Establishment of a new pig model for auxiliary partial orthotopic liver transplantation

AIM: To establish a new pig model for auxiliary partial orthotopic liver transplantation (APOLT).METHODS: The liver of the donor was removed from its body. The left lobe of the liver was resected in vivo and the right lobe was used as a graft. After the left lateral lobe of the recipient was resected, end-to-side anastomoses of suprahepatic inferior vena cava and portal vein were performed between the donor and recipient livers,respectively. End-to-end anastomoses were made between hepatic artery of graft and splenic artery of the host.Outside drainage was placed in donor common bile duct.RESULTS: Models of APOLT were established in 5 pigs with a success rate of 80%. Color ultrasound examination showed an increase of blood flow of graft on 5th d compared to the first day after operation. When animals were killed on the 5th d after operation, thrombosis of hepatic vein (HV) and portal vein (PV) were not found. Histopathological examination of liver samples revealed evidence of damage with mild steatosis and sporadic necrotic hepatocytes and focal hepatic lobules structure disorganized in graft. Infiltration of inflammatory cells was mild in portal or central vein area. Hematologic laboratory values and blood chemical findings revealed that compared with group A (before transplantation), mean arterial pressure (MAP), central venous pressure (CVP), buffer base (BB), standard bicarbonate (SB) and K+ in group B (after portal vein was clamped) decreased (P＜0.01). After reperfusion of the graft, MAP, CVP and K+ restored gradually.CONCLUSION: Significant decrease of congestion in portal vein and shortened blocking time were obtained because of the application of in vitro veno-venous bypass during complete vascular clamping. This new procedure,with such advantages as simple vessel processing, quality anastomosis, less postoperative hemorrhage and higher success rate, effectively prevents ischemia reperfusion injury of the host liver and deserves to be spread.

EFFECTS OF PSEUDOFYPE RETROVIRUS CONTAINING HUMAN N-RAS ANTISENSE GENE ON THE GROWTH OF HUMAN LIVER CANCER LTNM4 TRANSPLANTED IN NUDE MICE

An amphotropic pseudotype retrovirus containing human N-ras antisense gene was constructed and packaged with helper cells. It has been previously demonstrated that the virus did inhibit the growth of human hepatocarcinoma cell line PLC PRF/5 in vitro accompanied with the blockage of p21 expression. Based on these results, further study was carried on to examine the effect of these viruses on the growth of human hepatoma transplanted LTNM4 in nude mice. It has been shown that the retrovirus containing human antisense N-ras gene could inhibit the hepatoma in nude mice at a rate of 78% (P<0.05) as compared with saline control. No inhibition was observed in group treated with retrovirus which contained no N-ras sequence. These results in vivo lend further support that human N-ras antisense gene mediated by retrovirus could block the expression of the relevant oncogene and lead to the inhibition of cancer growth. It also provided the basis for further approaches of gene therapy for human cancer.

Orthotopic liver transplantation as a rescue operation for recurrent hepatocellular carcinoma after partial hepatectomy

AIM: To compare post-orthotopic liver transplantation (OLT) survival between patients with recurrent hepatocellular carcinoma (HCC) after partial hepatectomy and those who received de novo OLT for HCC and to assess the risk factors associated with post-OLT mortality. METHODS: From July 2003 to August 2005, 77 consecutive HCC patients underwent OLT, including 15 patients with recurrent HCC after partial hepatectomy for tumor resection (the rescue OLT group) and 62 patients with de novo OLT for HCC (the de novo OLT group). Thirty-three demographic, clinical, histological, laboratory, intra-operative and post-operative variables were analyzed. Survival was calculated by the Kaplan- Meier method. Univariable and multivariable analyses were also performed. RESULTS: The median age of the patients was 49.0 years. The median follow-up was 20 mo. Three patients (20.0%) in the rescue OLT group and 15 patients (24.2%) in the de novo OLT group died during the follow-up period (P = 0.73). The 30-day mortality of OLT was 6.7% for the rescue OLT group vs 1.6% for the de novo OLT group (P = 0.27). Cox proportional hazards model showed that pre-OLT hyperbilirubinemia, the requirement of post-OLT transfusion, the size of the tumor, and family history of HCC were significantly associated with a higher hazard for mortality. CONCLUSION: There are no significant differences in survival/mortality rates between OLT as de novo therapy and OLT as a rescue therapy for patients with hcc. Pre-OLT hyperbilirubinemia, post-OLT requirement of transfusion, large tumor size and family history of HCC are associated with a poor survival outcome.

