雷帕霉素等免疫抑制剂影响肝癌生长及复发转移的实验和临床研究
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摘要
原发性肝癌是亚洲及非洲大陆最常见的恶性肿瘤之一，手术切除是其首选治疗方法。但由于肝癌多合并伴有不同程度的肝硬化，使手术切除受到极大的限制，在很多情况下肝移植成为治愈肝癌的唯一选择。从某种程度来说，肝移植也可能是治疗肝癌的最佳手段之一，因为肝移植能最大限度的切除肿瘤及移除硬化的肝脏，从根本上消除肝癌产生的土壤，预防肿瘤的复发；同时可避免出现肝切除术后肝功能衰竭等致死性并发症。
由于供肝的短缺，肝癌肝移植术后肿瘤的转移复发引起了人们格外的关注，对其疗效的评价也更为谨慎。既往认为，肝癌肝移植术后免疫抑制剂的应用可能促进了术后肿瘤的转移复发；但事实上，并不是每一种免疫抑制剂都会促进肿瘤的生长。不同免疫抑制方案对肝癌肝移植术后肿瘤的转移复发可能有不同的影响，但目前此方面的系统研究还较少。在此，我们将探讨三种主要的免疫抑制剂(他克莫司，雷帕霉素和环孢素)对肝癌生长及转移复发的影响及相关机制，为肝癌肝移植术后免疫抑制方案的选用提供可借鉴的依据。
第一部分：雷帕霉素等免疫抑制剂对肝癌生长及转移的实验研究
体内试验中，利用复旦大学肝癌研究所(以下简称我所)建立的高转移人肝癌裸鼠模型LCI-D20，探讨目前临床上常用的三种免疫剂(他克莫司，雷帕霉素和环孢素)对裸鼠移植瘤生长及自发肺转移的影响。用免疫组化及TUNEL法分别检测移植瘤组织中细胞增殖核抗原(PCNA)、微血管密度(MVD)及细胞调亡水平的改变。结果表明，与对照组相比，雷帕霉素抑制了LCI-D20模型移植瘤的生长(瘤重0.76+0.38g vs．2.09+0.75g，P=0.001；肿瘤体积0.65±0.31 cm~3 vs．2.11±1.02 cm~3，P=0.003)及肺转移的发生(3/7 vs．7/7，P=0．007)；环孢素组肺部转移灶的数目较对照组明显增多(6±2 vs．4±1，P=0.046)；他克莫司组肺转移发生率虽较对照组减少，但无显著性差异(5/7 vs．7/7，P=0.078)；环孢素及他克莫司均对移植瘤的生长无影响(P＞0.05)。进一步的研究表明，与对照组相比，雷帕霉素抑制了移植瘤组织中PCNA的表达(免疫组化阳性指数244932.2±20998.6 vs．252766.7±29221.4，P=0.029)；降低了肿瘤组织中的微血管密度(29±5 vs．37±4，P=0.009)；同时促进了肿瘤细胞的调亡(25.3％±2.7％vs．13.9％±2.6％，P=0.000)；而他克莫司及环孢素对上述三方面均无显著影响。
体外实验中，采用扫描电镜、肌动蛋白聚合实验、MTT、流式细胞仪、Boyden小室、明胶酶谱法分别研究各免疫抑制剂对人肝癌高转移细胞株MHCC97H增殖、凋亡、细胞周期、运动、黏附及侵袭的影响。用real-time PCR、western blot等方法探讨免疫抑制剂对与肿瘤进展相关的分子，如E-cadherin，、ICAM-1，、MMP-2等表达的影响。结果表明，与对照组相比，环孢素促进了MHCC97H细胞侵袭性表型的改变，运动、侵袭能力的增强及F型肌动蛋白的聚合。另外，环孢素还促进了细胞及肿瘤组织中MMP-2的表达。雷帕霉素则抑制了MHCC97H细胞的增殖，并使细胞周期停滞于G0-G1期，但并未促进细胞调亡。雷帕霉素及他克莫司均抑制了MHCC97H细胞的运动、侵袭和黏附，同时两者还抑制了ICAM-1和MMP-2在细胞及肿瘤组织中的表达。
从上述结果中我们可以得到初步的结论：环孢素促进了肝癌细胞的转移；他克莫司至少不促进肝癌细胞的转移；而雷帕霉素则抑制了肝癌细胞的增殖及转移。
第二部分：雷帕霉素抗肝癌血管形成机制的初步研究
前述的研究发现，雷帕霉素抑制肿瘤血管形成是其抑制移植瘤生长与肺转移的重要机制之一。然而，雷帕霉素抗肝癌血管形成的具体分子机制仍未得到阐明。在此，我们先用基因芯片技术进行初步地筛选，接着用实时定量荧光PCR、Western blot和免疫组化来进一步验证，结果发现雷帕霉素抑制了移植瘤组织中VEGF、HIF-1a和Angiopoietin-2的表达。体外实验中，用CoCl_2模拟缺氧环境，发现随着时间的推移，雷帕霉素抑制了MHCC97H细胞中VEGF及HIF-1a的表达，且两者随时间变化的趋势是完全一致的。而HIF-1α是目前公认的能与VEGF基因启动子结合并调节VEGF表达的细胞内转录因子。据此推测，雷帕霉素下调VEGF的表达可能是通过下调HIF-1α的表达实现的。我们的结果亦提示，雷帕霉素抑制肝癌细胞中VEGF蛋白及Ang-2蛋白的分泌，可能是其抗肝癌血管生成的重要机制之一。
在雷帕霉素与干扰素α的联合干预实验中发现，对照组、雷帕霉素治疗组(2mg/kg/day)、干扰素α治疗组(1×10~6U/kg/day)和联合用药组的肿瘤体积分别为：4.10cm~3±0.58cm~3、1.82cm~3±0.90cm~3、0.70cm~3±0.39cm~3和0.20cm~3±31cm~3；肺转移发生率分别为：100％、83.3％、50％和0％。联合用药组的抑瘤效果较干扰素α治疗组明显增强(P=0.000)，但与雷帕霉素治疗组相比无显著性差异(P=0.128)。与单独用药组相比，联合用药组明显抑制了肿瘤肺转移的发生(与雷帕霉素组比较P=0.023，与干扰素α组比较P=0.000)。因此，雷帕霉素与干扰素α合用在抗肿瘤方面具有协同效应。目前干扰素α已被用于治疗肝移植术后丙肝的复发，可以预见，两者合用在肝移植术后、尤其是在肝癌肝移植术后用于预防
