ObjectiveTo analyze the death causes of postoperative early-stage after orthotopic liver transplantation (OLT) in rats, and to provide appropriate treatment strategies. MethodsThree hundreds of rat OLT models were performed by modified Kamada two-cuff technique. Operation time of each stage during OLT and postoperative survival time of rats were recorded and analyzed. According to survival time, the rats were divide into 4 groups:intraoperative death group (rats died during operation), < 6 hours group, 6-24 hours group, and > 24 hours group. Then comparison of operation time of each stage during OLT in rats of 4 groups was performed, and reasons of death during 24 hours after OLT were analyzed. ResultsOf the 300 OLT models, 37 rats died during operation (12.33%), 51 rats died within 6 hours after operation (17.00%), 76 rats died during 6-24 hours after operation (25.33%), and 136 rats survived longer than 24 hours (45.34%). The most common death causes of < 6 hours group were as follows:lose too much blood during the operation (27.45%, 14/51), postoperative bleeding (27.45%, 14/51), and vascular embolization (15.69%, 8/51). However, the most common death causes of 6-24 hours group were as follows:angiostenosis (27.63%, 21/76), postoperative bleeding (21.05%, 16/76), and pulmonary edema (19.74%, 15/76). There were significant differences in the cold ischemia time and anhepatic phase among the 4 groups (P < 0.05). The cold ischemia time and anhepatic phase of intraoperative death group were longer than those of other 3 groups (P < 0.05), in addition, the cold ischemia time of > 24 hours group was shorter than those of other 3 groups (P < 0.05). ConclusionsThere are many reasons leading to the early death after OLT. The long time of anhepatic phase and the cold ischemia time, intraoperative and postoperative bleeding, thrombosis, angiostenosis, and pulmonary edema are key factors for the improvement of prognosis in rats after OLT operation. Improvements of the reasons above are helpful to improve the successful rate of modeling and quality of OLT rats.
Objective To study the mechanism of immune hyporesponsiveness of allograft rejection induced by transfusion nonpufsed allopeptide syngeneic immature dendritic cell (imDC) generated from recipient bone marrow progenitors and to explore a possible strategy for liver allograft protection in clinic. Methods Forty experimental rats were randomly divided into 4 group: control group, cyclosporine A (CsA) group, mature DC (mDC) group and imDC group. In control group, Wistar rats only received liver transplantation. In CsA group, Wistar rats underwent liver transplantation plus CsA treatment 〔10 mg/（kg·d)〕. In mDC group, recipient-derived mDC 1×106 were infused intravenously through the penile vein to Wistar rats. In imDC group, ImDC with the dose of 1×106 were injected into Wistar rats via the dorsum vein of penile. In each group, five recipients were killed on the 10th day after transplantation, the other five recipients were left to observe survival time. The levels of ALT, AST, TBIL, IL-2, IFN-γ, IL-4 and IL-10 were detected. The acute rejection and the expression of FasL/Fas in the grafts were detected by HE and immunohistochemical staining. Western blot was used to detect Scurfin protein expression of CD4+ CD25+ T cells. Results The median survival time of the liver allografts in CsA group and imDC group were significantly longer than that in control group and mDC group ( P ＜ 0.05). The levels of ALT and TBIL in control group and mDC group were significantly higher than those in CsA group and imDC group ( P ＜ 0.05). Compared with CsA group and imDC group, the levels of IL-2 and IFN-γ were higher but the levels of IL-4 and IL-10 were lower in control group and mDC group ( P ＜ 0.01). Slightly or no rejection reaction was found in CsA group and imDC group ( P ＜ 0.05). The Scurfin protein expressions of CD4+ CD25+ T cells of imDC group were significantly higher than those of other three groups. Conclusion Application of nonpufsed allopeptide syngeneic recipient-derived imDC is an effective way to induce immune hyporesponsiveness by blocking indirect recognition in rat liver transplantation model. Survival span is significantly prolonged by its protective effect. The mechanism of immune hyporesponsiveness induced by imDC transfusion might be involved in some aspects: T cell apoptosis, immune deviation of Thl/Th2 cytokine net and inhibition of T lymphocytes responsiveness by regulatory T cells.