Orthotopic liver transplantation for giant liver haemangioma: A case report

In liver haemangiomas, the risk of complication rises with increasing size, and treatment can be obligatory. Here we present a case of a 46-year-old female who suffered from a giant haemangioma causing severe portal hypertension and vena cava compression, leading to therapy refractory ascites, hyponatremia and venostasis-associated thrombosis with pulmonary embolism. The patients did not experience tumour rupture or consumptive coagulopathy. Surgical resection was impossible because of steatosis of the non-affected liver. Orthotopic liver transplantation was identified as the only treatment option. The patient's renal function remained stable even though progressive morbidity and organ allocation were improbable according to the patient's lab model for end-stage liver disease(lab MELD) score. Therefore, non-standard exception status was approved by the European organ allocation network "Eurotransplant". The patient underwent successful orthotopic liver transplantation 16 mo after admission to our centre. Our case report indicates the underrepresentation of morbidity associated with refractory ascites in the lab MELD-based transplant allocation system, and it indicates the necessity of promptly applying for non-standard exception status to enable transplantation in patients with a severe clinical condition but low lab MELD score. Our case highlights the fact that liver transplantation should be considered early in patients with non-resectable, symptomatic benign liver tumours.

LIVER TRANSPLANTATION FOR HEPATIC CANCER

Controversy still exists in the liver transplantation as one of the surgical treatments for hepatic cancer.Of the 14cases of orthotopic liver transplantation performed in our institute since 1977,12 cases were primary liver cancer(PLC).No intraoperative and immediate postoperative death occured.Four cases survived for over 3 months,in whom 2 survived for over half a year,and 1 nearly 9 months.The main causes of death were liver failure,rejection, tumor recurrence and infection.The results and the indication of liver transplantation for PLC are discussed.The liver cancer classification more suitable for PLC and selection of the surgical treatment are suggested. It is hold that the liver transplantation is justified for the PLC at stageⅠ,type B and stageⅡ.The reasons why the results of the liver transplantation were poor in China and in our institute are analyzed.It is believed that better results will be gained by means of more strict case selection,immprovement of surgical technique and Cyclosporin A(CsA)application.

Targeting Effect Study of ￣(3) H-Mitoxantrone Nanosphereson Hepatocellular Carcinoma(HCC) Model in Nude Mice

The distribution of ￣(3)H-mitoxantrone polybutyl cyanoacrylate nanospheres(￣(3)H-DHAQ-PBCA-NS)in the viscera,muscle and tumors of human hepatocellular carcinoma (HCC)model in nude mice was studied with liquid scintillation counting techniique. The results showed that the ￣(3)H-DHAQ-PBCA-NS had remarkable liver targeting effect. The content of ￣(3)H-DHAQ-PBCA-NSin liver and heterotopic liver tumor was found to be 71.31±10. 49% of total amount of drug in animal body. It was also found that the content of ￣(3)H-DHAQ-PBCA-NS in liver was higher than that in liver tissue, and the content of ￣(3)H-DHAQ-PBCA-NS in annpit tumor was higher than that in armpit muscle tissue,but had no significant difference;It provides an ideal preparation for the DHAQ admini-stration.

