输注转染人IL-10基因的骨髓间充质干细胞减轻大鼠异种移植肝脏的细胞凋亡

Objective To observe the influence of hIL-10-MSCs on apoptosis of rats subject to discordant liver xenotransplantation. Methods The model of guinea pig to rat discordant liver xenotransplantation was set up. The orthotopic liver transplantation model was established by using modified two-cuff technique. Following groups were designed: control group, MSCs group and hIL-10MSCs group. Before and after operation, the recipients in control group were injected with normal sodium (2 ml) and decaesadril (0.5 ml) ;Those in MSCs group were injected with MSCs (4.0× 106/ml)and decaesadril (0.5 ml); Those in hIL-10-MSCs group were injected with hIL-10-MSCs (4.0 × 106/ ml)and decaesadril (0.5 ml). Twelve h after operation, the livers of recipient were observed by HE staining and electron microscope, and apoptosis of the livers was detected by using TUNEL. The expression levels of hIL-10, caspase-3, Fas and FasL were assayed by Western blot or immunohistochemistry. Results As compared with control group and MSCs group, the pathological changes was alleviated, the expression of IL-10 was significantly increased, the expression of FasL was significantly reduced in hIL-10-MSCs group. The positive area size of Fas and Caspase-3 in hIL-10MSCs group was 11.5 % and 25.1 %, respectively, significantly smaller than those in control group (35.3 % and 70.8 % respectively, P＜0.05. Apoptosis index in hIL-10-MSCs group was 32.5 %,which was significantly lower than in control group (74.1%) and MSCs group (50. 3 % ). ConclusionhIL-10-MSCs can notably decrease apoptosis of the transplanted liver probably by inhibiting theexpression of Fas/FasL.     

输注转染人IL-10基因的骨髓间充质干细胞减轻大鼠异种移植肝脏的细胞凋亡

Objective To observe the influence of hIL-10-MSCs on apoptosis of rats subject to discordant liver xenotransplantation. Methods The model of guinea pig to rat discordant liver xenotransplantation was set up. The orthotopic liver transplantation model was established by using modified two-cuff technique. Following groups were designed: control group, MSCs group and hIL-10MSCs group. Before and after operation, the recipients in control group were injected with normal sodium (2 ml) and decaesadril (0.5 ml) ;Those in MSCs group were injected with MSCs (4.0× 106/ml)and decaesadril (0.5 ml); Those in hIL-10-MSCs group were injected with hIL-10-MSCs (4.0 × 106/ ml)and decaesadril (0.5 ml). Twelve h after operation, the livers of recipient were observed by HE staining and electron microscope, and apoptosis of the livers was detected by using TUNEL. The expression levels of hIL-10, caspase-3, Fas and FasL were assayed by Western blot or immunohistochemistry. Results As compared with control group and MSCs group, the pathological changes was alleviated, the expression of IL-10 was significantly increased, the expression of FasL was significantly reduced in hIL-10-MSCs group. The positive area size of Fas and Caspase-3 in hIL-10MSCs group was 11.5 % and 25.1 %, respectively, significantly smaller than those in control group (35.3 % and 70.8 % respectively, P＜0.05. Apoptosis index in hIL-10-MSCs group was 32.5 %,which was significantly lower than in control group (74.1%) and MSCs group (50. 3 % ). ConclusionhIL-10-MSCs can notably decrease apoptosis of the transplanted liver probably by inhibiting theexpression of Fas/FasL.     

微透析技术连续监测大鼠肝移植后吲哚胺2,3-双加氧酶在体活性

目的 观察肝移植后肝内吲哚胺2,3-双加氧酶(IDO)在体活性,探讨肝移植免疫排斥对IDO酶活性的影响.方法 施行假手术、同品系(SD→SD)及异品系(Wistar→SD)大鼠肝移植各10例.术后在肝内植入微透析探针,连续收集透析液,HPLC测定色氨酸及犬尿氨酸浓度,以表明IDO酶在体活性.结果 假手术、同品系及异品系组在术后2周内色氨酸浓度均值分别为97.14、85.71、53.07nmol/L(P＜0.05),犬尿氨酸浓度均值分别为3.46、14.58、46.79 nmol/L(P＜0.05),异品系组术后第2天犬尿氨酸浓度即开始增加(20.03nmol/L),第7天达到峰值(70.75nmol/L),此后保持在40.00nmol/L水平以上.结论 同品系或异品系均能诱导移植后肝内IDO酶活性增加,且异品系大鼠肝移植后IDO酶活性表现早而强烈.

