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Objective To study the protective effect of minocycline against ischemia-reperfusion injury after liver transplantation and its molecular mechanism after circulatory death in rats.Methods The rat donation after circulatory death (DCD) liver transplantation model was established by using magnetic-ring method.The donor and recipient were male SD rats.The rats were divided into sham operation group (SHAM group),liver transplantation group,minocycline group (MIN group),atractyloside + minocycline group (ATR + MIN group),24 rats in each group.In the MIN group,10 mg/kg minocycline was injected through the dorsum vein of the penis after reperfusion.The ATR + MIN group was injected with 2 mg/kg atractyloside.The open status of mitochondrial permeability transition pore (mPTP) was detected at 2,6,and 24 h after operation.Western blotting and immunohistochemistry were used to detect the expression of caspase3 and cyt c.Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined.Liver tissues were stained with hematoxylin-eosin (HE) and pathological changes were evaluated by Suzuki's standard.The survival of each group was calculated.Results As compared with liver transplantation group and ATR+ MIN group,the mPTP opening of MIN group decreased (P＜0.05),the expression of caspase3 and cyt c and the serum ALT and AST levels decreased significantly (P＜0.05),liver tissues injury was alleviated (P＜0.05),and the survival rate increased significantly after 30 days (P ＜0.05).There was no significant difference between liver transplantation group and ATR + MIN group (P ＞ 0.05).Conclusion Minocycline reduces ischemia-reperfusion injury in DCD liver transplantation in rats probably by inhibiting the mPTP opening,and preventing cyt c release and caspase3 activation to reduce hepatocyte apoptosis.This effect can be blocked by mPTP opener.
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Objective To explore the influence of abnormal donor hepatic artery on hepatic artery and biliary complications after liver transplanta?tion,and summarize the hepatic artery reconstruction procedures during transplantation. Methods The clinical data of 210 cases of liver transplan?tation conducted in our hospital from May 2005 to April 2015 were retrospectively searched for the study,including 42 with abnormal donor hepatic artery. Results Among the 210 liver transplantation,42 cases exhibited abnormal donor hepatic artery,and the aberration rate was 20.0%. Mean volume of blood flow of abnormal group and normal group was 4.7±95.1 mL/min and 190.9±101.6 mL/min,respectively. There was no statistic differ?ence(P=0.519). Twelve cases had arterial complications,the incidence rate was 5.71%,and there was no statistic difference between each group (χ2=0.72,P>0.05). Twenty five cases got biliary complications,the incidence rate was 11.9%,and there was no statistic differences between each group(χ2=0.05,P>0.05). Conclusion There was no statistic difference of mean volume of blood flow after arterial reconstruction between two groups. Liver transplantation with abnormal arterial reconstruction will not increase the incidence rate of arterial and biliary complications.
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Objective To isolate,purify and identify pancreatic duct derived stem cells (PDSCs) from the pancreatic duct of rats,and culture in the three-dimension cell culture system.Methods Adult male Wistar rats underwent perfusion with collagenase V via the pancreatic duct,then the pancreas was surgically procured,digested,followed by discontinued density gradient centrifuge to isolate ductal tissue from islets.The acinar and ductal tissue was cultivated in serumcontaining medium in the three-dimension cell culture to obtain adherent cells,as PDSCs,which were expanded by consecutive passages.The morphology and characterization of PDSCs on phenotype were examined.Results PDSCs could be obtained through in situ collagenase V digestion,discontinued density gradient centrifuge,and culture in the three dimension cell culture system.Morphologically,PDSCs had remarkable size,most with one nucleus.PDSCs grew in many layers in three-dimension cell culture system.PDSCs was revealed to express CD29,CD73,CD90,CD105,but not CD14,CD19,CD34,CD45 by FACS,in agreement with MSCs.Conclusion PDSCs of rats could be obtained through in situ collagenase V digestion,discontinued density gradient centrifuge,and culture in the three-dimension cell culture system.PDSCs lines were successfully established.