Post-operative imaging in liver transplantation: State-of-the-art and future perspectives

Orthotopic liver transplantation(OLT)represents a major treatment for end-stage chronic liver disease,as well as selected cases of hepatocellular carcinoma and acute liver failure.The ever-increasing development of imaging modalities significantly contributed,over the last decades,to the management of recipients both in the pre-operative and post-operative period,thus impacting on graft and patients survival.When properly used,imaging modalities such as ultrasound,multidetector computed tomography,magnetic resonance imaging(MRI)and procedures of direct cholangiography are capable to provide rapid and reliable recognition and treatment of vascular and biliary complications occurring after OLT.Less defined is the role for imaging in assessing primary graft dysfunction(including rejection)or chronic allograft disease after OLT,e.g.,hepatitis C virus(HCV)recurrence.This paper:(1)describes specific characteristic of the above imaging modalities and the rationale for their use in clinical practice;(2)illustrates main imaging findings related to post-OLTcomplications in adult patients;and(3)reviews future perspectives emerging in the surveillance of recipients with HCV recurrence,with special emphasis on MRI.

BUILDING OF A SIMPLE MODEL OF AUTO-OLT IN DOGS AND ITS SIGNIFICANCE

AIM: we recommend a method of Simple auto-oLT model in dogs.METHODS:The model was ligated all ligaments or connective tissues of the liver,only reserved the vascular construction,that was the suprahepatic and infrahepatic inferior vena cava,portal vein,hepatic artery or common bile duct.the operation was similar to the orthotopic liver transplantation except vascular anastomoses,the dog liver underwent the warm or cold ischemia and the reperfusated injurous process.RESULTS: The imitability was exactly good and the operation was simple and safe. Because the hepatic vessels of the going out or coming in was clamped block and might open or blind the blood flow whenever necessary,the model might control the warm or cold ischemic time accurately,and eliminate the influence or the complications due to vascular anastomoses.CONCLUSIONS: The model avoided many-sided Influences of the traditional OLT and was a good method to study hepatic artery or portal vein ischemic injury and created a new way to explore the pathogenesis or some complications in the OLT.

不同方法建立小鼠肝癌原位移植瘤模型差异性的探讨

目的 比较肿瘤细胞注射和肿瘤组织块移植方法建立小鼠肝癌原位模型的差异,为小鼠肝癌原位模型的建立提供技术参考。方法 取健康雄性KM小鼠,随机分为4组:A组注射小鼠肝癌H22细胞;B组注射腹水型肝癌H22细胞;C组采用小鼠肝癌组织块移植法;D组肝部注射生理盐水作为假手术组。定期观察各组小鼠活动情况、体重变化。记录4组小鼠的生存时间。观察各组小鼠肝部成瘤状况、肿瘤程度、与腹腔脏器粘连程度和转移情况,并进行B超成像,检测血清中甲胎蛋白(alpha-fetoprotein, AFP)、异常凝血酶原(des-gamma-carboxyprothrombin, DCP)的浓度及苏木素-伊红(HE)染色观察肝组织病理学变化。结果 A组、B组、C组小鼠造模操作时间分别为(3.36±0.44)min、(3.30±0.41)min、(5.68±0.65)min。建模第25天,A组、B组、C组小鼠成瘤率均100.0%,A组腹腔重度粘连率为40.0%,B组腹腔重度粘连率为60.0%,C组与D组均未出现重度腹腔粘连。A组、 B组、C组小鼠腹水出现率分别为40.0%、100.0%、0.0%;腹壁瘤出现率分别为30.0%、60.0%、0.0%。B组小鼠还存在肝转移情况(40.0%)。B超成像、血清AFP和DCP水平检测、组织病理学检查结果显示,与D组小鼠相比,A组、B组、C组小鼠肝边缘均不光滑,回声欠均匀,可见稍低回声包块;保持AFP、DCP高分泌;存在大量炎症细胞与肿瘤细胞。结论 在第25天时,3种方法均可以建立肝癌原位移植模型,其中,相较于肝癌组织块移植,肿瘤细胞注射具有操作简单、肝内存在多个转移结节的特点。而肝癌组织块移植相较于肿瘤细胞注射,则具有对腹腔等脏器影响较小等特点。

