近交系LEWIS BN大鼠原位肝移植急性排斥模型的建立

目的 建立近交系LEWIS→BN大鼠原位肝移植急性排斥模型,分析手术成功率的影响因素及该模型的稳定性,并总结该模型区别于常规使用的SD、Wistar等封闭群大鼠间原位肝移植的特点。方法 实验组选择近交系雄性LEWIS及BN大鼠各30只分别作为供、受体,对照组选择雄性BN大鼠各9只作为供、受体。采用Kamada“二袖套”法实施原位肝移植术,不吻合肝动脉;于术后3、5、7、9、11、13、15 d处死受体获取肝脏组织,用中性甲醛固定后制作石蜡切片进行HE染色以判定急性排斥的程度。结果 原位肝移植术成功率约为74%,导致手术失败的原因依次为：肝上下腔静脉出血,门静脉出血,麻醉意外和其他;LEWIS→BN组出现不同程度的徘斥现象,而BN BN组则无排斥现象。与封闭群大鼠肝移植比较,该模型具有自身特点,即在排斥出现的时间、程度和结果转归上表现并非完全一致,然而所有受体均出现了排斥现象。结论 大鼠肝移植是目前研究肝移植理想模型,本研究采用Kamada的“二袖套”法成功建立了大鼠肝移植急性排斥模型。     

建立近交系大鼠肝移植急性排斥模型的体会

目的:探讨近交系大鼠原位肝移植模型的建立并判断排斥反应发生的强度.方法:采用Kamada二袖套法,利用封闭群大鼠SD和Wistar进行建模技能训练,在此基础上建立近交系大鼠DA→LEW肝移植模型,根据临床表现和Banff标准判断排斥反应发生的强度.结果:共施行DA→LEW大鼠肝移植模型15例,手术成功率86.7%,死亡原因为肝上下腔静脉漏血、肝下下腔静脉血栓、胆道梗阻.术后第3天开始出现Ⅰ级排斥反应,7 d以后逐渐达到高峰,除术后并发症致死外,剩余均在12 d内死于Ⅲ级排斥反应.结论:DA→LEW为稳定、强烈的大鼠肝移植急排模型,是研究肝移植排斥及免疫耐受的理想动物模型.但近交系大鼠在组织结构上有其自身特点,给建模带来一定难度.     

对DA－Lewis大鼠肝移植模型建立的探讨

[摘要]目的 探讨近交系大鼠肝移植的手术技巧,建立稳定的急性排斥模型。方法 DA大鼠做为供体,雄性Lewis大鼠做为受体,采用改良后的Kamada“二袖套管法”,建立大鼠原位肝移植模型。观察术后存活情况,并对其生存时间进行统计分析。结果 60例大鼠肝移植手术中,供体手术时间(30±8)min,修肝时间(15±2)min,受体手术时间（60±7）min,无肝期时间（21±3）min,手术成功率88.3％。术后4至5天开始出现黄疸,精神差,睁眼困难,反应迟钝,并于术后11天内全部死亡。结论 根据近交系大鼠自身结构特点,对大鼠原位肝移植模型建立方法进行了改进,取得了一定的成效。DA-Lewis为稳定、可靠的大鼠肝移植急性排斥模型,是研究肝移植排斥反应及免疫耐受的理想动物模型。     

大鼠原位肝移植急性排斥反应模型的建立

目的 建立大鼠原位肝移植急性排斥反应模型.方法 采用改进的Limmer和Kamada的二袖套法建立大鼠肝移植模型.将大鼠分为2组:①实验组:急性排斥反应组(Wistar→SD);②对照组:免疫耐受组(SD→SD).结果 免疫排斥组肝存活时间(7.4±1.7) d低于对照组(18.9±7.6)d,差异有统计学意义0.05 (P<0.05). 结论 Wistar→SD大鼠之间的原位肝移植模型可产生中、重度的免疫排斥反应,是一种较理想的可作为研究急性排斥反应的动物模型.     