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Objective Pancreas perfusion is an essential step in human islet isolation.To develop the new methods for introductal canulation,collagenase infusion and to observe their effects on islets isolation.Methods A total of 17 pancreases were digested from March 2005 to April 2010.The pancreases were distended by three methods:the standard method (n =3),the one-cannula method (n =11) and three-cannula method (n =3).In the standard group,the pancreases were completely cut into half at the mid-body.Two catheters were inserted into the main duct:one directed toward the tail and the other to the head.In the one-cannula method group,a long tube was inserted into the duct at the head,advancing to the tail In the three-cannula method group,pancreatic parenchyma was then minimally cut at the mid-body and three catheters were inserted into the main pancreatic duct:one at the head (the first catheter) and two at the mid-body,one toward the tail (the second catheter) and the other toward the head (the third catheter).The pancreases were digested by improved Ricordi technique.Ficoll continuous density/grads centrifuge method was performed to purify the islets.DTZ staining was adopted to identify islets and count islet equivalent (IEQ). AO/EB fluorescence examination was used to count active islet percentage.Static glucose stimulating test (SGS) in vitro was designed to estimate islet function and calculate SI.Results The distension volume of the threecannula method group was 1.24 rnl/g pancreas,and higher than the other groups (for the standard group:0.71 ml/g pancreas; for one-cannula method group:0.96 ml/g pancreas,P＜0.05).The yield of islet in the three-cannula method group and the one-cannula method group was 2514 and 2270 IEQ/g,which was significantly more than that in the standard group (1914 IEQ/g pancreas,P＜0.05).The purity and viability of the islets were 74 ％/79.3 ％,75.6 ％/79.4 ％ and 78.3 ％/84.0 ％ respectively in the three groups with the difference being not significant among the groups.SI in the one cannula method group (4.74) and the three-cannula method group (5.27) was significantly higher than that in the standard group (3.46).ConclusionThe three-cannula method improved collagenase infusion and the islet yields.
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Objective To summarize the clinical experience of harvesting and using the kidneys from donation after cardiac death (DCD) donors.Methods Fourteen kidney transplantations were successfully performed on 14 patients with end-stage renal diseases.The kidneys were harvested from 7 volunteer donors (age 30～53 years) diagnosed with cardiac death,who were scored 19～23according to the University of Wisconsin donation after cardiac death evaluation.Primary diseases of the donors were cerebral hemorrhage,brain injury,ischemic cerebral vascular disease and brain tumor.Warm ischemia time ranged from 5 to 45 min,and cold ischemia time was 4.5 ～ 12.5 h.Results After transplantation,three patients had delayed graft function (DGF),one had primary non-function (PNF),and two patients developed acute rejection.In the patient with PNF,the transplanted kidney was removed one day after operation and the patient went back to hemodialysis.One patient with DGF was still in recovery with serum creatine 149 μmnol/L (within 3 months after operation).The above two cases both utilized the kidneys with 45 min of warm ischemia time.The rest 12 patients were discharged with normal renal function.Conclusion Under the condition of our country,kidneys strictly harvested from DCD donors can be used as one of the main sources of kidney grafts for kidney transplantation.
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Objective To summarize our experience in the liver transplantation from the donation after cardiac death (DCD).Methods The livers from three DCD donors (2 cases of brain trauma and 1 case of cerebral hemorrhage) were harvested according to the Guidelines for Donation after Cardiac Death in China.These grafts were orthotopically transplanted into three recipients including 2 cases of decompensative hepatic cirrhosis and 1 case of primary liver cancer.The warm ischemic time ranged from 7.5 to 10 min and the cold ischemic time was 4.5,8.2 and 6.5 h respectively.Postoperative immunosuppressive regimens included prednisone,FK506 and mycophenolate mofetil (MMF).Antibiotics and anticoagulatants were used accordingly.Results All of the 3 recipients obtained normal liver function within 3 weeks since the grafts were implanted without PNF,thrombosis and rejection.No postoperative complications occurred in 3 recipients during the follow-up period of 2 to 9 months with normal liver function.Conclusion The liver transplant from DCD donor showed good results in our center.Chinese group Ⅲ of DCD donor,UW score above the middle level and the short warm ischemic time are three keys ensuring the success of the liver transplant from DCD donors.
