| Objective:DVT incidence of orthopedic surgery, clinical symptoms occult, heavy damage. Thrombosis natural healing mechanism is unclear, rabbit model to detect DVT and blood clots in patients with DVT subside subside during Plat, expression Plau explore Plat, Plau thrombus regression in the correlation.Technical analysis of genetic information science Plat, Plau in fibrinolysis, extracellular matrix degradation caused by thrombus regression effect. Provide a theoretical basis and experimental research platform for further research on the mechanism of thrombus regression.Method:Part1:Test the expression of Plat、Plau protein in DVT rabbit model1. Modeling and grouping:51Japanese white rabbits, male or female, irrespective of age, adaptive feeding3-5days.Normal group (8):Normal feeding, blood drawn, the survivors of rabbits are not drawn randomly assigned to two other groups.Model group(24):Anesthesia in ear vein, laparotomy, separation and expose of the inferior vena cava,4-0silk bypassing the inferior vena cava, while placing a4-0silk thread in the vein surface. Ligation of the wire to bypass the inferior vena cava, playing on the surface of the silk knot within. After tightening the surface of the thread drawn through ligation of the blood still visible. Off abdomen, normal feeding.Sham group (24):Randomly laparotomy, do not separation and expose of the inferior vena cava. Off abdomen, normal feeding.2.Venous blood collection and biopsy:According to preliminary experiments the specific time point of the set, respectively, after modeling2h,6h,24h,7d,14d,21d, each8rabbits of randomly selected sham group and model group.Anesthesia laparotomy was inferior vena cava (ligation point above0.5cm)5ml blood,placed into medical anticoagulant vacuum tube,3000rpm centrifuge15min, separated plasma,-80℃stored frozen;3rabbits were sacrificed at each time point, take of the inferior vena cava and thromboembolic body under ligature.3. Expression of Plat, Plau by ELISA:Plasma samples were measured at different points of time and groups, detected to the expression changes.of Plat, Plau protein by ELISA in plasma.4. Statistical analysis:Data analysis using SPSS17.0, measurement data were expressed as mean±standard deviation, groups were compared using ANOVA, P<0.05was statistically significant.Part2The expression changes of Plat、Plau protein in DVT patients1. Thrombotic state observation and grouping:In accordance with the diagnostic criteria (see Table2), Select the First Affiliated Hospital of Medical University Kunming from August2013to January2014selected30patients with thrombosis(no use of fibrinolytic drugs), observed the Changes of thrombotic in patients with B-mode ultrasound every day, B-mode ultrasonic examination every two weeks after discharge, according to whether thrombus regression divided into three groups:Normal group (10cases):10cases were randomly selected from20healthy volunteers;Thrombus regression group (10cases):19patients appeared to dissolve blood clots, randomly selected10cases;Thrombosis did not subside group (nine cases):Nine patients were observed after12weeks showed no change in blood clots, thrombosis did not subside for the group.2. Sample collection:Collected upper peripheral blood subjects in each group, placed into medical anticoagulant vacuum tube,3000rpm centrifuge15min, separated plasma,-80℃stored frozen.3. Expression of Plat, Plau by ELISA:Plasma samples were measured at different points of time and groups, detected to the expression changes.of Plat, Plau protein by ELISA in plasma. Carry blood coagulation indicators hematology.4. Statistical analysis:Data analysis using SPSS17.0, measurement data were expressed as mean±standard deviation, groups were compared using ANOVA, P<0.05 was statistically significant.Part3The use of genetic information science to explore the key role in thrombus subsided of Plat, Plau1. Applications2014Pubmed NCBI database, enter "Plat and DVT","Plat and arterial thrombosis","Plat and pulmanory embolis","Plau and DVT","Plau and arterial thrombosis","Plau and pulmanory embolis", Looking Plat, Plau with arterial and venous thrombosis and pulmonary embolism contact;2. In the above premise, Retrieved again" Plat and endothelial cell","Plau and endothelial cell", Looking for association of Plat, Plau and endothelial cells in the thrombus regression;3. Continue retrieval "Plat and endothelial cell"."Plau and endothelial cell", Find the specific role form of Plat, Plau in the process of thrombus regression and endothelial cell membrane;4. Using2014Kegg Pathway Database, enter "Plat and fibrinolysis"、"Plau and fibrinolysis", analysis, summarized the role of Plat, Plau in thrombus regression methods and specific signaling pathways.Result:Part1:Test the expression of Plat、Plau protein in DVT rabbit model1. Animal modeling successful, in the plasma of the corresponding time, the protein expression of Plat in the normal group10850.55±1350.29, sham