低体重大鼠二袖套法原位肝移植模型的建立

目的 尝试用低体重大鼠建立原位肝移植模型。方法 用LEWIS大鼠做供体．BN大鼠做受体．共30对。供体平均体重199go受体平均体重173g．采用二袖套法行原位肝移植手术。结果 用低体重大鼠做肝移植模型，效果确切。手术成功率达93．3％．两周存活率达92．9％。结论 该模型稳定可靠．可以用于肝移植方面的研究。     

大鼠原位肝移植模型的技术改进

目的:探讨大鼠原位肝移植模型的技术改进.方法:选取体质量相近的SD大鼠作为供受体,分别用Kamada"二袖套法"和改良"二袖套法"(改良的方法包括:提高肝上下腔静脉的吻合质量、改进门静脉和肝下下腔静脉套管的吻合及胆总管的重建等)建立大鼠原位肝移植模型,比较两组在总的手术时间﹑无肝期时间﹑受体手术时间以及手术成功率上的差异.结果:两组手术成功率分别是78.9%(71/90)和91.7%(55/60)(P＜0.05),而两组在总的手术时间﹑无肝期时间﹑受体手术时间上无统计学差异.结论:改良的大鼠原位肝移植模型是一种简单﹑易行﹑稳定的肝移植研究模型.     

异种鼠原位肝移植模型的改进

目的:建立金黄地鼠到大鼠异种原位肝移植模型,使异种肝移植模型能够普及应用. 方法:共施行99例金黄地鼠到大鼠原位肝移植,其中44例采用Kamada方法,55例采用改进方法(即受体肝分步切除的二袖套法原位肝移植),对两种术式的操作时间、无肝期及手术成功率等进行了比较. 结果:Kamada方法受体手术时间(44.1±4.5) min、无肝期(17.1±2.2) min、肝下下腔静脉和门静脉套接时间均3.5 min、手术成功率88.6% (39/44),改进方法所需时间分别为(39.2±3.7) min, (13.9±1.2) min, 1 min,手术成功率87.3% (48/55). 结论:改进的受体肝分步切除的二袖套法金黄地鼠到大鼠异种原位肝移植术,能缩短无肝期和手术时间,是异种肝移植研究较简单、易行、稳定的动物模型.     

大鼠原位肝移植模型的建立和术式改进

目的探讨双袖套法建立稳定大鼠原位肝移植模型及术式改进.方法在Kamada等的袖套法大鼠肝移植模型的基础上改进,施行原位肝移植95例,建立稳定的大鼠原位肝移植模型.结果供体手术时间(33.1±2.4)min,受体手术时间(45.7±2.1)min,无肝期(15.9±1.9)min.手术死亡9例,手术成功率90.5%,其中出血5例,空气栓塞2例,血管扭曲2例.术后1周存活率87.2%,术后1周内死亡的主要原因:术后胆漏和腹腔感染各1例,肝上下腔静脉狭窄2例和肝下下腔静脉血栓1例、胆管梗阻和袖套脱落出血各3例.结论改进的大鼠原位肝移植模型稳定可靠,手术成功率高,为肝移植提供理想的研究手段.高质量完成手术每一步骤,缩短无肝期是手术成功的关键.     

大鼠原位肝移植手术技巧探讨

目的:探讨建立稳定的大鼠原位肝移植模型的外科技巧. 方法:在Kamada二袖套法基础上,着重对供肝分离、灌注、肝上下腔静脉切取,肝上下腔静脉吻合,袖套和胆道处理等手术方法作了进一步改进. 结果:共建立40例大鼠原位肝移植模型,手术成功率80%,7d存活率达70%. 结论:在娴熟细致的外科操作基础上进一步缩短无肝期,可顺利完成大鼠肝移植模型的建立.     

泡状棘球蚴感染大鼠肝移植模型的建立

目的研究泡状棘球蚴感染大鼠原位肝移植模型的手术技巧及术后并发症的预防与处理。方法 50只BN大鼠作供体,50只泡状棘球蚴感染的Lewis大鼠为受体,通过"二袖套法"施行大鼠原位肝移植。用改进的二袖套法对大鼠行原位肝移植,移植中门静脉、肝下下腔静脉用袖套法进行吻合,肝上下腔静脉用缝合法吻合,胆道采用支架法进行胆道重建。结果建立大鼠原位肝移植模型共50例,术中未出现死亡,存活时间最短为5h,48h存活率为96%(48/50)。结论只有熟练地掌握手术技巧,细致耐心的操作,最大程度减少各种并发症的发生,才能得到良好的肝移植模型。     