人肝肿瘤细胞的裸小鼠原位癌建模条件优化及评价

目的优化通过注射人肝肿瘤细胞株构建原位癌裸小鼠模型的条件,并探索适宜的给药治疗时间。方法选用稳定表达萤光素酶报告基因(LUC)的人肝细胞癌Hep3B与肝母细胞瘤HepG2细胞株,使用小动物活体成像系统分析萤光素酶发光强度与肝肿瘤细胞数量之间的线性相关性,验证人源肝肿瘤细胞的发光效率。在5周龄雌性BALB/c裸小鼠的肝叶原位接种不同浓度(8×10^(6)、2.4×10^(7)、7.2×10^(7)个/mL)、不同重悬介质(PBS、Matrigel)的人肝肿瘤细胞悬液HepG2-LUC和Hep3B-LUC(共12组,每组7只),分别构建人肝肿瘤裸小鼠原位癌模型。每7 d为1个周期记录各组小鼠体重,用小动物活体成像系统定期监测原位肿瘤的生长过程,观察肿瘤生长趋势。接种肿瘤细胞后第35天剖取小鼠肝脏,制备病理切片,进行苏木精-伊红(hematoxylin and eosin,HE)染色观察组织病理学变化。结果两种人肝肿瘤细胞株的发光强度均与细胞数量呈正相关(R^(2)=0.9831,R^(2)=0.9705),适宜用于原位癌模型的构建。HepG2-LUC高浓度组,HepG2-LUC+Matrigel低、中、高浓度组,Hep3B-LUC中、高浓度组与Hep3B-LUC+Matrigel低、中、高浓度组均成功造模。HepG2-LUC+Matrigel高浓度组较低浓度与中浓度组小鼠的体重显著下降(P<0.05),Hep3B-LUC+Matrigel高浓度组较低浓度与中浓度组小鼠的体重也显著下降(P<0.05)。成功造模组小鼠的荧光发光强度随时间呈指数型增长(R^(2)>0.9500),且在移植后14 d发光强度至少可达到1.0×10^(7) p/(s·cm^(2)·sr)。HepG2-LUC低、中浓度组和Hep3B-LUC低浓度组小鼠肝脏未见明显的病理学变化,其余组肝脏肿瘤和肝细胞病变明显。结论对于HepG2-LUC细胞株,推荐肝叶原位注射2.4×10^(7)个/mL(50μL)且与Matrigel重悬的混合细胞液体造模,并于造模后第7天给药或采取预后措施;而对于Hep3B-LUC细胞株,推荐肝叶原位注射7.2×10^(7)个/mL(50μL)(不与Matrigel重悬混合)造模,并于造模后的第14天给药或采取预后措施。

小鼠肺原位移植瘤及淋巴结转移模型的建立

目的建立一种小鼠肺原位移植瘤及淋巴结转移模型,模拟肺癌发生发展过程中淋巴结转移,进行肺癌转移前微环境中免疫细胞的研究。方法培养小鼠Lewis肺癌细胞,经胸壁细胞注射法构建小鼠肺原位移植瘤模型,分别于肿瘤移植后第7、14、21和28天取材,苏木素-伊红(HE)染色和免疫组化法(immunocytochemistry,IHC)观察原位移植瘤、淋巴结组织结构及B细胞变化。结果HE染色观察到随着肺癌的发展,淋巴结内生发中心、淋巴管及血管增多,发生肿瘤转移的淋巴结增大变形并有肿瘤细胞浸润。IHC结果同样表明原位肺癌模型小鼠淋巴结B细胞密度增加。结论成功构建C57BL/6J小鼠原位肺癌模型,该模型的建立为今后肺癌淋巴结转移前微环境中免疫细胞的相关研究奠定了基础。

Methodologies for the establishment of an orthotopic transplantation model of ovarian cancer in mice