Abstract：

Objective To observe the activity of indoleamine 2, 3-dioxygenase (IDO) enzyme in vivo after rat liver transplantation, and the effects of liver transplantation rejection on IDO enzyme activity.Methods Ten SD rats were subjected to othotopic liver transplantations respectively in allogenic OLT group (SD→SD) and heterogenic OLT group (Wistar→SD). In the other 10 SD rats, the blood supply to the liver was obstructed for 15 min for simulating the anhepatic period in the OLT group ( sham group). An in vivo rat liver microdialysis-HPLC system was established on the day I after OLT. The concentrations of kynurenine and tryptophan in dialysate samples were continuously monitored quantitatively and reproducibly for more than 2 weeks in the same animal. Results The average concentrations of tryptophan in dialysate were 97. 14, 85.71, 53.07 nmol/L respectively in the sham, allogenic OLT and heterogenic OLT groups respectively. The concentrations of kynurenine were 3.46, 14. 58, 46.79 nmoL/L in the sham, allogenic OLT and heterogenic OLT groups respectively. The concentration of kynurenine was increased significantly on the day 7 after surgery in heterogeneic OLT group (70. 75 nmol/L). Conclusion The transplantation surgery can induce the activity of IDO enzyme in vivo and accelerate the metabolism of tryptophan in the liver allografts.     

大鼠肝脏胆管及门静脉铸型扫描图像分析

目的 建立大鼠去胆管肝叶和去门静脉肝叶自身对照模型,观察两肝叶之间胆管及门静脉是否存在交通支及其大体形态变化.方法 SD大鼠40只,分为S、BL、PL和BPL共4组,分别应用氰基丙烯酸酯对肝右叶胆管进行栓塞结扎制备去胆管肝叶;对肝方叶行门静脉结扎制备去门静脉肝叶.通过测量肝重/体重和方叶重/右叶重及对各组大鼠胆管和门静脉分别灌注硫酸钡明胶混悬液制备铸型标本,并运用Micro-CT扫描来观察两叶肝脏胆道和门静脉形态变化.结果 (1)大鼠手术后在本观察期内存活率达到100%,无黄疸表现.肝叶大体形态观察和两叶肝重量比指标显示,S、BL、PL组肝重/体重为3.5%,与BPL组比较差异有统计学意义(P<0.01).S、BL组方叶/右叶重量比为60%～70%,PL及BPL组则为20%左右,提示去胆管和去门脉肝叶之间的重量比差异有统计学意义(P<0.05或P<0.01).(2)Micro-CT铸型扫描可以直观地显示胆管和门静脉形态变化,未发现两个肝叶之间存在交通支或侧枝循环.结论 去胆管肝叶无明显萎缩.胆管及门静脉灌注造影显示两叶胆管及门静脉无明显的侧枝循环.Micro-CT扫描可以直观地显示胆管及门静脉形态变化,硫酸钡明胶灌注铸型为小动物肝脏Glissons系统形态学研究提供了一种借鉴方法.

Abstract：

Objective To establish a rat self-control model with the bile duct deprived (BDD) and the portal vein deprived (PVD) hepatic lobe and to observe whether there were communicated branches between the two lobes.Methods Forty SD rats were divided into four groups: group S with sham operation as an undisposed blank control, group BL with the right lobe bile duct embolized and ligated, group PL with the quadrate lobe portal vein ligated, and group BPL with the right lobe bile duct embolized and ligated and meanwhile the quadrate lobe portal vein ligated. The right hepatic bile ducts were embolized with cyanoacrylate and then ligated to prepare the BDD lobe. The portal vein of quadrate hepatic lobes was ligated as the PVD lobes. The observation period was 1 month after the bile duct or portal vein ligated. The values of liver weight/body weight and the quadrate lobe weight/the right lobe weight were recorded. The bile duct and portal vein casting specimens of these four groups were prepared by a perfusion with barium and gelatin solution. Three-dimensional micro-computerized tomography (Micro-CT) data sets were acquired to observe the morphological changes of bile duct and portal vein of the livers and whether there were communicated branches between the right and quadrate lobes in order to estimate the feasibility of the model.Results (1) The survival rate of rats after operation was 100%. No jaundice was observed. The ratio of liver/body weight in groups S, BL and PL was about 3.5%, significantly lower than that in group BPL (P<0.01). The ratio of quadrate/right lobe weight in groups S and BL was about 60%-70%, while that was about 20% in groups PL and BPL (P<0.05, or P<0.01); (2) Micro-CT images exhibited directly the morphological changes of the hepatic bile duct and portal vein, and no communicated branches or side circulation situation were observed between the two lobes.Conclusion No collateral branches were found between the two lobes and the model was successfully established. The barium casting liver specimen scanned by micro-CT provided a useful method for the morphological observation of rat liver Glissons system.     