group10703.65±1430.35before thrombosis group10534.14±1440.05, thrombosis early8165.86±1050.40, thrombosis fully formed8489.00±987.56, thrombosis subsided early14042.17±1275.77, thrombosis dissipating16319.64±1563.14, thrombus completely subsided10997.25±1750.80;the protein expression of Plau in normal group0.588±0.173, sham group0.589±0.177, thrombosis,0.567±0.136, thrombosis early0.347±0.097, thrombosis fully formed0.367±0.084,0.816thrombosis subsided early±0.138, thrombosis dissipating1.181±0.156, thrombus completely subsided0.589±0.154.2. There was no statistically significant difference compared Plat (P>0.05) in the sham group and normal control group;Compared with the control group, in model group, thrombosis table before significant difference (P>0.05); Thrombosis early after thrombosis expression were reduced (P<0.05), Thrombosis subsided early and thrombosis subsided expression were increased (P<0.05), Expression of thrombus disappeared completely down to the level of the control group (P>0.05);There was no statistically significant difference compared Plau (P>0.05) in the sham group and normal control group; Compared with the control group, model group, thrombosis table before significant difference (P>0.05); Thrombosis early after thrombosis expression were reduced (P<0.05); Thrombosis subsided early and thrombosis subsidedexpression were increased (P<0.05); Expression of thrombus disappeared completely down to the level of the control group (P>0.05). Part2The expression changes of Plat、Plau protein in DVT patients30Cases of thrombosis in patients with19patients by B-ultrasound thrombus subsided, dissolved discharge occurs after10weeks of the experiment one case, one case of lost, nine cases no dissolution,12weeks after blood collection.Plat expressed in normal human plasma group14.22±2.80, thrombosis did not subside group14.46±3.07, thrombus regression group19.95±4.99; Plau expression in normal group266.79±138.46, thrombosis did not subside group309.95±131.19in human blood, thrombus regression group511.32±215.65.Thrombus regression group and the normal group and thrombosis did not subside group Plat, Plau increased, the difference was statistically significant (P<0.05), Compare with thrombosis did not subside group and the normal group the expression of Plat and Plau no significant difference (P>0.05).Part3The use of genetic information science to explore the key role in thrombus subsided of Plat, Plau1. In the arterial and venous thrombosis and pulmonary suppository, Plat, Plau were expressed by endothelial cells, fibrinolytic, thrombolytic role to play, these studies investigate the clinical efficacy and more, and less for protein level detection.2. After stimulation of the endothelial cells are released thrombus Plat, Plau, primary endothelial cells on the active plasmin to generate plasmin, hydrolyzed polypeptide chain of fibrin and extracellular matrix dissolution, vascular regression. 3. Plat, Plau binding endothelial cell membrane receptor, can activate plasminogen, plasmin generation increased, the fibrinolytic protein polypeptide chain hydrolysis, Plat via ERK and p38signaling pathways (ERK can also activate p38), prompting endothelial cells to produce (vascular endothelial growth factor) VEGF, inhibition of vascular smooth muscle proliferation, inhibition of platelet aggregation, induce angiogenesis, and promote blood vessel lumen recanalization, thrombus; Plau by ERK, p38signaling pathway expression of MIP-1α, stimulated monocytes/macrophages secrete MMP-9, MMP-12, MMP-13degradation of extracellular matrix type Ⅰ, Ⅲ collagen, promote thrombolysis.4. Endothelial cells were stimulated, may be Complement and cogulation cascades Pathway, that complement and coagulation pathway signaling pathways, which plays an important role in that Plat, Plau increased expression promoting thrombolysis; Plau increased regulation by Heparan sulfate proteoglycans (HSPGs) Pathway that heparan sulfate proteoglycans enzyme signaling pathways, activation of MMP-2, MMP-9secretion, participate in angiogenesis and extracellular matrix degradation promoting thrombolysis.Conclusion:1. Plat, Plau expression in plasma of rabbits and DVT patient were raised in thrombus regression, Trend basic biological processes consistent with thrombosis, fibrinolysis may reflect the state of the body, effectively guide treatment.2. Plat, Plau mainly through the complement and coagulation pathway and endothelial cell receptor binding, activation of plasminogen hydrolysis of fibrin polymers; Plat via ERK and p38signaling pathway, induce endothelial cells to produce VEGF, inhibit vascular smooth muscle proliferation, inhibition of platelet aggregation, induce angiogenesis, and promote blood vessel lumen recanalization, thrombus; Plau pathway activation via HSPGs MMPs degrade extracellular matrix system Ⅰ, Ⅲ collagen-induced thrombus regression. |
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