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Objective To evaluate the effect of islet transplantation for patients with type 2diabetes mellitus (DM). Methods Since December 2007, 4 cases of islet transplantations were performed on 3 patients with type 2 DM and end-stage renal disease (ESRD). Two patients received simultaneous islet-kidney transplant from single-donor (SIK), and one received 2 consecutive islet transplants 5 months following kidney transplantion (IAK). All recipients given insulin with a dose of percutaneous transhepatic portal catheterization. Anti-CD25 monoclonal antibody was used as induction. For SIK, low-doses of Tacrolimus and sirolimus were used as maintenance immunosuppression protocol. For IAK, the maintenance protocol included cyclosporine and MMF.Insulin dose, the level of blood glucose, C-peptide and the value of HbA1 were observed. Results The first patient of SIKhad normal glucose level 3 days after surgery and became insulin independent within the first month, but insulin was administered gradually and the dose reduced to 1/3. The second patient of SIK died of bleeding and secondary infection of liver puncture site 5 days following operation, the blood glucose level recovered to normal 24 h after operation. The insulin dose of the patient of IAK was reduced to 1/2 after the first transplant. The patient became insulin free after the second operation. The level of fasting and postprandial C-peptide of the surviving recipients increased by 600 pmol/L. The value of HbA1 of the SIK was 6.7 %～7.3 %, while that of the IAK was 5. 5 %～ 5. 9 %. Conclusion Islet transplantation is an effective treatment for patients with type 2 DM.
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Objective To summarize experience for hepatic artery reconstruction in orthotopic liver transplantation(OLT).Method A retrospective analysis was made for 183 cases of orthotopic liver transplantation performed in our institute from May 1995 to december 2006.All the arterial reconstructions were performed with 6-0 polypropylene sutures in an interrupted fashion under a 3.5 magnification surgical loupe.Donor hepatic arteries were anastomosed at the origin of the celiac artery with a Carrel's patch or at the level of splenic artery confluence.Extra-anatomic arterial reconstruction was based on recipient aorta using donor iliac artery graft.OLT with routine anatomic arterial construction served as control.Heparin or low-molecule-weight heparin as a prophylactic anticoagulation therapy was maintained during and after operation if prothrombin time is less than eighteen seconds.Follow-up Doppler ultrasonography was used daily in the early postoperative period.Results Overall incidence of hepatic artery complications was 3.28%.Hepatic artery thrombosis(HAT)was observed in 5 cases.Hepatic artery stenosis(HAS)occurred in 1 patient.Routine anatomic arterial construction was performed in 89.07%(163)of cases,and HAT developed in 3(1.84%)cases.Extra-anatomic arterial reconstruction was carried out in 10.93%(20)of the patients;the presence of HAT was identified in 3(15.0%)cases(X2=9.73,P＜0.01).Thrombolysis,balloon angioplasty,and vascular stenting via hepatic artery were performed.One patient suffering from identified hepatic artery thrombosis died of liver failure 19 days post-op.The other 5 patients were cured successfully with patent blood flow by interventional therapy.Mortality related to hepatic artery complication was 16.7%.Conclusion HAT and HAS may be minimized by using gastroduodenal branch-patch anastomosis and postoperative anticoagulation.Close follow-up by Doppler ultrasonography helps to make a prompt diagnosis and reduce HAT-and HAS related graft loss.