雌激素在大鼠肝移植缺血再灌注损伤中的保护作用

目的 了解雌激素对大鼠肝移植缺血再灌注损伤是否具有保护作用及其可能的作用机制.方法 将大鼠分为三组:雄性组、雌性组以及给予外源性雌激素的雄性组,分别应用两袖套法实施肝移植手术.术后6 h麻醉后处死,取血及肝组织标本.观察外周血丙氨酸氨基转移酶(ALT)和谷氨酸氨基转移酶(AST)以及一氧化氮(NO)水平,免疫组化染色分析内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)在各组肝组织中的表达.结果 与雄性组和雌性组比较给予外源雌激素的雄性大鼠血清ALT水平和AST水平显著降低(分别为561.69 U/L比730.78 U/L和678.82 U/L,726.44 U/L比914.21 U/L和861.86 U/L);血清NO水平明显升高,平均为63.54 μmol/L;肝组织eNOS表达显著增加,iNOS表达显著降低.结论 雌激素对于大鼠肝移植缺血再灌注损伤具有保护作用,这种保护作用一部分是通过调节eNOS和iNOS表达、提高NO水平实现的.

Abstract：

Objective To investigate the protective effects of 17β-estradiol on ischemia reperfusion injury in rat liver transplantation. Methods The rats were divided into three groups: male to male group(MG), female to female group (FG), and male to male group which were given 17β-estradiol 4000 μg/kg 24 hours before liver transplantation intraperitoneally (M + EG). Then transplantation was performed. At 6 hours after portal vein reperfusion, blood samples were obtained to determine the levels of alanine aminotransferase(ALT), aspartate aminotransferase (AST), and nitric oxide(NO). The expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in liver were observed by immunohistochemistry. Results ALT and AST levels in the M+EG group were lower than those in MG and FG (561. 69 U/L vs 730. 78 U/L and 678. 82 U/L; 726. 44 U/L vs 914. 21 U/L and 861. 86 U/L). The NO level (63. 54 μmol/L) was much higher than those in the MG and FG groups. The expression of eNOS in the M+EG group was higher than those in MG and FG, while the expression of iNOS in M+EG were lower than those in MG. and FG. Conclusion 17β-estradiol can attenuate ischemia reperfusion injury in rat liver transplantation by improving the balance of eNOS and iNOS.     

脂肪干细胞侧脑室定向移植改善脑出血大鼠的神经功能

Objective To study improvement of neural function by stereotaxic transplantation of adipose-derived stem cells (ADSC) into lateral cerebral ventricle after intracerebral hemorrhage in rats and its mechanism. Methods ADSC were cultured and proliferated in vitro, which had been marked with Brdu for 48 h before transplantation. The rat caudate nucleus hemorrhage (ICH) models were divided into 2 groups. ADSC were stereotaxically transplanted into the right lateral ventricles in ADSC group, and equal volume of saline was transplanted into control group. The score of neurological behavior were evaluated at modeling and 1, 3, 7, 14, 28 days after transplantation respectively.Double-staining immunofluorescence technique was used to detect Brdu-positive cells and the differentiation of neurons and astrocytes. In accordance with the instructions of TUNEL kit, cell apoptosis, and the expression of VEGF and angiogenesis were assayed. Results In vitro ADSC expressed undergo osteogenic and adipogenic differentiation. Compared with the control group, ADSC group had better motor function at 3, 7, and 14 days (P＜0. 05). Double-staining immunofluorescence showed mostly grafted Brdu-reactive ADSC had migrated to the hematoma zone, and some survivedand expressed Neun of Gfap. TUNEL analysis revealed that, 3 days after transplantation, the number of apoptotic cells in ADSC group was significantly less than in the control group (P＜0. 05). Three days after transplantation, VEGF expression levels in ADSC group were significantly higher than in the control group (P＜0. 05). Conclusion ADSC stereotaxially transplanted into the lateral ventricle can survive and differentiate into neuron-like cells. ADSC transplantation may reduce apoptosis and secret VEGF to promote the angiogenesis, and improve neural functional in intracerebral hemorrhage rats.     

