| Part 1: The influence of TF changes on coagulation system in AP patientsObjective: Dynamic monitor the blood coagulation indexes and the change of TF and TF-MPs in patients with acute pancreatitis,evaluate the correlation between the blood coagulation system and TF in patients,explore the function of TF as an early diagnostic index of AP microcirculation dysfunction.Methods: 80 patients with acute pancreatitis were selected from May2013 to May 2014 in the second affiliated hospital of Chongqing Medical University,40 patients with MAP,and 40 patients with SAP,MAP group consisted of 40 patients,men and women were 18 and 22,patients with a median age(63.3±5.6)years;SAP group consisted of 40 patients,16 men and 24 women,with a median age(64.2±6.8)years.Selected 40 cases of normal healthy elderly persons as control group in the same time period in the court(CON group),20 males and 20 females,mean age(63±6.5)years.peripheral venous blood was extracted in l,2,7 days after admission,and to detect coagulation index: 1)PT(prothrombin time);2)FIB(fibrinogen);3)APTT(clotting thromboplastin time);4)DD(D-dimer).The blood was also used to detect BAMY and WBC.The change of plasma TF was detected by ELISA.Flow cytometry(FCM)was used to detect the tissue factor microparticles(TF-MPs).Results: PT,FIB,APTT and DD in MAP group were significantly increased compared to the control group in day 1 and day 2,the difference is more significant(P<0.05),on the 7th day,there were no significantly difference(P> 0.05).On 1,2 and 7 days,the PT,FIB,APTT and DD levels in SAP group patients had a significant increase compared with the control group of patients(P<0.05).On 1,2 and 7 days,the rise of WBC and BAMY levels in SAP group compared to MAP group was more obvious(P<0.05).The serum TF level was detected by ELISA,it was moderately increased in MAP group compared to CON group on the 1,2,and 7th days(P<0.05),the serum TF level was significantly increased in SAP group compared to CON group and MAP group(P<0.05).The TF-MPs level was increased in MAP group compared to CON group on the 1,2,and 7th days(P<0.05),the serum TF-MPs level was significantly increased in SAP group compared to CON group and MAP group(P<0.05).Conclusions: There was an obvious influence on blood coagulation and fibrinolysis system in patients with AP especially in SAP patients.TF and TF-MPs levels were positively correlated with the severity of the patients,if we can inhibit the TF and TF-MPs,presumably blood coagulation and fibrinolysis function have a certain improvement.By balancing the body’s microcirculation disorder,it helps regulate and improve the prognosis of acute pancreatitis.Part2: Effects on the blood coagulation system by blocking KCs with GdCl3 on the state of APObjective: To observe the changes of TF,TF-MPs,coagulation and fibrinolytic system on rats with AP,and to probe into the effects of blood coagulation and fibrinolysis system by the expression of TLR4 and TF on the state of AP.Metheds: A total of 168 SD rats,divided into seven groups by using the random method: 1)control group(CON);2)sham operation group(SO);3)mild acute pancreatitis group(MAP);4)severe acute pancreatitis group(SAP);5)SO+gadolinium chloride(Gd Cl3)group(SO+Gd Cl3);6)MAP+Gd Cl3 group;7)SAP+Gd Cl3 group.By injection with Gd Cl3(10mg/kg,0.1 m L)through vena caudalis 24 h before the establishment of AP models,the pancreatic duct injection of 1.5% or 5% taurocholate acid were received to prepare MAP or SAP models.The rats were sacrificed at 6,12,24 h after AP models were established or sham operation.Serum amylase,WBC and the coagulation makers such as PT、APTT、FIB、D-D levels were detected;TF lever was checked by ELISA;FCM was used to detect serum TF-MPs.KCs were isolated and cultured,and then the expression of TF protein and TLR4 protein in KCs were detected by Western Blot;The TF expression in liver tissues was measured by immunohistochemistry.The TF m RNA and TLR4 m RNA in KCs were detected by real-time PCR.Results: The PT,APTT,FIB,D-D,BAMY,and WBC levels were moderately increased in MAP group compared to CON group and SO group(p<0.05),while it was significantly increased in SAP group compared to other groups(p<0.01).The PT,APTT,FIB,D-D,BAMY,and WBC levels were decreased in MAP+Gd Cl3 group compared with MAP group(p<0.05),and it was significantly detected in SAP+Gd Cl3 group compared with SAP group(p<0.01).The serum TF levels were markedly increased in MAP group and SAP group compared with SO group and CON group(p<0.05),but were distinctively decreased in MAP+Gd Cl3 and SAP+Gd Cl3 group compared with MAP group and SAP group(p<0.05).The TF-MPs levels were moderately increased in MAP group compared to CON group and SO group(p<0.05),while it was significantly increased in SAP