Clinical And Experimental Study On Restenosis Of Graft-Venous Anastomosis After Artificial Vessel Shunt In Combined Budd-Chiari Syndrome

BackgroundBudd-Chiari syndrome (BCS) is a group of clinical symptom complex characterized by blocked hepatic venous trunk and/or partial or complete inferior vena cava at various levels from retrohepatic to right atrium, resulting from venous refluence obstruction.It is a kind of disease with badly prognosis,threatening human health seriously.Recently, with the deepening research of this kind of disease,great progress of medical diagnosis technology and widely application of imaging techniques, a large numbers of clinical cases have been reported. Researchers from all over the world are carrying out more and more investigation on epidemiology, pathogenesis, pathology and molectroics of BCS,and reasonally surgical as well as multi-subject compositive treatments,which highly improving the treatment levels of BCS. Nevertheless, there are still something kept unknowned and deficient.China is a high incidence region of BCS, with the annually morbidity of4-6/100thousand. Adult men are the most patients and the mainly type is membranous occlusion and short range thrombotic occlusion or obstruction of IVC. The initial treatment is intervention, that is percutaneous angioplasty (PTA) or simultaneous embedded metal stent (EMS). Interventional therapy has been widely applied in the treatment on BCS cause of its micro invasion, quick effect and easily operation. However, in case of combined BCS and interventional interdictions caused of combined blocked hepatic venous trunk, long range occlusion of IVC, secondary long thrombosis under septum or recurrence of postoperational restenosis and voided intervention, all kinds of shunt and bypass surgical operation are the preffered remedy and candidate methods. The key determining the surgical methods is the long term potency of shunt or bypass vessels. The past research work showed, restenosis or occlusion of graft anastomosis was the main cause of dysfunction of shunt or bypass vessels, whatever the type of vessels made of self venous or other kind of artificial vessels. The past research works mainly aimed at peripheral artery occlusive disease (PAOD) and coronary arteriosclerotic heart disease (CASHD). However, there are no recent systemic experimental and clinical reports on study of prevent and therapy for restenosis or occlusion of graft venous anastomosis after shunt/bypass operation in BCS.This research collected and retrospectively analysed a group of clinical data of complex combined BCS, and carried out two mainly shunt with expanded polytetrafluoroethylene (ePTFE),which showed better clinical effect resulting from sufficient decompression. The mainly recent and long term complication was restenosis or occlusion of graft venous anastomosis, resulted in dysfunction of shunt vessel and furtherly liver failure and even death. It is an important and needing resolved question and one of focus of surgical basic research as well that how to reduce the incidence rate of restenosis or occlusion of graft venous anastomosis after shunt in BCS and keep long term potency of artificial vessels. Based on the clinical effects of two kinds of shunt with ePTFE in combined BCS, all kinds of risk factors of restenosis or occlusion of graft venous anastomosis were seriously analyzed and assumed, which provided contraposed researching way of subsequent experiments and basic investigation.Recent study shows that the pathogenesis of venous restenosis is more compiex than of artery, which is mainly associated with injury stress of endothelial cells and hemodynamics, especially the endothelial cells injury and subsequent series reactions are the key factors in the cause of anastomosis restenosis. Previous study focused on the subsequent reactions after endothelial cells injury, i.e. hyperplasia of smooth muscle cells and activation of fibrinolysis system. And there are no systemic basic experimental research on self changes of endothelial cells and how to prevent and repair the injury. As the first wall of inner membrance of blood vessels, endothelial cells not only keep the whole and slippy of blood vessels wall but excrete many kinds of cell genes to protect good function of self and neighbor cells. Of all, nitri oxide (NO) is one of most important adjust genes. It can expand blood vessel, inhibit blood platelet, white blood cell adhesion, multiplication and transfer of smooth muscle cells, advance living and repairment of endothelial cells. It is the key gene by which endothelial cells effect. Endothelial Nitric Oxide Synthase (eNOS) is the key enzyme in synthesise of NO, which is expressed in the endothelium of blood vessels and the mainly source of endogenesis NO of blood vessel system. By building the rat model of acute BCS with blocked IVC, we kept watching on the level of NO in venous blood and expression of eNOS in the local blood vessel wall, and analysized the changing rules of NO and eNOS in the occurrence of BCS, evaluated the various self changes after injury of