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Objective To investigate the energy metabolism state and the posttransplantive function under different warm ischemia time (WIT). Methods The left lobe of pancreas was resected, and according to WIT (30, 60, 90, 120 min), the canines were randomized into 4 groups. The graft was perfused and preserved by the UW solution (0-4 ℃) cold storage method for 24 h. And then the heterotopic segmental autotransplantation of pancreas was performed, while the normal control group and no warm ischemia(NWI)group were also set up. Blood sugar (BG) and intravenous glucose tolerance test (IVGTT) K value and the plasma insulin level were examined in the first week after pancreas transplantation. The amylase level of pancreatic juice 24 h and one week after transplantation were detected either. The tissue concentrations of adenosine triphosphate (ATP) and total adenine nucleotides (TAN) were determined using high performance liquid chromatography (HPLC). The pancreas cortex was taken for morphological analysis and ultrastructure study 1 h after transplantation. Results Plasma glucose in groups of 30 min and 60 min was returned to normal 3-5 days after transplantation, while groups of 90 min and 120 min could not one week after transplantation. IVGTT K values in groups of NWI, 30 min and 60 min were more than 1, while those in groups of 90 min and 120 min were less than 1 one week after transplantation. The plasma insulin levels in groups of 90 min and 120 min were much lower than those in groups of NWI, 30 min and 60 min (P
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ObjectiveTo investigate the methods of hepatic arterial reconstruction in orthotopic liver (transplantation)(OLT), and the prevention and management of associated complications, so as to improve the therapeatic results and donor survival rate of OLT.MethodsThe clinical data of 85 cases of orthotopic liver transplantation performed in our institute from May 1995 to May 2004 were retrospectively analysed. Hepatic artery reconstruction was made by use of Carrels patch of donor celiac artery or bifurcation of donor common hepatic artery and splenic artery anastoniosed to left and right hepatic artery of recipient in 16 cases((18.82)%), to bifurcation of gastroduodenal and proper hepatic arteries of recipient in 61 cases((71.76)%), or use of donor iliac artery interposition graft to abdominal aorta in 8 cases(9.42%). Regular heparin or low-molecular-weight heparin as a prophylactic anticoagulation therapy was maintained during and after (operation) as indicated by prothrombin time. Intra-and postoperative Doppler ultrasonography was used to (monitor) hepatic arterial blood supply.ResultsHepatic artery thrombosis(HAT) was observed in 1 case (intraoperatively)(overall incidence 1.2%).Thrombectomy and reconstruction of the arterial anastomosis were performed immediately.This patient currently has a survival time of 13 months without HAT. HAT was not observed in any of the other 84 cases during the follow up of 2 to 52 months.Conclusions Hepatic artery thrombosis may be minimized by proper selection of anastomotic site of hepatic artery reconstruction and (effective) use of postoperative anticoagulation.Close follow-up by Doppler ultrasonography may make a prompt diagnosis of HAT, salvage grafts and avoid re-transplantation.
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Objective To investigate the role of neutrophils and protease inhibitor in the pathogenesis of hepatic ischemia reperfusion injury. Methods The models of liver ischemia reperfusion (LIR) and orthotopic liver transplantation (OLT) were established in rats. The contents of serum ALT and LDH and liver myeloperoxidase(MPO) were determined. Meanwhile, the hepatic pathohistological changes were observed and the number of infiltrating neutrophils counted. As for the LIR model, the animals were administered with aprotinin (AP) before ischemia. Then reperfusion was achieved 45 min after ischemia. The samples were collected after reperfusion for 4 h. In OLT model, the animals were administered with AP 1 h before the transplantation and the samples were collected 4 h postoperatively. Results The contents of serum ALT and LDH and liver MPO were significantly higher in the control group than in the experimental one (P
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Objective To study the effects of cholecystokinin (CCK) on canine Oddi sphincter(SO)function after pancreas transplantation with bladder drainage. Methods Normal canine and transplant canine SO manometry before and after CCK administration was carried out. Results SO basal pressure in control group was(18 5?2 8) mm Hg, frequency was(9 7?1 5)per min, amplitude was (47?6) mm Hg, motility index was(236?56). After CCK administration, basal pressure, frequency, amplitude and motility index decreased significantly to(10 2?2 2) mm Hg ,(5 0?1 2)per minute,(19?5) mm Hg and(50?17), all P
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Objective To study the effects of L-arginine on the reperfusion injury of hypothermic perfusion and on preservation of isolated pancreas(ISP) in canines . Methods After segmental pancreatic transplantation(SPTS) model were set up,the animals were randomly divided into 3 groups:(1)ISP irregulated and preserved with Euro Colln Solution(ECS)plus L-arginine(L-arg group);(2)IPS irregulated and preserved with ECS plus N-nitro-L-arginine methy ester(L-NAME group);and(3)IPS irregulated and preserved with ECS plus normal saline(Control group).The IPS were irregulated and preserved at 0-4℃ for 24h.Serum concentration of amylase and lipase were detected. NO,NOS and myeloperoxidase (MPO) and expression of iNOS mRNA in the pancreas graft were measured. Histological examination was performed. Results The level of NO in the L-arg group was significantly higher than that in the control group(P
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8-PT group[(2.05?0.66)?mol/g, ( 3.98 ?0.41)?mol/g] 24h after transplantation(P