Thrombin Time

Leech has a good anticoagulant activity and is one of the raw materials for treatment of many cardiovascular and cerebrovascular diseases. This study was based on in vitro anticoagulant experiments( APTT and PT) to investigate the effects of lead contamination on the anticoagulant effect of leech. At present,the Hirudo circulating in the market are dominated by Whitmania pigra,therefore Wh. pigra were cultivated under a different lead pollution for 50 days. Then,the effects of Wh. pigra extract,extracting from different cultivating environment,on activated partial thrombin time( APTT) and prothrombin time( PT) were determined by automatic coagulation instrument. The results showed that the Wh. pigra extract significantly prolonged the APTT compared with the saline group.The APTT of the lead-high residual Wh. pigra was shorter than that of the blank Wh. pigra. The Wh. pigra extracts from different treatment groups had little effect on PT. The results showed that the lead residue in the Wh. pigra increased with the increase of lead in the cultured soil,the lead residual of the Pb-H group was( 10. 66±2. 79) mg·kg~(-1),which exceeded the lead limit specified in the 2015 edition of the Chinese Pharmacopoeia. The results indicated that growth environment pollution is one of the important factors causing excessive lead in Wh. pigra. Lead pollution will reduce the anticoagulant effect of Wh. pigra and affect its clinical efficacy.
Animals ; Anticoagulants ; Biological Products/pharmacology* ; Blood Coagulation ; Environmental Pollution ; Lead/toxicity* ; Leeches/drug effects* ; Prothrombin Time ; Thrombin Time