肿瘤转移复发方面可能有一定的临床应用前景。
第三部分：雷帕霉素等免疫抑制剂对荷瘤肝移植大鼠肿瘤生长及肝癌肝移植大鼠术后肿瘤转移复发的实验研究
我们已在本文第一、第二部分中详细探讨了雷帕霉素、环孢素和他克莫司三种免疫抑制剂在联合免疫缺陷裸鼠体内对肿瘤生长的影响。在这部分，我们将进一步探讨这三种免疫抑制剂在免疫功能正常的大鼠体内对肿瘤生长的影响。
在第一组模型中，以Wistar大鼠作为供体，SD大鼠作为受体，建立急性排斥大鼠肝移植模型，术后即开始服用免疫抑制剂：雷帕霉素(1mg/kg/d，灌胃)、他克莫司(1mg/kg/d，灌胃)、环孢素(20mg/kg/d，灌胃)；术后3天，受体大鼠左肩胛区皮下注射腹水瘤Wlaker-256细胞。同法建立同基因肝移植大鼠(以SD大鼠作为供体及受体)皮下荷瘤模型，以生理盐水灌胃作对照，每周3次记录各组受体大鼠皮下肿瘤的生长情况，绘制成瘤曲线；并于皮下种植肿瘤术后14天处死大鼠，检测受体大鼠的肝功能、移植肝病理、脾细胞的NK活性及皮下肿瘤大小。
在第二组模型中，我们将大鼠皮下Wlaker-256瘤块接种于SD大鼠肝左叶上，建立移植性大鼠肝癌模型。造模成功后14天再行原位肝移植术(以SD大鼠作为供体及受体)，术后应用不同的免疫抑制剂(用法同上)，以生理盐水灌胃作对照，观察肝癌肝移植术后各组大鼠肿瘤转移复发的情况。
结果表明，与急性排斥大鼠相比，第一模型组中的受体大鼠在应用各免疫抑制剂后肝功能明显改善、移植物未出现严重的急性排斥反应、脾细胞的NK活性均受到明显的抑制；同时，与生理盐水对照组相比，雷帕霉素明显抑制了大鼠皮下肿瘤的生长(1.41±0.87cm~3 vs．3.65±0.87cm~3，P=0.047)，环孢素及他克莫司则促进了皮下肿瘤的生长(9.56±2.81cm~3 vs．3.65±0.87cm~3，P=0.000；8.11±1.69cm~3 vs．3.65±0.87cm~3，P=0.001)。在第二组模型中，肝癌肝移植大鼠术后分别接受了生理盐水、环孢素、他克莫司和雷帕霉素的治疗，各组大鼠肝内肿瘤的复发率分别为：0％、20％、16.7％、0％；肺转移的发生率分别为：42.9％、60％、33.3％、0％。与对照组相比，雷帕霉素抑制了肝癌肝移植大鼠术后肺转移的发生；术后肝内复发灶的出现仅见于他克莫司及环孢素组。
综上所述，雷帕霉素在有效的保护移植物的同时抑制了移植受体体内肿瘤的生长；环孢素和他克莫司虽然抑制了机体的排斥反应，但也同时促进了受体体内肿瘤的生长。
第四部分：我所134例肝癌肝移植的临床回顾分析及雷帕霉素的临床应用评价
为了了解影响肝癌肝移植预后的一些重要因素及评价雷帕霉素在肝癌肝移植中的应用价值，我们回顾分析了复旦大学肝癌研究所从2004年1月到2005年10月共134例肝癌肝移植的临床资料(包括年龄、性别、肝炎病史、术前Child-Pugh分级、术中输血量、AFP、肿瘤大小、数目、包膜、微血管侵犯(MVI)、肿瘤分化程度、是否符合Milan标准及术后免疫抑制方案等)，和术后随访资料(包括肿瘤转移复发情况、生存情况等)。
134例肝癌肝移植患者术后1年、2年累积生存率分别为82.75％±3.47％、68.80％±7.07％，1年、2年累积无瘤生存率分别为69.13％±4.42％、41.33％±17.20％。单因素分析发现，术前Child-Pugh评分(P=0.000)、术中输血量(P=0.037)、肿瘤大小(P=0.032)、MVI(P=0.019)、是否符合Milian标准(P=0.007)是影响肝癌肝移植患者生存率的重要因素；术前Child-Pugh评分(P=0.009)、肿瘤数目(P=0.041)、肿瘤大小(P=0.002)、MVI(P=0.000)、pTNM分期(P=0.001)、是否符合Milian标准(P=0.000)是影响肝癌肝移植患者无瘤生存率的重要因素。多因素分析发现，术前Child-Pugh评分(P=0.000)、是否符合Milian标准(P=0.001)是影响肝癌肝移植患者生存率的独立预后因素；术前Child-Pugh评分(P=0.009)、是否符合Milian标准(P=0.004)、MVI(P=0.004)是影响肝癌肝移植患者无瘤生存率的独立预后因素。
以是否符合Milan标准为依据，将134例肝癌肝移植患者分为2组。在符合Milan标准的61例肝癌肝移植患者中，多因素分析发现，术前Child-Pugh评分(P=0.043)是影响患者生存率的独立预后因素；MVI(P=0.014)是影响患者无瘤生存率的独立预后因素。在不符合Milan标准的73例肝癌肝移植患者中，与术后使用以他克莫司为基础的免疫抑制方案的患者(46例)相比，术后应用以雷帕霉素为基础的免疫抑制方案的患者(27例)其生存率(P=0.011)及无瘤生存率(P=0.037)均明显提高，且复发时间明显延迟(263.0±100.0天vs．140.1±123.5天，P=0.035)；单因素及多因素分析均提示，术后使用的免疫抑制方案是影响患者预后的重要因素；服用雷帕霉素的患者随访期间出现急性排斥反应5例、血小板减少2例、贫血6例、口腔溃疡7例，均得到及时的治疗而痊愈。