Objective To explore improvement of orthotopic liver transplantation model in rhesus monkey. Methods Healthy rhesus monkeys were chosen to perform orthotopic liver transplantation for 10 cases. The model was established by drawing on a variety of animal model methods, and the portal vein cuff method was used to establish stable model of orthotopic liver transplantation in rhesus monkeys. Results Ten orthotopic liver transplantation models in rhesus were performed, and the achievement ratio of operation was 10/10. The time of donor hepatectomy and donor preparation was (20±5) min and (30±7) min, respectively. The operation time of recipient and anhepatic phase were (180±35) min and (17±4) min, respectively. After 24 h of operation 9 cases survived, one case died of intra-abdominal hemorrhage after 9 h of operation. After 72 h of operation 8 cases survived, and one case died of upper gastrointestinal bleeding after 38 h of operation. After one week of operation 5 cases survived, and 3 cases died of rejection after 9, 11, and 11 d of operation, respectively. The longest survival time was 32 d, but all of them also died of rejection. No portal vein thrombosis and biliary complications were found in all recipients.Conclusion The improved rhesus monkey model of orthotopic liver transplantation is easy to perform with high achievement ratio of operation. It is an ideal animal model for pre-clinical studies of liver transplantation.
Objective To explore the methods of hepatic artery reconst ruction with iliac arterial interpositiongraf t in orthotopic liver t ransplantation (OL T) and influential factor of relevant complications postoperatively.Methods Analyzed ret rospectively 8 OL T , the hepatic artery reconst ruction with arterial inflow based on recipientinf rarenal aorta using donor iliac artery graf t tunneled through the t ransverse mesocolon and pancreas. Results Thetime required for hepatic artery reconst ruction with iliac arterial interposition graf t was 52 - 126 minutes. Amongthe 8 patient s , 2 patient s developed postoperative bililary t ract complications , 1 with biliary fistula , 1 with int rahepatic biloma , the others were recovered smoothly and liver function returned to normal about one week af ter livert ransplantation. No complications of hepatic artery were observed. Conclusion Iliac arterial interpositional graft is aneffective and reliable method of revascularization in liver transplantation when the use of hepatic artery is not possible.
Objective To observe the dynamic histopathologic changes of acute rejection in rat orthotopic liver transplantation (OLT) model after tacrolimus discontinued and provide some prediction and evaluation data for clinical acute rejection after liver transplantation. Methods Kamada two-cuff technique was used to establish 60 rat OLT model， and male DA rats, male Lewis rats were used as donors and recipients respectively. Therapeutic amount of tacrolimus (0.05 mg/kg, twice per day, continued for 8 d, 1 d before operation and 7 d after operation, intragastric administrated) was administrated to recipients, then continuously half dose was decreased every day beginning from day 8 after operation and tacrolimus administration was stopped on day 13. Liver tissues were collected on day 7, 14, 21, and 28 after liver transplantation. Histopathologic changes and rejection activity index (RAI) of liver tissues were observed, survival time of recipients was calculated. Results Owing to protection effects of tacrolimus, liver tissues displayed no significant histopathologic changes of acute rejection in 7 d after OLT, while typical acute rejection histopathologic changes began to be observed on day 14 after OLT due to tacrolimus discontinuation. On day 14, 21, and 28, RAI were 3.7±0.9, 6.3±0.9, and 8.1±0.7 respectively. Survival time of recipients was (20.85±0.71) d with a median of 21 d. Conclusion Acute rejection could be induced in rat OLT model after tacrolimus discontinuation, and data collected from this model shows some extent of predictive value and assessment value for clinical liver acute rejection.
ObjectiveTo clone full-length cDNA of rat galectin-9 and construct recombinant adenovirus granule containing rat galectin-9 gene. MethodsThe galectin-9 gene was amplified by RT-PCR from rat liver tissue and inserted orientationally into plasmid pDC316-GFP digested by restriction endonucleases NotⅠ and HindⅢ. The recombinant pDC316-GFP-galectin-9 shuttle plasmid was identified by PCR, restriction endonuclease digestion and sequencing, and then co-transfected with rescue plasmid pBHGlox△E1.3Cre into HEK-293 cells by liposome reagent. Recombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) was generated by sitespecific recombination and confirmed by PCR, and then Ad5-galectin-9 was propagated in HEK-293 cells and purified. The infectious titer of viral stock was determined by TCID50 assay. ResultsConstruction of pDC316-GFP-galectin-9 shuttle plasmid was confirmed to be correct by PCR, restriction endonuclease digestion and sequencing. Construction of recombinant adenovirus Ad5-galectin-9 was confirmed to be correct by PCR. The infective titer of Ad5-galectin-9 was 1.4×109 U/ml. ConclusionRecombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) is successfully constructed, which provides the foundation of further research on the function of galectin-9 gene.