脂肪干细胞侧脑室定向移植改善脑出血大鼠的神经功能

Objective To study improvement of neural function by stereotaxic transplantation of adipose-derived stem cells (ADSC) into lateral cerebral ventricle after intracerebral hemorrhage in rats and its mechanism. Methods ADSC were cultured and proliferated in vitro, which had been marked with Brdu for 48 h before transplantation. The rat caudate nucleus hemorrhage (ICH) models were divided into 2 groups. ADSC were stereotaxically transplanted into the right lateral ventricles in ADSC group, and equal volume of saline was transplanted into control group. The score of neurological behavior were evaluated at modeling and 1, 3, 7, 14, 28 days after transplantation respectively.Double-staining immunofluorescence technique was used to detect Brdu-positive cells and the differentiation of neurons and astrocytes. In accordance with the instructions of TUNEL kit, cell apoptosis, and the expression of VEGF and angiogenesis were assayed. Results In vitro ADSC expressed undergo osteogenic and adipogenic differentiation. Compared with the control group, ADSC group had better motor function at 3, 7, and 14 days (P＜0. 05). Double-staining immunofluorescence showed mostly grafted Brdu-reactive ADSC had migrated to the hematoma zone, and some survivedand expressed Neun of Gfap. TUNEL analysis revealed that, 3 days after transplantation, the number of apoptotic cells in ADSC group was significantly less than in the control group (P＜0. 05). Three days after transplantation, VEGF expression levels in ADSC group were significantly higher than in the control group (P＜0. 05). Conclusion ADSC stereotaxially transplanted into the lateral ventricle can survive and differentiate into neuron-like cells. ADSC transplantation may reduce apoptosis and secret VEGF to promote the angiogenesis, and improve neural functional in intracerebral hemorrhage rats.     

脂肪干细胞侧脑室定向移植改善脑出血大鼠的神经功能

Objective To study improvement of neural function by stereotaxic transplantation of adipose-derived stem cells (ADSC) into lateral cerebral ventricle after intracerebral hemorrhage in rats and its mechanism. Methods ADSC were cultured and proliferated in vitro, which had been marked with Brdu for 48 h before transplantation. The rat caudate nucleus hemorrhage (ICH) models were divided into 2 groups. ADSC were stereotaxically transplanted into the right lateral ventricles in ADSC group, and equal volume of saline was transplanted into control group. The score of neurological behavior were evaluated at modeling and 1, 3, 7, 14, 28 days after transplantation respectively.Double-staining immunofluorescence technique was used to detect Brdu-positive cells and the differentiation of neurons and astrocytes. In accordance with the instructions of TUNEL kit, cell apoptosis, and the expression of VEGF and angiogenesis were assayed. Results In vitro ADSC expressed undergo osteogenic and adipogenic differentiation. Compared with the control group, ADSC group had better motor function at 3, 7, and 14 days (P＜0. 05). Double-staining immunofluorescence showed mostly grafted Brdu-reactive ADSC had migrated to the hematoma zone, and some survivedand expressed Neun of Gfap. TUNEL analysis revealed that, 3 days after transplantation, the number of apoptotic cells in ADSC group was significantly less than in the control group (P＜0. 05). Three days after transplantation, VEGF expression levels in ADSC group were significantly higher than in the control group (P＜0. 05). Conclusion ADSC stereotaxially transplanted into the lateral ventricle can survive and differentiate into neuron-like cells. ADSC transplantation may reduce apoptosis and secret VEGF to promote the angiogenesis, and improve neural functional in intracerebral hemorrhage rats.     

IGFBPrP1对小鼠肝组织胶原含量的影响及其机制的研究

目的 明确外源性胰岛素样生长因子结合蛋白相关蛋白1(IGFBPrP1)对肝组织胶原含量的影响及可能的机制.方法 24只雄性C57BL/6野生型小鼠随机分为3组:正常对照组、rmIGFBPrP1 2周组和rmIGFBPrP1 4周组,每组8只.取肝组织行HE、苦味酸-天狼猩红、免疫组织化学染色及Western blot检测.结果 HE和苦味酸-天狼猩红染色显示外源性rmIGFBPrP1可使肝组织发生病理改变,并导致肝组织中胶原含量显著增多(P＜0.05).Western blot检测显示rmIGFB-PrP1 2周组及4周组肝组织中IGFBPrP1、纤维连接蛋白(FN)、Ⅰ型胶原(Collagen Ⅰ)的表达明显增强,且4周组表达量较2周组高(P＜0.01).免疫组织化学染色显示Ⅲ型胶原(Collagen Ⅲ)在rmIG-FBPrP1 2周组及4周组表达量明显增高,且4周组高于2周组(P＜0.01).Western blot及免疫组织化学染色共同显示转化生长因子β1(TGF-β1)、Smad3、p-Smad2/3在rmIGFBPrP1 2周组及4周组表达显著增强,且4周组强于2周组(P＜0.01).结论 外源性IGFBPrP1可通过TGF-β1/Smad3信号通路导致肝组织中胶原含量明显增加、ECM过度沉积.