因此我们可得出以下的结论，对于不符合Milan标准的肝癌肝移植患者，术后转换为以雷帕霉素为基础的免疫抑制方案可能有助于提高患者的生存期，同时患者对雷帕霉素也有良好的耐受性。对于肝癌肝移植术后肿瘤转移复发的预测，术前只能依据一些影像学的资料(如肿瘤的大小、数目等)来判断，术后则必须依据病理情况(如MVI等)重新修正原有的判断，制定治疗方案。同时，肝癌肝移植患者的预后不仅与肿瘤情况有关，与患者手术时的肝功能也密切相关。
Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Asia and Africa. Although the first-line surgical therapy for HCC is liver resection, the concomitant cirrhosis most often leaves liver transplantation (LT) and not liver resection as the only potentially curative option. Live transplantation may also be the best curative treatment for HCC since it removes the tumor with the widest margin as well as the underlying cirrhosis that is responsible for both postoperative hepatic decompensation and tumor recurrence after partial hepatectomy.
Because of the scarcity of donor livers, there is low tolerance within the organ allocation system for posttransplantation HCC recurrence and metastasis. Although it is known that the pharmacologic immunosuppression required after liver transplantation for HCC may be accelerated tumor recurrence and metastasis, recent reports suggest that not all immunosuppressive drugs necessarily promote HCC recurrence in transplant recipients. However, the possible influence of different immunosuppressive schedules on HCC recurrence and metastasis had been poorly investigated until recently. In the following study, we will focus the discussion on the pro- and anti-recurrence properties and mechanics of three key immunosuppressive drugs (FK506, rapamycin, and cyclosporine) on HCC. The results may provide evidence for immunosuppressive protocols introduction after OLT for HCC.
Section I: Experimental studies of rapamycin and other immunosuppressive agents on growth and metastasis of hepatocellular carcinoma
The effects of three immunosuppressive agents (rapamycin, FK506, and cyclosporine) on tumor growth and metastasis were investigated in LCI-D20 nude mice model with a highly-potentially human hepatocellular carcinoma. Immunohistochemistry and terminal deoxynucleotidyl transferas-mediated dUTP nick end labeling (TUNEL) assay were used to detect the levels of PCNA, microvessel
density (CD31 stained) and tumor cell apoptosis in vivo, respectively.