This study used different methods to establish an animal model of orthotopic transplantation for ovarian cancer to provide an accurate simulation of the mechanism by which tumor occurs and develops in the human body. We implanted 4T1 breast cancer cells stably-transfected with luciferase into BALB/c mice by using three types of orthotopic transplantation methodologies： （1） cultured cells were directly injected into the mouse ovary; （2） cell suspension was initially implanted under the skin of the mouse neck; after tumor mass formed, the tumor was removed and ground into cell suspension, which was then injected into the mouse ovary; and （3） a subcutaneous tumor mass was first generated, removed, and cut into small pieces, which were directly implanted into the mouse ovary. After these models were established, in vivo luminescence imaging was performed. Results and data were compared among groups. Orthotopic transplantation model established with subcutaneous tumor piece implantation showed a better simulation of tumor development and invasion in mice. This model also displayed negligible response to artificial factors. This study successfully established an orthotopic transplantation model of ovarian cancer with high rates of tumor formation and metastasis by using subcutaneous tumor pieces. This study also provided a methodological basis for future establishment of an animal model of ovarian cancer in humans.

神经母细胞瘤肝及肾上腺移植模型比较

目的 建立神经母细胞瘤(neuroblastoma, NB)肝移植模型,并与肾上腺原位移植模型进行比较,探索模型的特征。方法 采用微量注射针将含有5×10^(5)(SK-N-SH)细胞溶液沿肝叶长轴种植于小鼠肝左叶,造模结束后检测肿瘤的生长、转移情况、相关基因表达及肿瘤组织病理学变化。结果 小鼠接种肿瘤细胞21 d后成瘤率达100%,肿瘤的生长、转移、相关基因表达及病理特征明显。结论 本文通过更为简单的手术造模方法,成功构建了一种神经母细胞瘤肝移植模型,为NB研究者今后科学实验提供了更多理想的模型选择。

两套袖法大鼠原位肝移植模型的建立及改进

目的:改进两套袖法大鼠原位肝移植模型,研究其手术成功率和远期存活率.方法:在Kamada两套袖法基础上,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,胆管吻合等手术方法作了进一步改进.结果:改进方法共施行手术80次,与以往92次传统手术法相比,供体手术时间(24.3±3.2)min和受体手术时间(56.4±5.0)min缩短(P<0.05),48h存活率91.3%(73/80)和1wk存活率87.5%(70/80)明显提高(P<0.05).结论:改进的两套袖法大鼠原位肝移植能明显提高供肝的质量和受体手术成功率.

蚯蚓纤溶酶抗肝癌转移作用的实验研究

目的探讨蚯蚓纤溶酶(earthworm fibrinolytic enzyme,EFE)对人肝癌细胞转移的影响。方法建立裸鼠人肝癌高转移原位移植瘤模型,造模后7d将裸鼠随机分为模型对照组和EFE高、低剂量组,共3组,每组7只,每天分别给予生理盐水和1600,800uku/kgEFE灌胃,连续30d。实验结束时完整剥除种植瘤并称重;肉眼直接观察种植瘤的肝内播散及腹腔种植情况,计算肝内播散率及腹腔种植率;通过病理切片观察肺转移肿瘤灶的数目;采用RT-PCR及Western blot方法检测移植瘤中FAK(focus adhesion kinase)及β1-整合素蛋白的表达。结果与模型对照组相比,EFE低、高剂量组原位种植瘤瘤重减轻(P<0.05或P<0.01);种植瘤肝内播散率和腹腔种植率降低,其中,EFE高剂量组与模型对照组比较,具有显著性差异(P<0.05);肺转移灶数目明显减少,与模型对照组相比,具有显著性差异(P<0.05或P<0.01)。在原位种植瘤和移植瘤中FAK及β1-整合素在mRNA及蛋白水平的表达方面,EFE高、低剂量组的表达均明显下降,与模型对照组比较具有非常显著性差异(P<0.01)。结论EFE具有抗肝癌转移的作用,其机理可能与EFE能够抑制FAK及β1-整合素的表达有关。