组蛋白去乙酰化酶11在诱导大鼠肝移植免疫耐受中的作用

目的通过观察组蛋白去乙酰化酶11(histone deacetylase 11,HDAC11)及IL-10表达水平在3种大鼠肝移植模型中的变化,探讨肝移植免疫耐受形成的相关机制。方法纯系Lewis和BN大鼠各30只,建立肝移植排斥模型后将受体分为排斥组(Lewis-BN)、免疫抑制剂干预组(Lewis-BN)、耐受组(BN-lewis),每组10只。干预组于术后1~7 d以1 mg/kg肌肉注射他克莫司(FK506);分别于术后7 d处死受体。移植物排斥反应程度采用Banff评分标准;免疫荧光组织化学观察HDAC11表达情况;实时荧光定量PCR及Western blot测定肝组织HDAC11 mRNA和蛋白表达水平;酶联免疫吸附法检测血清IL-10及IFN-γ的含量。结果术后7 d,排斥组为严重的急性排斥反应,Banff评分Ⅲ级,干预组为Ⅱ级,耐受组为0~I级。排斥组HDAC11 mRNA及蛋白表达的水平明显高于干预组和耐受组(P<0.05),耐受组表达量最低,与干预组有统计学差异(P<0.05)。排斥组IL-10表达明显低于干预组和耐受组(P<0.05);而IFN-γ含量排斥组明显高于干预组和耐受组(P<0...     

Preliminary study on the suppression of acute rejection following orthotopic liver transplantation in experimental rats infected with Echinococcus multilocularis

Background: Hepatic alveolar echinococcosis (AE) is a parasitic disease in humans and caused by the Echinococcus multilocularis (E.m). Orthotopic liver transplantation (OLT) may be the only effective treatment for end-stage hepatic AE . however, in some AE patients, extrahepatic E.m cannot be completely eliminated after OLT. it is not known whether the immunological changes caused by E.m evasion may influence the rejection. Methods: The rat molde of AE was established by injection the E.m suspension into abdomen of Brown Norway (BN) rats after Three months later.,.In the experimental group, the liver were transplanted from a LEW rats to an E.m-infected BN rat. In the control group, transplantation from a LEW rats to a healthy BN rats. Liver tissue and peripheral blood (PB) samples were collected on days 1, 3, 5, and 7 after OLT. Liver tissue was analyzed after hematoxylin and eosin (H&E) staining; number of CD4, CD8, and CD28 on peripheral blood cells was detected by flow cytometry; and expression of the chemokine fractalkine (Fkn) was detected by reverse RT-PCR. IL-10 was measured in the serum by ELISA.Eight BN rats was retained for observeing survival time in every group. Results: The survival time of recipients in the experimental group were prolonged compared with those in the control group. the rejection occurred later and was milder in the experimental group. percentage of CD4, CD8, , CD28 T-cells and Fkn mRNA expression were lower ,The serum IL-10 levels were higher in the experimental group than that in the control group,respectively. Conclusions: Acute rejection after OLT was attenuated in the rats with E.m infection, and the recipients` survival time was prolonged. E.m may play a role in this process by elevating IL-10 secretion,decreasing the effector T cells,Inhibiting the expression of Fkn, which lead to reduce the inflammatory cells infiltration into the liver .     

Changes in systemic and splanchnic hemodynamics after orthotopic liver transplantation in cirrhotic rats

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DBcAMP对大鼠肝脏冷缺血再灌注损伤的保护作用

目的:探讨DBcAMP对大鼠移植肝脏在冷缺血再灌注损伤的保护作用。方法:应用SD大鼠原位肝移植(OLT)动物模型,根据保存液的不同,80只大鼠随机分为3个组:HC-A液对照组(A组,n=30);DBcAMP+HC-A液实验组(B组,n=30);UW液对照组(C组,n=20)。所有组在单纯低温保存8h再行原位肝移植术,测定肝组织ATP含量和肝细胞内游离Ca2+浓度变化情况、血清转氨酶,进行组化染色。结果:B组与A组相比,低温保存8h末ATP含量(μmol/g湿重)较高,分别为0.53±0.10,0.48±0.08组间有显著性差异(P<0.05)。B组与A组相比,门静脉开放6h后肝细胞内游离Ca2+浓度(nmol/L)低,分别为419.30±51.12,472.20±57.93,组间有显著性差异(P<0.05);血清ALT、AST(U/L)均较低,分别为3153±741.79,2208±450.34;3790±1029.82,2527±662.20组间有显著性差异(P<0.01)。PAS染色示门静脉开放后,B组较A组肝组织糖原含量少。HE染色及电镜下观察B组较A损伤明显减轻,但是仍比C组重。结论:DBcAMP对大鼠肝脏冷缺血再灌注损伤具有保护作用。     