endolial cells. So that, we intend to afford certain theory evidences and experimental bases of prevention and therapy for injury of endolial cells and protection of its functions.It is the recent hot problem in clinical and basic research of injury and repairment of blood vessel endothelium. Previous study aimed at improving and building the self function of endolial cells through inner ways (i.e. gene amending and cell structure reconstruction of tissue engineering). However, this method was proved time wasting, process fussy, and needing high professional employee and equipment, and huge expenses. Ectogenous way has been proved able to abate the above difficulties and receive better experimental effects. In our research, we studied the influences of ectogenous Nitric Oxide on the changes of morphology and function index of invitro cultured human umbilical vein endothelial cells (HUVEC), and analysized the mainly ways of endothelial cells injury repairment by NO, explored the possible molecular biology pathogenesis, so that we may provide new ideas and ways to invent effective drugs and better prevention and therapy on restenosis or occlusion of graft venous anastomosis after shunt in BCS. Part OneStudy and Comparison of Clinical Effects in Treatment of Complex Combined Budd-Chiari Syndrome by Two Types of Artificial Vessel Shunts and Correlative Factor Analysis of Restenosis of Graft Venous AnastomosisAims:To investigate and compare the therapeutic effects of Meso-Atrial Shunts (MASs) and Meso-Cavo-Atrial Shunts (MCASs) as treatment for complex combined Budd-Chiari Syndrome (BCS) with ePTFE, and analysize the causes and correlative pathogenesis of shunt dysfunction resulting from restenosis of graft venous anastomosis.Methods:We retrospectively gathered29cases of complex combined BCS with all or bilateral hepatic vein occlusion and long range occlusion or obstruction of retrohepatic IVC from Oct1998toAug2003. Of them,12were treated with a MAS and17with a MCAS.We analyzed pre-and postoperative clinical symptoms, IVC pressure (IVCP) and portal venous pressure (PVP), occurrence rates of postoperative complications, restenosis of graft venous anastomosis, patency rates of artificial vessels and survival rates.Results:One patient in the MAS group died in the preoperative period. During the follow-up period from3d to60mo,96.6%were included. The effective rates were54%in MAS and90.8%in MCAS. The occurrence rates of postoperative complications were12.5%and1.8%, respectively. The average decreases in IVCP and PVP were15.5mm saline and62.4mm saline for MAS, and12.3mm saline and184.7mm saline for MCAS.4patients in the MAS group died of liver function failure caused of blocked artificial shunt within one year of the postoperative period while only one case in the MCAS4.5years after the operation. Intima hyperplasia at the graft venous anatomosis was found in two groups. In MAS, the total restenosis rates of graft-superior mesenteric vein (G-SMV) anatomosis at1,3, and5y were66.7%,75%and83.3%. While in MCAS, the numbers were47.1%,58.8%and64.7%, respectively. And the total restenosis rates of G-IVC anatomosis at1,3, and5y were35.3%,41.2%and58.8%. The survival rates at1,3, and5y were41.7%,41.7%, and 16.7%for MAS, and94.1%,88.2%, and82.4%for MCAS. The5-y patency rates were41.7%and94.1%. Comparing these two groups, all of the studied factors, with the exception of PV pressures, were significantly different (P<0.05). Thus, the therapeutic effects of MCASs were better than those of MASs.Conclusion:1, MCAS can simultaneously relieve high IVC and PV pressure in combined BCS. Compared with MASs, MCASs showed a decreased postoperative complication rate, a higher5-y survival and artificial vessel patency rate.2, The primary reason of impacting the patency of artificial vessels was restenosis of graft venous anastomosis, and also the key factor determining the effects of shunts.3, Intima hyperplasia was the primary reason of restenosis of graft venous anastomosis. Part TwoExperimental Research on Level of NO and Expression of eNOS in Rat Model of Acute Budd-Chiari SyndromeAims:To build steady animal model of acute BCS with blocked IVC in rats, and measure the level of NO in venous blood and expression of eNOS in the local blood vessel wall.Methods:Adult male Wistar rats were used as experimental animals, and divided into three groups:normal control group (10rats, unoperated), training group (10rats), and experimental group (60rats,20pre group). We used the method of modified suprahepatic vena cava reduction and killed the rats at1,7,14day after operation. We observed changes of abdomen organs, measured pressures of IVC and PV, liver function index, organ indexes of liver and spleen and their HE changes. Also, we measure the level of NO in venous blood plasma and expression of eNOS in the local suprahepatic vena cava wall by immunohistochemistry.Results:Four rats of training group died in perioperative period, others lived well beyond two weeks after operation. No rats died in normal control group and experimental group and all rats lived very well. The