group compared to other groups(p<0.01).The TF-MPs levels were decreased in MAP+Gd Cl3 group compared with MAP group(p<0.05),and it was significantly decreased in SAP+Gd Cl3 group compared with SAP group(p<0.01).TF protein and TLR4 protein expression derived from KCs was distinctively increased compared with SO group and CON group(p<0.05).And TF protein and TLR4 protein expression of KCs in MAP+Gd Cl3 group and SAP+Gd Cl3 group was obviously lower than MAP group and SAP group(p<0.05).TF m RNA and TLR4 m RNA expression of KCs was distinctively increased compared with SO group and CON group(p<0.05).And TF m RNA and TLR4 m RNA expression of KCs in MAP+Gd Cl3 group and SAP+Gd Cl3 group was obviously lower than MAP group and SAP group(p<0.05).The pathological section of liver showed that MAP group characterized by mild to moderate damage in liver cells,accomplished with cytoplasmic vacuolation,and infiltration of inflammatory cells,MAP+Gd Cl3 group showed pathological damage was better than MAP group.SAP group showed severe damage in liver cells,appeared in scattered liver cell necrosis,fuzzy boundaries between cells,and a large number of inflammatory cells.The pathological damage was obviously alleviated in SAP+Gd Cl3 group compared with SAP group.The pancreas pathological section of rats showed that oedematous in the pancreatic interstitial in MAP group,the pathological damage of MAP+Gd Cl3 group was alleviatived compared with MAP group;Coagulation necrosis in pancreas was showed in SAP group,accompanied with a large number of inflammatory cells;it was obviously showed that the pathological damage of SAP+Gd Cl3 group was alleviatived compared with SAP group.Conclusion: TF and TLR4 expression in KCs was significantly increased in rats with AP which lead to increased release of TF-MPs,then leaded to the change of blood coagulation and fibrinolysis.Inhibition of KCs could effectively reduce the expression of TF.TLR4 pathway could be blocked by blocking of KCs with Gd Cl3,then obviously decreased the expression of TF,improve microcirculation on early AP,slow or stop process of change from MAP to SAP,so as to inhibit the occurrence and progress of AP.Part3: TF and TF-MPs expression regulated by TLR4 signaling pathways in KCsObjective: To probe into if TF-MPs can be released under the LPS stimulation in KCs by imitiating the body inflammation stimulation,and to explore if TF expression can be regulated by blocking the TLR4 pathway in k Cs.Methods: KCs were separated and clultured from wild type of C57 mice were assigned to WT group,while KCs were separated and clultured from TLR4-/-mice were assigned to TLR4-/-group.Each group were stimulated by LPS(300 ng/ml),detected at 0 min,15 min,30 min,60 min and 120 min.ELISA was used to detect the TF concentration at each time point.TF-MPs concentration of culture supernatant was detected by flow cytometry instrument.Western Blot was used to detect TF protein expression and TLR4 protein expression in KCs;TF m RNA and TLR4 m RNA expression in KCs was detected by Real-time PCR.Results: The TF levels were significantly increased in WT group compared with TLR4-/-group at 30 min to 120 min(p<0.05).The rising trend was showed in WT group from 0 min to 120 min,and the rising trend was also showed in TLR4-/-group from 0 min to 60 min,but that was no obvious difference between 60 min and 120 min(p>0.05).The TF-MPs levels were significantly increased in WT group compared with TLR4-/-group at 30 min to 120 min(p<0.05).The rising trend was showed in WT group from 0 min to 120 min,and the rising trend was also showed in TLR4-/-group from 0 min to 60 min,but that was no obvious difference between 60 min and 120 min(p>0.05).The TLR4 protein expression and TLR4 m RNA expression was significantly increased in WT group compared with TLR4-/-group at each time point(p<0.05).The rising trend was showed in WT group from 0min to120 min,while there was almost no expression in TLR4-/-group(p>0.05).The TF protein expression was significantly increased in WT group compared with TLR4-/-group at each time point(p<0.05).TF m RNA expression was significantly increased in WT group compared with TLR4-/-group at 30 min to 120 min(p<0.05).The rising trend was showed in WT group from 0 min to 120 min,and the rising trend was also showed in TLR4-/-group from 0 min to 60 min,but that was no obvious difference between 60 min and 120 min(p>0.05).Conclusions: Our study confirmed that TLR4,TF and TF-MPs were positively correlated,LPS may regulate the expression of TF in KCs through TLR4 pathway.LPS may active TLR4 pathway through TLR4 receptor,promote KCs to express TF,release TF through TF-MPs,then affect inflammatory response and blood coagulation. |
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