双 mTORC1/2 抑制剂 AZD2014 可抑制大鼠肝移植后的急性排斥反应

目的 在同种异基因大鼠肝移植模型中验证AZD2014是否具有抑制肝移植术后急性排斥反应的作用。方法 采用Kamada 提出的“二袖套”法建立Lewis→BN 同种异基因大鼠肝移植急性排斥反应模型,随机分成对照组和AZD2014组,各4只。AZD2014组腹腔内注射AZD2014药物,5 mg/kg,1次/d;对照组腹腔内注射药物溶剂2.5 mL/kg,1次/d。不同时间点取外周血检测肝功能（丙氨酸氨基转移酶、天门冬氨酸氨基转移酶和总胆红素）。记录生存时间,进行生存分析。移植肝脏行免疫组化检测CD3和Foxp3的表达水平,评估T淋巴细胞和Treg淋巴细胞浸润的程度;移植肝脏行HE染色,采用Banff方案评估肝移植术后排斥反应的严重程度。结果 对照组在术后14 d内有3/4 的大鼠死亡,而AZD2014组在术后14 d内无大鼠死亡,AZD2014组与对照组相比生存时间明显延长（χ2=4.213,P=0.040）。对照组血清中ALT、AST和TBIL进行性升高,上述指标均高于同时间AZD2014组（P<0.05）。病理检查显示对照组移植肝内排斥反应明显重于AZD2014组（排斥指数P<0.01）,对照组中T淋巴细胞（CD3阳性）浸润相较于AZD2014组更为严重（P<0.01）,而Treg细胞（Foxp3阳性）明显少于AZD2014组（P<0.01）。结论 双mTORC1/2抑制剂AZD2014可以有效地抑制同种异基因大鼠肝移植术后的急性排斥反应。     

The role of vasoactive substances in hyperhemodynamics after orthotopic liver transplantation in cirrhotic rats

Objective To evaluate the role of endogenous vasoactive substances in hyperdynamic circulation after orthotopic liver transplantation (OLT) in cirrhotic rats. Methods Male SD rats were randomly divided into 4 groups: normal controls (NL, n=10), rats with intrahepatic portal hypertension (IHPH, n=10), normal rats with OLT (NL-OLT, n=9), and IHPH rats with OLT (IHPH-OLT, n=16). IHPH-OLT rats were divided into 2 subgroups: Group A (3 days after OLT, n=9) and Group B (7 days after OLT, n=7). IHPH was induced by injection of CCI(4) and OLT was performed using cuffs for the anastomosis of suprahepatic inferior vena cava, infrahepatic vena cava and portal vein. Radioactive microsphere method was used for hemodynamic study. The concentrations of plasma glucagon (Glu), nitric oxide (NO), prostaglandin (PGI(2)), thromboxaneA(2) (TXA(2)) and endothelin (ET) were measured by radioimmunoassay. Results No significant difference in hemodynamic changes was observed between NL-OLT and NL rats, except for mean arterial blood pressure. No significant changes in NO and PGI(2) were seen between NL-OLT and NL rats. Glu, ET and TXA(2) were significantly elevated in NL-OLT rats compared with NL rats (P&lt;0.05). Characteristics of systemic and splanchnic hyperdynamic circulatory states were observed in IHPH, IHPH-OLT A, IHPH-OLT B rats. Both the magnitude of hyperhemodynamics and increasing concentrations of Glu and NO occurred in the order of IHPH&gt;IHPH-OLT A&gt;IHPH-OLT B rats. The level of plasma PGI(2) in IHPH rats was significantly elevated compared with NL rats, while PGI(2) in IHPH-OLT A and B rats was found to be lower than in IHPH rats (P&lt;0.05). There was no obvious difference in PGI(2) between IHPH-OLT B and NL rats. Vasoconstrictors including ET and TXA(2) were found elevated in IHPH-OLT rats. Conclusions OLT does not induce postoperative hyperhemodynamics per se. Vasodilators including NO and Glu, especially NO, play an important role in the hyperhemodynamics of IHPH and IHPH-OLT rats. The results of the present study demonstrate that the persistence of systemic and splanchnic hyperkinetic circulation in the early stages after OLT may result from those non-eliminated factors that caused hyperhemodynamics in liver cirrhosis patients with portal hypertension before OLT.     