Three immunosuppressive agents effects on phenotypic and cytoskeleton changes, proliferation, apoptosis, cell cycles, migration, adhesion, and invasiveness on MHCC97H cell line were also studied in vitro by scanning electron microscopy, F-actin polymerization, MTT, flow cytometry, Boyden chamber methods, and gelatin zymograph, respectively. And the expression of molecules (E-cadherin, ICAM-1, and MMP-2) implicated in tumor progression were analyzed by real-time PCR, western blot analysis in vitro, and immunohistochemistry, ELISA in vivo, respectively.
The results showed that, in vivo, rapamycin inhibited tumor growth and lung metastasis compared with control in nude mice (0.76±0.38 g vs. 2.09±0.75 g, P=0.001, 3/7 vs. 7/7, P=0.007). The number of metastatic nodules in lung was increased in cyclosporine group as compared with control group (6±2 vs. 4±1, P=0.046). There was no significant difference between FK506 and control in lung metastasis rate (5/7 vs. 7/7, P=0.078). Rapamycin but not FK506 or cyclosporine inhibited tumor cell proliferation, induced apoptosis, and decreased tumor angiogenesis in LCI-D20 model.
In vitro, treatment of MHCC97H resulted in striking morphological alterations in cyclosporine group, including numerous pseudopodial protrusions, increased cell motility, and metastasis abilities. Confocal laser scan microscopy of MHCC97H cells stimulated in cyclosporine showed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. Cyclosporine could also evidently increase the secretion of MMP-2 by MHCC97H and tumor cells in LCI-D20 model. Rapamycin blocked proliferation of MHCC97H and induced cell cycle arrest at the G1 checkpoint, but did not induce apoptosis. FK506 and rapamycin inhibited the ability of migration, adhesion, and invasiveness of MHCC97H. They also resulted in a significant decrease in the levels of expression of ICAM-1 and MMP-2.
Our findings suggest that cyclosporine can promote hepatocellular carcinoma cell progression by a direct cellular effect, FK506 may not promote hepatocellular carcinoma cell invasiveness, and rapamycin seems to inhibit the growth, motility and invasiveness of hepatocellular carcinoma cell.
Section II. Preliminary study on the molecular mechanisms of antiangiogenic effects of rapamycin in hepatocellular carcinoma
The first section demonstrated that rapamycin inhibited tumor growth and recurrence in the LCI-D20 xenograft model in nude mice by suppressing tumor angiogenesis. However, the underlying molecular mechanism was not fully elucidated. In this study, a cDNA microarray analysis followed by Real-time PCR, Western blot, and Immunohistochemistry analysis revealed that VEGF, Hypoxia-inducible factor 1 a (HIF-1a) and Angiopoietin-2 might be inhibited by rapamycin in vivo. Then we demonstrated that rapamycin could also downregulate VEGF expression in protein levels in a time-dependent manner, as well as downregulating HIF-1a expression in MHCC97H cells treated with the hypoxia mimetic agent, CoCl_2, in vitro. While HIF-1a is a well characterized regulator of VEGF expression, we may lead to the initial conclusion that rapamycin could suppress VEGF synthesis and secretion by downregulating HIF-1a expression. Our results also suggest that the anti-angiogenesis treatment of rapamycin may be associated with suppression of VEGF and Angiopoietin-2.
In LCI-D20 nude mice model, when comparison was made among control, rapamycin 2mg/kg/day, interferon alpha (IFNα) 1×10~6 U/kg/day, and combined treatment of rapamycin and IFNa for treated groups; tumor volume was 4.10cm~3±0.58cm~3, 1.82cm~3±0.90cm~3, 0.70cm~3±0.39cm~3, and 0.20cm~3±31cm~3, and incidence of lung metastasis being 100%, 83.3%, 50%, and 0%, respectively. The combined treatment did not increase the inhibition effect on the growth of tumor compared with rapamycin group (P=0.128). However, the combined therapy significantly improved the effect on lung metastasis with single treatment with either rapamycin (P=0.023) or IFNa (P=0.000). Considering the fact that IFNa is used in liver transplant patients with chronic hepatitis C and rapamycin acts as a primary immune suppressant, combination therapy of rapamycin plus IFNa has the potential clinical implication, particularly for the prevention of recurrence and metastasis after liver transplantation for hepatocellular carcinoma.
Section HI: Experimental studies on the effects of immunosuppression on tumor growth, recurrence and metastasis in rats
The pro- and anti- tumor effects of three key immunosuppressive drugs (FK506, rapamycin, and cyclosporine) had been discussed in severe combined
immunodeficient BALB/c mice in section I and II. Here we investigated the effects of rapamycin, FK506, and cyclosporine on established tumors in rats simultaneously bearing a liver allograft.