150例大鼠原位肝移植手术方法比较

目的：探索大鼠原位肝移植最佳手术方法与模型。方法：采用雄性ＳＤ大鼠３００只，分三袖套法（ＴＣ）、改良三袖套法（ＭＴＣ）和二袖套法，即缝合肝上下腔静脉法（ＡＳＶＣ）行原位肝移植１５０次。结果：各组无肝期：ＴＣ组（１５．５±５．５）ｍｉｎ；ＭＴＣ组（１３．５±３．５）ｍｉｎ；ＡＳＶＣ组（２１±４．０）ｍｉｎ。手术时间：ＴＣ组（９０±２５）ｍｉｎ；ＭＴＣ组（８０±１８）ｍｉｎ；ＡＳＶＣ组为（９５±２０）ｍｉｎ。术后血管并发症发生率：ＴＣ组为４２％，ＭＴＣ组为５６％，ＡＳＶＣ组为６％。２４ｈ存活率：ＴＣ组为４６％，ＭＴＣ组为２０％，ＡＳＶＣ组为７０％。结论：二袖套法可作为大鼠肝移植首选手术方法。

LJH-OS人骨肉瘤裸鼠原位移植模型的建立及其生物学特性

用人成骨肉瘤细胞系LJH- OS的传代移植瘤组织作为移植材料,进行胫骨原位移植及皮下移植。结果发现胫骨原位移植的潜伏期较短,生长快。皮下移植瘤呈局限性膨胀性生长,有不完整的纤维包膜,未见肺转移,观察7 周无明显消瘦;而胫骨原位移植瘤浸润基层,无纤维包膜,且发生肺转移,7 周时有明显消瘦。原位移植的裸鼠血清ALP水平高于皮下移植者。说明裸鼠胫骨微环境较皮下组织更适于人骨肉瘤的浸润及转移表达,裸鼠胫骨原位移植模型的恶性生物学行为更接近临床骨肉瘤患者的实际情况,该原位移植模型的建立为骨肉瘤的研究提供了良好的实验模型。

工程化人胃癌裸鼠原位模型的建立及其活体荧光成像观察

目的建立工程化人胃癌裸鼠原位模型,并利用活体荧光成像技术进行观察。方法以胶原为支架材料,表达绿色荧光的人胃癌BGC823-EGFP细胞为种子细胞,建立人胃癌体外模型;倒置显微镜观察肿瘤细胞生长状态;通过注射将体外模型移植到裸鼠体内;活体荧光成像评估胃癌原位模型的建立;解剖观察原位肿瘤的移植成功率,生长及转移情况;冰冻切片及HE染色进行组织学观察。结果工程化人胃癌体外模型中肿瘤细胞呈立体生长;原位肿瘤成瘤率100%,可见腹腔重要脏器转移;组织学特征呈典型低分化腺癌;活体荧光观察可见原位肿瘤形成,但效果不理想。结论建立了工程化人胃癌裸鼠原位模型;绿色荧光成像效果较差,活体荧光成像观察应选用穿透力较强的荧光。

“二袖套法”制备大鼠原位肝移植模型的方法改进

目的在Kamada“二袖套法”的基础上对大鼠肝移植手术方法进行改进,提高大鼠肝移植模型的稳定性和动物存活率。方法在Kamada“二袖套法”大鼠肝移植基础上对供肝切取、受体手术及术中管理进行改进行大鼠肝移植60例,Kamada“二袖套法”行大鼠肝移植20例。比较两组大鼠肝移植术后并发症的发生率和1周生存率。结果在Kamada“二袖套法”基础上改进的肝移植大鼠并发症发生率较少,1周存活率91.7%,经典Kamada“二袖套法”肝移植大鼠1周存活率70%。结论改进的大鼠原位肝移植术操作简便,手术成功率高,模型稳定性好。