Abstract：

Objective To investigate the effect and mechanism of exogenous IGFBPrP1 on collagen content in liver tissue. Methods Twenty-four male C57BL/6 wild-type mice were randomly divided into three groups: Control group (n=8), rmIGFBPrP1 2 weeks group (n=8) and rmIGFBPrP1 4weeks group (n=8). Both hematoxylin-eosin (HE) staining and picric acid-Sirius red staining were performed. The protein expression of IGFBPrP1, Collagen Ⅰ , Collagen Ⅲ, FN, TGF-β1, Smad3 andp-Smad2/3 was evaluated by immunohistochemistry or Western blot. Results Exogenous IGFBPrP1 can cause pathological changes in liver tissue. Collagen content was significantly increased by hematoxylin-eosin (HE) staining and picric acid-Sirius red staining (P＜0.05). The protein expression of IGFBPrP1, FN and Collagen Ⅰ was gradually increased after rmIGFBPrP1 injection for 2 weeks and 4 weeks by Western blot (P＜0.01). The protein expression of Collagen Ⅲ was obviously increased in the rmIGFBPrP1 2 weeks group and rmIGFBPrP1 4 weeks group by immunohistochemistry, and the level in the 4 weeks group was higher than that in the 2 weeks group (P＜0. 01). The protein expression of TGF-β1, Smad3 and p-Smad2/3 in liver tissue was significantly increased after rmIGFBPrP1 injection in a time-dependent manner by both immunohistochemistry and Western blot (P＜0. 01).Conclusion Exogenous IGFBPrP1 can cause a marked increase in collagen content and the excessive deposition of ECM through the TGF-β1/Smad3 pathway in liver tissue.     

重建肝动脉血供的小鼠原位肝移植模型的建立

目的 构建稳定的重建肝动脉血供的小鼠原位肝移植模型,初步探讨重建肝动脉血供在小鼠肝移植中的意义.方法 66只小鼠分为实验组(n=33)和对照组(n=33).实验组行重建肝动脉血供的原位肝移植术,在经典"双袖套法"的基础上,采用支架法重建肝动脉血供;对照组行非动脉化的原位肝移植.结果 共施行小鼠原位肝移植共66只.术后2周实验组生存率为84.85%(28/33),对照组生存率为81.82%(27/33);实验组的血清γ-GT和ALP水平显著低于对照组;病理学证据亦提示实验组胆管上皮细胞受损较对照组为轻. 结论肝动脉血供重建有助于减轻移植肝胆道冷保存-再灌注损伤.该模型可能成为肝移植分子生物学研究的理想动物模型.     

多烯磷脂酰胆碱对大鼠移植肝缺血再灌注损伤的保护作用

目的 探讨多烯磷脂酰胆碱对大鼠肝移植术中相对热缺血(relative warm ischemia time,RWIT)造成胆道损伤的保护作用.方法 将60只SD大鼠分为胆道相对热缺血0 min(A组)、胆道相对热缺血60 min(B组)、胆道相对热缺血60 min并于术后每日腹腔注射多烯磷脂酰胆碱注射液(C组).建立大鼠自体原位肝移植胆道外引流模型,检测各组术后2 h及术后第1、3、5天胆汁中总胆汁酸(total bilirubin,TBA)浓度、磷脂(phospholipid,PL)浓度、TBA/PL值,并留取胆道标本进行组织病理学检测,并检测胆汁碱性磷酸酶(alkaline phosphatase,ALP)及谷氨酰基转移酶(gamma glutamyltransferase,γ-GT)水平作为观察胆管损伤的指标.结果 A组术后TBA、PL及TBA/PL均稳定,未出现明显变化;TBA浓度:术后早期B、C组明显低于A组(F=19.662,P＜0.05),此后逐渐升高,到第3天各组之间已无明显差异(F=1.244,P＞0.05).PL浓度:术后B组较C组下降明显,此后缓慢升高,至术后第5天仍低于C组(t=2.832,P＜0.05).TBA/PL值:术后早期B组明显高于A组,并于术后逐渐升高,至术后第3天达到最高,术后第5天开始下降.C组术后未出现明显变化,在各个时间点A、C组之间比较差异无统计学意义(t=0.307,P＞0.05).胆道损伤评分(胆汁ALP水平、γ-GT水平、胆道病理形态学评分、线粒体平均体积及胆道上皮细胞微绒毛密度)组间两两比较差异有统计学意义,A组(对照组)大致正常,B组(相对热缺血组)最重,C组(干预组)较轻.结论 胆道相对热缺血再灌注损伤使肝移植术后早期分泌的胆汁中胆盐及磷脂分泌均降低,其中胆盐早期恢复分泌,而磷脂恢复分泌较迟,导致了早期TBA/PL值增高,胆汁毒性增强,是导致胆道损伤的因素之一;多烯磷脂酰胆碱注射液可增加胆汁中磷脂浓度,并降低TBA/PL值,减少胆汁毒性,减轻因相对热缺血导致的胆道损伤.     