In one tumor-transplant model, SD rats received subcutaneous syngenic walker-256 carcinosarcoma cells 3 days after orthotopic liver transplantation (Wistar →SD) with character of acute rejection. Rapamycin (1 mg/kg/d), FK506 (1 mg/kg/d) or CsA (20 mg/kg/d) was initiated with transplantation. The controls received a similar volume of saline. The clinical characters, the liver function, the transplantated liver pathologic character and the cytotoxicity of spleen mononuclear from recipient against the Yac-1 cells were observed and measured on tumor postimplantative day 14. The treatment and control groups were evaluated for tumor volume thrice every one week.
In a second model system, a transplanted hepatoma model was made in rats by implantation of histologically intact walker-256 tumor fragment into the left lateral lobe of the liver. On the 14th days after implantation, the hepatic tubercle was excised to be examined by histological method to confirm the successful establishment of primary carcinoma of the livers in rats. Then the orthotopic liver transplantation (SD →SD) with character of no acute rejection was performed in different groups. Intrahepatic recurrence and lung metastasis were recorded.
In the first model system, results showed that the function of graft was improved, the cytotoxicity against the Yac-1 cells decreased, and the rejection reaction inhibited in the immunosuppressive treatment groups compared with control groups. The rapamycin treatment group showed a significantly decreased tumor volume (1.41±0.87cm3 vs. 3.65±0.87cm3, P=0.047), and CsA or FK506 group increased tumor size versus controls (9.56±2.81cm3 vs. 3.65±0.87cm3, P=0.000; 8.11±1.69cm3 vs. 3.65±0.87cm3, P=0.001) at the end of treatment.
In the second model system, when comparison was made among control, FK506, rapamycin, and cyclosporine, incidence of recurrent tumor was 0%, 20%, 16.7% and 0%; incidence of lung metastasis being 42.9%, 60.0%, 33.3% and 0%, respectively. The incidence of lung metastasis was decreased in the rapamycin-treated animals, and tumor recurrence in liver was seen only in the cyclosporine-treated and FK506-treated animals.
In conclusion, this study demonstrates that rapamycin simultaneously protects allografts from rejection and attacks tumors in a complex transplant-tumor situation.
Notably, CsA or FK506 protects allografts from rejection, but cancer progression is promoted in transplant recipients.
Section IV: Retrospective analysis of 134 cases of liver transplantation for hepatocellular carcinoma at the Liver cancer institute of Fudan University, china
A total of 134 patients received orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) from January 2004 to October 2005 in authors' institute were enrolled in this study to evaluate the clinical value and prognostic factors of OLT for HCC. Their clinicopathological characteristics [including age, gender, the history of hepatitis, Child-Pugh class, α-fetal protein (AFP), blood transfusion, tumor size, number, capsule, micro-vascular invasion, tumor differentiation, pTNM stage, Milan criteria, and immunosuppressive protocol], and follow-up data [including tumor recurrence, overall (OS) and disease-free survival (DFS) of patients] were retrospectively analyzed.
The 1- and 2-year OS rates of the 134 patients were 82.75%±3.47% and 68.80%±7.07% and the DFS rates were 69.13%±4.42% and 41.33%±17.20%, respectively. Univariate analysis revealed Child-Pugh class (P=0.000), tumor size (P=0.032), micro-vascular invasion (P=0.019), blood transfusion (P=0.037), Milan criteria (P=0.007) were statistically significant factors affecting OS; and Child-Pugh class (P=0.009) , tumor size (P=0.002), tumor number (P=0.041), micro-vascular invasion (P=0.000), Milan criteria (P=0.000), pTNM stage (P=0.001) were statistically significant factors affecting DFS. Multivariate analysis using Cox proportional hazards regression model demonstrated the Child-Pugh class (P=0.000) and Milan criteria (P=0.001) were the independent and statistically significant factors affecting OS; and the Child-Pugh class (P=0.009), Milan criteria (P=0.004), and micro-vascular invasion (P=0.004) were independent and statistically significant factors affecting DFS.
Taking the Milan criteria as a categorized variable, the 134 cases were classified into two subgroups: within the Milan criteria group (n= 61) and beyond group (n=73). In the Milan group, with the Cox regression, only Child-Pugh class (P=0.043) was identified as the independent predictors for OS, only micro-vascular invasion (P=0.014) had independent predictive significance for DFS.
In the beyond Milan criteria group, the patients with the sirolimus-based protocol (n= 27) were significantly associated with a better OS (P=0.011) as well as DFS (P=0.037), and later posttransplant recurrence time (263.0± 100.0 天 vs. 140.1± 123.5 天, P=0.035), compared with the FK506-based patients (n=46). Immunosuppressive protocol after OLT was one of the leading independent prognostic factors for both OS (P=0.015) and DFS (P=0.015) in multivariate Cox models analyses. Acute rejection (n=6), thrombocytopenia (n=3), anemia (n=12), and oral aphthous ulcers (n=7) were found in beyond Milan cohort and were easily manageable.