ObjectiveTo explore perioperative management model of ABO-incompatible liver transplantation. MethodsThe clinical data of ABO-incompatible caderveric liver transplantions without urgency performed in our center from July 2006 to May 2010 were analyzed retrospectively. Four patients had received an ABO-incompatible graft: AB to O in three, AB to A in one. All the cases were diagnosed as end-stage liver disese, one of them was primary hepatocellular carcinoma. ResultsFour survived to now （11 to 19 months） without severe infections and acute rejections. Two experienced coagulative disturbance and one of them had a second exploration. One developed acute renal failure and recovered with help under continuous veno-venous hemofiltration. All the cases were given 20 mg basiliximab two hours before revascularization and on day 4 after operation respectively. Splenectomy was performed in three, intravenous immunoglobulin was given in all more than seven days. Isohemagglutinin titers were basically stable and not relevant to the clinical manifestations. Antibiotic prophylaxis and immunosuppression protocol was same as the ABO compatible transplants except a 3-month-delay for steroid withdrawal. ConclusionABO-incompatible liver transplantation could be performed with appropriate perioperative management, such as basiliximab induction, splenectomy, intravenous immunoglobulin administration, and routine immunosuppression.
目的探讨胰腺损伤的诊断和治疗方法。 方法回顾性分析16例胰腺损伤患者的临床资料。 结果胰腺损伤属Ⅰ级3例，Ⅱ级5例，Ⅲ级4例，Ⅳ级3例，Ⅴ级1例；系单纯胰腺损伤3例，合并其他器官损伤13例。术前确诊为胰腺损伤者9例，另7例分别诊断为：脾破裂1例，肝破裂1例，开放性腹部损伤2例，空腔脏器穿孔、弥漫性腹膜炎3例。8例胰腺Ⅰ～Ⅱ级损伤者行清创、缝扎止血及胰腺周围双套管引流术；4例Ⅲ级损伤者行胰体尾切除+脾切除术；3例Ⅳ级损伤者，行近端胰腺断端缝合、胰管缝扎加胰体尾空肠Roux-en-Y吻合术；对1例Ⅴ级损伤者行胰十二指肠切除术。术后发生胰瘘5例；治愈13例，死亡3例。 结论早期诊断、及时手术探查以及术中选择合理的手术方式，对降低胰腺损伤的并发症和死亡率和改善胰腺损伤的预后均十分重要。
ObjectiveTo investigate the early diagnostic value of transforming growth factor-β1(TGF-β1) on acute rejection after liver transplantation in rhesus by detecting the expression of TGF-β1 in the liver tissue. MethodsLiver transplantation models in rhesus were constructed by the improved vascular dual cuff, supporting tube of biliary tract, and artery anastomosis method.The successful models were randomly divided into experimental group (no immunosuppressant treatment in perioperative period) and control group (treated by immunosuppressant in perioperative period).Then the blood samples and liver tissues were collected at 6, 12, 24, and 72 hours after surgery.Allograft rejections of liver tissue after liver transplantation were monitored by liver function test, hematoxylin-eosin staining and Banff score.Finally, the expression level of TGF-β1 was detected by Western blot analysis or immunohistochemistry technique. Results①The acute rejection happened in all the rhesus at 12 h, 24 h and 72 h after liver transplantation, especially at 72 h after liver transplantation in the experimental group, the Banff grade levels of acute rejection in the liver tissue was more severe than that in the control group (P < 0.05).②The levels of ALT, AST, and TBIL after liver transplantation was gradually increased, which were similar at 6 h and 12 h after transplantation between the two groups, but which at 24 h and 72 h after transplantation in the experimental group were significantly higher than those in the control group (P < 0.05).③The results of TGF-β1 protein expression using immunohistochemical detection:The percentage of positive area of TGF-β1 of liver tissue at 12 h in the experimental group was significantly higher than that in the control group (P < 0.05).With the extension of time, it was gradually increased and significantly higher than that in the control group at 24 h or 72 h (P < 0.05).④The semi-quantitative results of TGF-β1 protein expression using Western blot detection:The TGF-β1 protein expressions began to increase at 6 h after liver transplantation in the experimental group and the control group, and the magnitude of increase was more obvious in the experimental group.The TGF-β1 protein expressions at different time (6 h, 12 h, 24 h, and 72 h) in the experimental group were significantly higher than those in the control group (P value was 0.003, 0.001, 0.001, and 0.001, respectively). ConclusionsThe elevated level of TGF-β1 of liver tissue after liver transplantation might suggest the enhanced cellular immune function, it might have certain significance for early diagnosis of acute rejection after liver transplantation.