蛋白酶体抑制剂bortezomib对胆道梗阻大鼠肝脏的保护作用

目的探讨蛋白酶体抑制剂bortezomib对胆道梗阻大鼠肝脏的保护作用.方法将30只大鼠随机分为假手术组(SO组)、胆道梗阻对照组(Con组)和bortezomib实验组(Bor组).Con组通过胆总管结扎建立大鼠胆道梗阻模型,Bor组同法结扎胆总管并于术前1 d、术后第3天腹腔注射bortezomib,假手术组仅行剖腹和胆总管游离.所有大鼠均于术后7 d处死,处死前采集血清测定血清丙氨酸转氨酶(ALT),总胆红素(TB)和总胆汁酸(TBA)水平.免疫组化染色测定肝组织NF-κBp65含量.逆转录-聚合酶联反应测定肝脏组织中TNF-α mRNA水平.结果Con组与Bor组的TB和TBA水平并差异无统计学意义(P＞0.05),而Bor组的ALT水平[(92.4±21.4)μmo1/L]明显低于Con组[(145.7±33.5)μmol/L],P＜0.05.Bor组的NF-κB p65亚基阳性染色率(11.6%±2.7%)明显低于Con组(15.5%±4.3%),P＜0.05.而逆转录-聚合酶联反应发现,Bor组TNF-αmRNA相对表达量(1.0±0.2)明显低于Con组(1.3±0.4),P＜0.05.结论bortezomib可以通过抑制NF-κB的活化减少炎症反应的发生,从而减轻因胆道梗阻引起的肝脏损害.     

雷帕霉素对大鼠胆管缺血术后肝功能及生存的影响

目的 探讨雷帕霉素对大鼠肝内胆管缺血术后肝功能、营养状态及生存率的影响.方法 120只雄性SD大鼠随机分为4组,A组为对照组(假手术组)28只;B组为假手术+雷帕霉素组28只;C组为缺血组32只,D组为缺血+雷帕霉素组32只.雷帕霉素按每天2.0 mg/kg胃内注入.术前、术后7d及术后14 d分别测量实验大鼠体质量.各实验组于术后第1、3、7天分别处死6只大鼠,术后14天处死全部大鼠.处死前下腔静脉抽血2～3ml,分别检测血清总胆红素(TBIL)、碱性磷酸酶(ALP)、γ－谷氨酰胺转移酶(GGT)及谷丙转氨酶(ALT).多个独立样本比较采用Kruskal-Wallis H检验,两独立样本间的比较经秩转换后行One Way Anova检验.Kaplan-Meier方法分析各实验组动物生存率.结果 缺血组与缺血+雷帕霉素组术后血清TBIL升高,其中缺血+雷帕霉素组术后TBIL水平升高更为显著,术后14 d达(46.99±2.68) mmol/L明显高于缺血组(P＜0.01).术后1～7d,缺血组ALP明显升高,随后趋于平稳;缺血+雷帕霉素组术后血清ALP水平持续上升,术后14 d达(588.74±14.95) U/L,明显高于缺血组(P＜0.05);术后14 d缺血+雷帕霉素组血清GGT为(10.78 ±0.97)U/L,明显高于缺血组(P＜0.01).缺血组术后体质量下降明显,给予雷帕霉素后,术后7、14d体质量下降幅度增加(P＜0.01).Kaplan-Meier生存分析结果显示:缺血组术后14d累积生存率为68.3％,与照组差异无统计学意义,缺血+雷帕霉素组累积生存率下降明显(55.5％,P＜0.05).结论 雷帕霉素加重胆管缺血后肝内胆汁淤积及胆管损伤,影响肝功能恢复,并对大鼠术后营养状态及生存率造成影响.     

肝移植术变异胆管胆漏的预防与处理

目的 探讨肝移植术中变异肝管胆漏的预防及治疗.方法 回顾性分析我院3例肝移植术后发生变异肝管胆漏的诊断及预防、治疗方法.3例供肝切取均采用肝肾联合切取的方法,胆管重建方式为胆总管端端吻合.结果 1例右后叶副肝管汇入胆囊管患者在胆管吻合后发现肝门处胆囊管残端胆汁漏出,立即拆除原胆管吻合口,成型后一期吻合,术后痊愈.1例Luschka胆管漏患者术后胆漏经过充分引流漏口自行闭合痊愈,但最后终因肝内外感染而于术后7个月再次肝移植.另一例右后叶副肝管汇入胆总管患者,术中遗漏断端导致术后胆漏.该患者因严重并发症行二次肝移植.结论 了解肝内外胆管的解剖和常见变异形式,供肝修整时仔细辨认肝门组织,提高对存在副肝管及迷走胆管变异的警惕性,对预防肝移植术后胆管断端胆漏的发生非常重要.     