We can draw the following theoretical considerations based on our results that The sirolimus based immunosuppression protocol appears to have beneficial effects on tumor recurrence and survival with an acceptable rate of rejection and toxicity in beyond Milan criteria group. The risk of recurrence and metastasis after OLT for HCC may be predicted preoperatively using imaging-based criteria (presence of macroscopic vascular invasion and size/number of tumors, which are surrogates for microscopic invasion), and the predictive model may be refined based on information gained on pathologic study of the explanted livers (e.g. microscopic invasion). And the prognosis of these patients depends not only on the stage of the tumor at the time of diagnosis, but also on the degree of deterioration in liver function.
由于供肝的短缺，肝癌肝移植术后肿瘤的转移复发引起了人们格外的关注，对其疗效的评价也更为谨慎。既往认为，肝癌肝移植术后免疫抑制剂的应用可能促进了术后肿瘤的转移复发；但事实上，并不是每一种免疫抑制剂都会促进肿瘤的生长。不同免疫抑制方案对肝癌肝移植术后肿瘤的转移复发可能有不同的影响，但目前此方面的系统研究还较少。在此，我们将探讨三种主要的免疫抑制剂(他克莫司，雷帕霉素和环孢素)对肝癌生长及转移复发的影响及相关机制，为肝癌肝移植术后免疫抑制方案的选用提供可借鉴的依据。
第一部分：雷帕霉素等免疫抑制剂对肝癌生长及转移的实验研究
体内试验中，利用复旦大学肝癌研究所(以下简称我所)建立的高转移人肝癌裸鼠模型LCI-D20，探讨目前临床上常用的三种免疫剂(他克莫司，雷帕霉素和环孢素)对裸鼠移植瘤生长及自发肺转移的影响。用免疫组化及TUNEL法分别检测移植瘤组织中细胞增殖核抗原(PCNA)、微血管密度(MVD)及细胞调亡水平的改变。结果表明，与对照组相比，雷帕霉素抑制了LCI-D20模型移植瘤的生长(瘤重0.76+0.38g vs．2.09+0.75g，P=0.001；肿瘤体积0.65±0.31 cm~3 vs．2.11±1.02 cm~3，P=0.003)及肺转移的发生(3/7 vs．7/7，P=0．007)；环孢素组肺部转移灶的数目较对照组明显增多(6±2 vs．4±1，P=0.046)；他克莫司组肺转移发生率虽较对照组减少，但无显著性差异(5/7 vs．7/7，P=0.078)；环孢素及他克莫司均对移植瘤的生长无影响(P＞0.05)。进一步的研究表明，与对照组相比，雷帕霉素抑制了移植瘤组织中PCNA的表达(免疫组化阳性指数244932.2±20998.6 vs．252766.7±29221.4，P=0.029)；降低了肿瘤组织中的微血管密度(29±5 vs．37±4，P=0.009)；同时促进了肿瘤细胞的调亡(25.3％±2.7％vs．13.9％±2.6％，P=0.000)；而他克莫司及环孢素对上述三方面均无显著影响。
体外实验中，采用扫描电镜、肌动蛋白聚合实验、MTT、流式细胞仪、Boyden小室、明胶酶谱法分别研究各免疫抑制剂对人肝癌高转移细胞株MHCC97H增殖、凋亡、细胞周期、运动、黏附及侵袭的影响。用real-time PCR、western blot等方法探讨免疫抑制剂对与肿瘤进展相关的分子，如E-cadherin，、ICAM-1，、MMP-2等表达的影响。结果表明，与对照组相比，环孢素促进了MHCC97H细胞侵袭性表型的改变，运动、侵袭能力的增强及F型肌动蛋白的聚合。另外，环孢素还促进了细胞及肿瘤组织中MMP-2的表达。雷帕霉素则抑制了MHCC97H细胞的增殖，并使细胞周期停滞于G0-G1期，但并未促进细胞调亡。雷帕霉素及他克莫司均抑制了MHCC97H细胞的运动、侵袭和黏附，同时两者还抑制了ICAM-1和MMP-2在细胞及肿瘤组织中的表达。
从上述结果中我们可以得到初步的结论：环孢素促进了肝癌细胞的转移；他克莫司至少不促进肝癌细胞的转移；而雷帕霉素则抑制了肝癌细胞的增殖及转移。
第二部分：雷帕霉素抗肝癌血管形成机制的初步研究
前述的研究发现，雷帕霉素抑制肿瘤血管形成是其抑制移植瘤生长与肺转移的重要机制之一。然而，雷帕霉素抗肝癌血管形成的具体分子机制仍未得到阐明。在此，我们先用基因芯片技术进行初步地筛选，接着用实时定量荧光PCR、Western blot和免疫组化来进一步验证，结果发现雷帕霉素抑制了移植瘤组织中VEGF、HIF-1a和Angiopoietin-2的表达。体外实验中，用CoCl_2模拟缺氧环境，发现随着时间的推移，雷帕霉素抑制了MHCC97H细胞中VEGF及HIF-1a的表达，且两者随时间变化的趋势是完全一致的。而HIF-1α是目前公认的能与VEGF基因启动子结合并调节VEGF表达的细胞内转录因子。据此推测，雷帕霉素下调VEGF的表达可能是通过下调HIF-1α的表达实现的。我们的结果亦提示，雷帕霉素抑制肝癌细胞中VEGF蛋白及Ang-2蛋白的分泌，可能是其抗肝癌血管生成的重要机制之一。