operative time was15.3±3.6mins,and there was no unexpected bleeding.Rats were killed at1,7,14days after operation.Abdomen organs adhesion,ascites and abdominal venous distention were obvious, as well as hepatomegaly and splenomegaly.Organs index,liver function index and IVCP/PVP rised gradually,and the datas were significantly different (P<0.05) compared with those of normal control group and within experimental groups each other. HE stained liver and spleen slices accorded with the pathological manifestation of liver cirrhotic portal hypertension. Plasma NO levels were67.18±4.23,84.70±3.26and132.90±8.05μmol/L at1,7,14day after operation, respectively.The datas were significantly different (P<0.05) compared with those of normal control group and within experimental groups each other. Expression of eNOS located at cell membrane and cytoplasma was lower in normal contrl group and rised obviously in experimental group, and increased with time. Compared the datas of two groups, there was significantly different (P<0.05). There was a positive correlation between the level of plasma NO and expression of eNOS in venous wall during the observed period of experiment.Conclusions:1, Steady rat model of acute BCS can be built by the way of modified suprahepatic vena cava reduction.This method was proved easily operated and short period, which was the perfect animal model of acute BCS recently and the base of subsequent research.2, During the occurrence of acute BCS with blocked IVC, stagnanted blood stream of IVC and injuried endothelia of local blood vessel wall led the rising of levels of NO in plasma and positive expression of eNOS in intima of local blood vessel wall, which showed hemodynamics and self functional injury of endothelia were concerned with the pathogenesis. 3, There was a positive correlation between the level of plasma NO and expression of eNOS in venous wall during the observed period of experiment, which showed a kind of possibly positive cross action within each other. Part ThreeInvitro Experimental Study on Ectogenous Nitric Oxide Advancing Repair of Endothelia Cell Injury in Prevention and Therapy for Restenosis of Graft Venous Anastomosis after Shunt in Combined Budd-Chiari SyndromeAims:To study the influences of ectogenous Nitric Oxide on morphologic and function index changes of invitro cultured normal and injury HUVEC, and explore relations of NO function and endothelial cells injury, and mainly ways and mechanisms of repair. So that we may provide reliable experimental datas for gene therapy and clinical medicine to prevent and treat restenosis of graft venous anastomosis after shunt in combined BCS.Methods:1, Cultrue and identification of HUVEC;2, Building of injuried HUVEC model induced by H2O2;3, Experiment of Ectogenous Nitric Oxide on injuried HUVEC model4,Assessment of intervention of Ectogenous Nitric Oxide on injuried HUVEC(1) Group dividing:Normal HUVEC (Normal Group), injuried HUVEC model induced by H2O2(Injury Group), Injuried HUVEC model intervened by SNP (Intervened Group);(2) Observation and comparation of morphologic changes in various groups by optical microscope;(3) Observation and comparation of nucleolus apoptosis changes in various groups by Hoechst33258staining; (4) Test and comparation of expression of eNOS in various groups by Western Blot, and the changes of eNOS synthesis adjusted by NO;(5) Test and comparation of expression of Caspase-7in various groups by Western Blot, and ability of resisting apoptosis of injuried cells;(6) Test and comparation of VEGF levels of cell culture medium in various groups by Western Blot, and ability of excreting of injuried cells;(7) Test and comparation of expression of PCNA by imrnunohistochemistry, and ability of proliferation of injuried cells.Results:1, Normal HUVEC were injuried by oxidative stress induced by H2O2, and the cells changed morphologically, apoptotic bodies appeared.The cells of intervened group kept almost normal morphologically, and apoptotic bodies decreased obviously.2, Expression of eNOS deceased in injury group,and increased again in intervened group.3, No expression of Caspase-7in normal group, and it could be detected in injury group, while in intervened group, it decreased again obviously.4, Level of VEGF was higher in intervened group than those in other two groups, and there was significantly different (P<0.05) between intervened and injury groups.5, Expression of PCNA in intervened group was almost aqual to that in normal group,and higher than that in injury group. There was significantly different (P<0.05) between intervened and injury groups.Conclusions:1, Oxidative stress induced endothelial cells injury and morphologic changes,and there were deceases in aspects of eNOS expression, resisting apoptosis ability and function of proliferation, synthesis and excreting.2, Intervention of a certain dose of ectogenous Nitric Oxide can raise self synthesis and expression of eNOS gene, advance repair of endothelial cells morphologically, resist apoptosis and protect function of proliferation, synthesis and excreting of endothelial cells.