熊去氧胆酸减轻移植肝缺血再灌注损伤的临床前瞻性随机对照研究

目的 探讨熊去氧胆酸(UDCA)减轻移植肝缺血再灌注损伤(IRI)的临床效果.方法 将80例终末期肝病成人患者随机分为两组,一组42例,肝移植术后第1天起给予UDCA 10～15 mg·kg-1·d-1,维持治疗3个月,为UDCA组;另一组38例肝移植患者不使用UDCA,作为对照.分析两组术后3周内的肝生化指标、"严重IRI致移植物功能恢复延迟"、急性细胞性排斥反应(ACR)及药物性肝损伤的发生情况,以及术后3个月内血管及胆管并发症、患者死亡率、病毒性肝炎和原发性肝病复发情况.结果 UDCA组供肝热缺血时间和冷缺血时间分别为(3.33±0.92)min和(10.3±1.9)h,对照组分别为(3.68±1.16)min和(9.8±2.4)h,两组间的差异无统计学意义.UDCA组术后第7、14及21天的血清丙氨酸转氨酶显著低于对照组(P＜0.05),第7天的天冬氨酸转氨酶(AST)和γ-谷氨酰转移酶(γ-GT)也显著低于对照组(P＜0.05);两组其它时段AST、γ-GT及各时段胆红素总量、直接胆红素及碱性磷酸酶水平的差异均无统计学意义.UDCA组"严重IRI致移植物功能恢复延迟"发生率为9.5%(4/42),对照组为26.3%(10/38),两组比较,差异有统计学意义(P＜0.05).两组术后3周内ACR发生率和严重程度、药物性肝损伤发生率及术后3个月内胆管和血管并发症发生率的差异无统计学意义,患者死亡率的差异也无统计学意义.结论 UDCA可有效减轻移植肝的缺血再灌注损伤,降低"严重IRI致移植物功能恢复延迟"的发生率;但在冷缺血时间＜12 h的情况下,未能显示出保护胆管的临床效果.     

Increased bile duct complications in liver transplantation across the ABO barrier.

OBJECTIVE: This study evaluated the outcome of liver grafts from ABO incompatible donors, focusing on biliary complications, and compared the results to an ABO compatible control group. Also, the expression of donor ABH antigens in the liver graft was analyzed. SUMMARY BACKGROUND DATA: The outcome of liver transplantation using an ABO incompatible graft is still debated. These blood group related (ABH) antigens are known to be expressed not only on the surface of the erythrocytes, but also on the epithelial cells of large bile ducts. Because the biliary epithelium of hepatic allografts may continue to express donor ABH antigens, it may be more susceptible to immunologic bile duct injury after transplantation across the ABO barrier. METHODS: Eighteen ABO incompatible grafts were compared with 18 ABO compatible grafts in patients who were matched according to medical urgency, primary liver disease (PLD), and recipient age. After transplantation, the grafts were analyzed with cholangiography, Doppler ultrasound, or arteriography and liver histology according to protocol. Immunoperoxidase staining for ABH antigens was performed on hepatic tissue. RESULTS: Biliary complications developed in 82% of the ABO incompatible donors, compared to 6% of the ABO matched controls. Hepatic artery thrombosis occurred in 24%. Cellular rejection was diagnosed in 65% versus only 28% in the control group. The 1-year actuarial graft survival rate was 44% versus 78% in the control group. ABH antigens of the donor were expressed on vascular endothelium and bile duct epithelial cells as long as 150 days after transplant. CONCLUSIONS: Using ABO incompatible allografts, a high incidence of biliary and hepatic artery complications and decreased graft survival in liver transplantation were found. An immunologic injury to the bile duct epithelium and/or to vascular endothelium is suspected.     