在雷帕霉素与干扰素α的联合干预实验中发现，对照组、雷帕霉素治疗组(2mg/kg/day)、干扰素α治疗组(1×10~6U/kg/day)和联合用药组的肿瘤体积分别为：4.10cm~3±0.58cm~3、1.82cm~3±0.90cm~3、0.70cm~3±0.39cm~3和0.20cm~3±31cm~3；肺转移发生率分别为：100％、83.3％、50％和0％。联合用药组的抑瘤效果较干扰素α治疗组明显增强(P=0.000)，但与雷帕霉素治疗组相比无显著性差异(P=0.128)。与单独用药组相比，联合用药组明显抑制了肿瘤肺转移的发生(与雷帕霉素组比较P=0.023，与干扰素α组比较P=0.000)。因此，雷帕霉素与干扰素α合用在抗肿瘤方面具有协同效应。目前干扰素α已被用于治疗肝移植术后丙肝的复发，可以预见，两者合用在肝移植术后、尤其是在肝癌肝移植术后用于预防
肿瘤转移复发方面可能有一定的临床应用前景。
第三部分：雷帕霉素等免疫抑制剂对荷瘤肝移植大鼠肿瘤生长及肝癌肝移植大鼠术后肿瘤转移复发的实验研究
我们已在本文第一、第二部分中详细探讨了雷帕霉素、环孢素和他克莫司三种免疫抑制剂在联合免疫缺陷裸鼠体内对肿瘤生长的影响。在这部分，我们将进一步探讨这三种免疫抑制剂在免疫功能正常的大鼠体内对肿瘤生长的影响。
在第一组模型中，以Wistar大鼠作为供体，SD大鼠作为受体，建立急性排斥大鼠肝移植模型，术后即开始服用免疫抑制剂：雷帕霉素(1mg/kg/d，灌胃)、他克莫司(1mg/kg/d，灌胃)、环孢素(20mg/kg/d，灌胃)；术后3天，受体大鼠左肩胛区皮下注射腹水瘤Wlaker-256细胞。同法建立同基因肝移植大鼠(以SD大鼠作为供体及受体)皮下荷瘤模型，以生理盐水灌胃作对照，每周3次记录各组受体大鼠皮下肿瘤的生长情况，绘制成瘤曲线；并于皮下种植肿瘤术后14天处死大鼠，检测受体大鼠的肝功能、移植肝病理、脾细胞的NK活性及皮下肿瘤大小。
在第二组模型中，我们将大鼠皮下Wlaker-256瘤块接种于SD大鼠肝左叶上，建立移植性大鼠肝癌模型。造模成功后14天再行原位肝移植术(以SD大鼠作为供体及受体)，术后应用不同的免疫抑制剂(用法同上)，以生理盐水灌胃作对照，观察肝癌肝移植术后各组大鼠肿瘤转移复发的情况。
结果表明，与急性排斥大鼠相比，第一模型组中的受体大鼠在应用各免疫抑制剂后肝功能明显改善、移植物未出现严重的急性排斥反应、脾细胞的NK活性均受到明显的抑制；同时，与生理盐水对照组相比，雷帕霉素明显抑制了大鼠皮下肿瘤的生长(1.41±0.87cm~3 vs．3.65±0.87cm~3，P=0.047)，环孢素及他克莫司则促进了皮下肿瘤的生长(9.56±2.81cm~3 vs．3.65±0.87cm~3，P=0.000；8.11±1.69cm~3 vs．3.65±0.87cm~3，P=0.001)。在第二组模型中，肝癌肝移植大鼠术后分别接受了生理盐水、环孢素、他克莫司和雷帕霉素的治疗，各组大鼠肝内肿瘤的复发率分别为：0％、20％、16.7％、0％；肺转移的发生率分别为：42.9％、60％、33.3％、0％。与对照组相比，雷帕霉素抑制了肝癌肝移植大鼠术后肺转移的发生；术后肝内复发灶的出现仅见于他克莫司及环孢素组。
综上所述，雷帕霉素在有效的保护移植物的同时抑制了移植受体体内肿瘤的生长；环孢素和他克莫司虽然抑制了机体的排斥反应，但也同时促进了受体体内肿瘤的生长。
第四部分：我所134例肝癌肝移植的临床回顾分析及雷帕霉素的临床应用评价
为了了解影响肝癌肝移植预后的一些重要因素及评价雷帕霉素在肝癌肝移植中的应用价值，我们回顾分析了复旦大学肝癌研究所从2004年1月到2005年10月共134例肝癌肝移植的临床资料(包括年龄、性别、肝炎病史、术前Child-Pugh分级、术中输血量、AFP、肿瘤大小、数目、包膜、微血管侵犯(MVI)、肿瘤分化程度、是否符合Milan标准及术后免疫抑制方案等)，和术后随访资料(包括肿瘤转移复发情况、生存情况等)。
134例肝癌肝移植患者术后1年、2年累积生存率分别为82.75％±3.47％、68.80％±7.07％，1年、2年累积无瘤生存率分别为69.13％±4.42％、41.33％±17.20％。单因素分析发现，术前Child-Pugh评分(P=0.000)、术中输血量(P=0.037)、肿瘤大小(P=0.032)、MVI(P=0.019)、是否符合Milian标准(P=0.007)是影响肝癌肝移植患者生存率的重要因素；术前Child-Pugh评分(P=0.009)、肿瘤数目(P=0.041)、肿瘤大小(P=0.002)、MVI(P=0.000)、pTNM分期(P=0.001)、是否符合Milian标准(P=0.000)是影响肝癌肝移植患者无瘤生存率的重要因素。多因素分析发现，术前Child-Pugh评分(P=0.000)、是否符合Milian标准(P=0.001)是影响肝癌肝移植患者生存率的独立预后因素；术前Child-Pugh评分(P=0.009)、是否符合Milian标准(P=0.004)、MVI(P=0.004)是影响肝癌肝移植患者无瘤生存率的独立预后因素。