Long-term outcomes of liver transplant patients with human immunodeficiency virus infection and end-stage-liver-disease: single center experience

Objective

Orthotopic-liver-transplantation (OLT) in patients with Human-Immunodeficiency-Virus infection (HIV) and end-stage-liver-disease (ESDL) is rarely reported. The purpose of this study is to describe our institutional experience on OLT for HIV positive patients.

Material and methods

This is a retrospective study of all HIV-infected patients who underwent OLT at the University Hospital of Essen, from January 1996 to December 2009. Age, sex, HIV transmission-way, CDC-stage, etiology of ESDL, concomitant liver disease, last CD4cell count and HIV-viral load prior to OLT were collected and analysed. Standard calcineurin-inhibitors-based immunosuppression was applied. All patients received anti-fungal and anti-pneumocystis carinii pneumonia prophylaxis post-OLT.

Results

Eight transplanted HIV-infected patients with a median age of 46 years (range 35-61 years) were included. OLT indications were HCV (n = 5), HBV (n = 2), HCV/HBV/HDV-related cirrhosis (n = 1) and acute liver-failure (n = 1). At OLT, CD4 cell-counts ranged from 113-621 cells/μl, and HIV viral-loads from < 50-175,000 copies/ml. Seven of eight patients were exposed to HAART before OLT. Patients were followed-up between 1-145 months. Five died 1, 3, 10, 31 and 34 months after OLT due to sepsis and graftfailure respectively. Graft-failure causes were recurrent hepatic-artery thrombosis, HCV-associated hepatitis, and chemotherapy-induced liver damage due to Hodgkin-disease. One survivor is relisted for OLT due to recurrent chronic HCV-disease but non-progredient HIV-infection 145 months post-OLT. Two other survivors show stable liver function and non-progredient HIV-disease under HAART 21 and 58 months post-OLT.

Conclusions

OLT in HIV-infected patients and ESLD is an acceptable therapeutic option in selected patients. Long-term survival can be achieved without HIV disease-progression under antiretroviral therapy and management of the viral hepatitis co-infection.     

肝移植术后缺血型胆道病变的病因及防治

目的 探讨原位肝移植术后缺血型胆道病变的病因、预防和治疗的措施.方法 回顾性地分析自2002年1月至2009年1月间的326例次原位肝移植的临床资料,对23例缺血性胆道病变的治疗经验的总结分析.结果 肝移植术后共发生缺血性胆道病变23例(7.05%),其中肝内胆管病变9例,肝外胆管病变12例,肝内外多发胆管病变2例.通过COX比例风险模型分析,重症肝炎(RR:3.204;P=0.014)和冷缺血时间超过11.5 h(RR:4.895;P=0.000)是与移植术后发生缺血性胆病相关的独立危险因素.对23例缺血型胆道病变患者依各自特点采用药物、经内镜和放射介入(10例)、外科手术(6例)及再次肝移植(7例)等方法治疗,有效率为73.9%(17/23).结论 针对胆管病变特点选择适宜的治疗方法,是原位肝移植术后ITBL患者获得良好疗效的关键.尽量缩短供肝冷缺血时间和对受体术前仔细评估是预防肝移植术后发生缺血型胆道病变的重要措施.

Abstract：

Objective To discuss the causes, diagnosis, prophylaxis and treatment of ischemictype biliary lesions (ITBLs) following orthotopic liver transplantation (OLT). Methods A retrospective analysis was performed for 326 OLT patients from January 2002 to January 2009. The post-OLT etiological factors and treatment of ITBL cases were analyzed. Results ITBL occurred in 23 patients (7. 05% ). It included intrahepatic biliary lesions ( n = 9 ), extrahepatic lesions ( n = 12 ) and diffuse extrahepatic and intrahepatic biliary lesions ( n = 2 ). Through a COX regression, the risk factors were independently associated with ITBL serious hepatitis as the primary disease( RR:3. 204; P = 0.014)and cold donor ischemic time beyond 11.5 hours ( RR: 4. 895; P = 0. 000). All ITBL patients underwent drug therapy,endoscopy(n = 10), operation (n = 6) or re-OLT(n = 7). And improvement was found in 17 patients. Conclusion Avoiding too long old ischemic time of donor liver and carefully evaluating the indications of recipients are effective preventive measures of ITBL. It is crucial to select a proper treatment according to the conditions of each individual patient.