DA与Lewis大鼠品系组合建立大鼠肝移植排斥与耐受模型

目的 建立大鼠原位肝脏移植急性排斥与自然耐受模型.方法 采用近交系雄性DA大鼠与Lewis大鼠互为供受体,改良"二袖套"法建立大鼠原位肝脏移植(rat orthotopic liver trans-plantation,ROLT)模型84例.实验分为4组:排斥组(DA→Lewis,n=12),FK506处理排斥组[DA→Lewis,n=24,术后1～7 d用FK506 0.2 mg/(kg·d)灌胃],耐受组(Lewis→DA,n=24),同基因组(DA→DA,n=24).各组中随机取6例观察生存期,其余分别于术后7、14、28 d处死6例,收集外周血及肝脏标本.检测血清标本天冬氨酸转氨酶(aspartate aminotransferase,AST)、总胆红素(total bilirubin,TBIL)浓度,病理学检查移植物排斥反应程度.结果 排斥组中位生存时间(median surviv-al time,MST)为12 d,而FK506处理排斥组MST为76 d,较排斥组明显延长(vs排斥组,P<0.01).耐受组与同基因组的MST均>120 d.术后7 d,排斥组血清AST、BILI浓度均明显高于其余3组(P<0.05);术后14 d,FK506处理组、耐受组和同基因组血清AST、TBIL浓度无明显差异.术后28 d,FK506处理组血清AST、TBIL浓度较耐受组和同基因组明显升高(P相似文献   

Immunohistochemistry of the liver and biliary tree in extrahepatic biliary atresia.

BACKGROUND: Progressive destruction of intrahepatic bile ducts may determine outcome in extrahepatic biliary atresia (EHBA) despite successful portoenterostomy. The aim of this study was to characterize the inflammatory infiltrate of a large series of cases of biliary atresia and relate these findings to clinical outcome. METHODS: Immunohistochemical analysis was performed on frozen tissue sections of extrahepatic biliary tree and liver biopsies obtained (August 1996 to March 1998) from 28 infants with EHBA and 8 liver biopsy specimens from age-matched controls with other cholestatic liver disorders. A semiquantitative scoring system was designed to evaluate the staining with a panel of antibodies to the CD4, CD8, CD25, CD56, CD68, CD71 antigens and to HLA-DR, ICAM-1, VCAM-1, E-selectin and LFA-1. The infants then underwent followup prospectively and divided into 2 prognostic groups at 12 months postoperatively: those who had cleared their jaundice (graded as a good outcome [n = 19]), and those who required liver transplantation or who had failed to clear their jaundice (defined as > 50 micromol/L; graded as poor outcome [n = 9]). RESULTS: CD4(+) lymphocytes and CD56(+) (NK cells) predominated in the liver of infants with EHBA as compared with controls. The infiltrating cells exhibited marked proliferation (CD71 expression) and activation (particularly LFA-1 but also CD25 expression). A smaller subpopulation of the cells also expressed VCAM and E-selectin. HLA-DR was strongly expressed on Kupffer cells and to a lesser extent on proliferating bile ducts and sinusoidal endothelium. Expression of the majority of markers was lower in the remnant bile duct tissue than in the liver of EHBA (P <.05) with only HLA-DR and LFA-1 (on infiltrating cells) and ICAM (on endothelium) expressed strongly in the remnant bile duct tissue. Although quantitatively less pronounced, all of these immunohistochemical features also were noted in non-EHBA cholestatic liver tissue. A good outcome at 12 months was associated with lower CD68 (macrophage) expression in both the liver (P <.05) and biliary tree (P <.05) and with reduced expression of ICAM-1 (P =.05) on infiltrating cells in the biliary remnant. CONCLUSIONS: Immunohistochemical patterns of immune-mediated liver injury and inflammation were prevalent features at the time of portoenterostomy. They were neither exclusive to nor characteristic of EHBA. A reduction in the expression of the macrophage marker (CD68) within the liver and biliary remnants and reduction of ICAM-1 expression on infiltrating cells in the biliary remnants appear to be associated with a better postoperative prognosis.     

Presence of multiple bile ducts in the liver graft increases the incidence of biliary complications in pediatric liver transplantation.

We studied the impact of multiple bile duct anastomosis on the development of biliary complications after liver transplantation in children. A total of 101 patients received a primary liver transplant and were divided into 2 groups: those with a single bile duct (n = 77) and those with multiple bile ducts (n = 24). Mean follow-up was 39.8 +/- 20.8 months. A total of 27 patients presented with biliary complications (26.7%), 18 patients (18.7%) presented with early complications (12 leaks and 6 strictures), and 9 patients (8.9%) had late strictures. Hepatic artery thrombosis (HAT) and multiple bile ducts were significant risk factors for the development of biliary complications, and the presence of multiple bile ducts was an independent risk factor. Patients with multiple bile ducts had a significantly greater incidence of total biliary complications compared to those with single ducts. Patients with multiple ducts had a higher incidence of leaks when compared to those in the single duct group, but the incidence of strictures, both early and late, was similar in both groups. One-year patient and graft survivals were not statistically different in the 2 groups. In conclusion, the presence of more than one bile duct in the graft is an independent risk factor for the development of biliary complications after pediatric liver transplantation.