以是否符合Milan标准为依据，将134例肝癌肝移植患者分为2组。在符合Milan标准的61例肝癌肝移植患者中，多因素分析发现，术前Child-Pugh评分(P=0.043)是影响患者生存率的独立预后因素；MVI(P=0.014)是影响患者无瘤生存率的独立预后因素。在不符合Milan标准的73例肝癌肝移植患者中，与术后使用以他克莫司为基础的免疫抑制方案的患者(46例)相比，术后应用以雷帕霉素为基础的免疫抑制方案的患者(27例)其生存率(P=0.011)及无瘤生存率(P=0.037)均明显提高，且复发时间明显延迟(263.0±100.0天vs．140.1±123.5天，P=0.035)；单因素及多因素分析均提示，术后使用的免疫抑制方案是影响患者预后的重要因素；服用雷帕霉素的患者随访期间出现急性排斥反应5例、血小板减少2例、贫血6例、口腔溃疡7例，均得到及时的治疗而痊愈。
因此我们可得出以下的结论，对于不符合Milan标准的肝癌肝移植患者，术后转换为以雷帕霉素为基础的免疫抑制方案可能有助于提高患者的生存期，同时患者对雷帕霉素也有良好的耐受性。对于肝癌肝移植术后肿瘤转移复发的预测，术前只能依据一些影像学的资料(如肿瘤的大小、数目等)来判断，术后则必须依据病理情况(如MVI等)重新修正原有的判断，制定治疗方案。同时，肝癌肝移植患者的预后不仅与肿瘤情况有关，与患者手术时的肝功能也密切相关。
Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Asia and Africa. Although the first-line surgical therapy for HCC is liver resection, the concomitant cirrhosis most often leaves liver transplantation (LT) and not liver resection as the only potentially curative option. Live transplantation may also be the best curative treatment for HCC since it removes the tumor with the widest margin as well as the underlying cirrhosis that is responsible for both postoperative hepatic decompensation and tumor recurrence after partial hepatectomy.
Because of the scarcity of donor livers, there is low tolerance within the organ allocation system for posttransplantation HCC recurrence and metastasis. Although it is known that the pharmacologic immunosuppression required after liver transplantation for HCC may be accelerated tumor recurrence and metastasis, recent reports suggest that not all immunosuppressive drugs necessarily promote HCC recurrence in transplant recipients. However, the possible influence of different immunosuppressive schedules on HCC recurrence and metastasis had been poorly investigated until recently. In the following study, we will focus the discussion on the pro- and anti-recurrence properties and mechanics of three key immunosuppressive drugs (FK506, rapamycin, and cyclosporine) on HCC. The results may provide evidence for immunosuppressive protocols introduction after OLT for HCC.
Section I: Experimental studies of rapamycin and other immunosuppressive agents on growth and metastasis of hepatocellular carcinoma
The effects of three immunosuppressive agents (rapamycin, FK506, and cyclosporine) on tumor growth and metastasis were investigated in LCI-D20 nude mice model with a highly-potentially human hepatocellular carcinoma. Immunohistochemistry and terminal deoxynucleotidyl transferas-mediated dUTP nick end labeling (TUNEL) assay were used to detect the levels of PCNA, microvessel
density (CD31 stained) and tumor cell apoptosis in vivo, respectively.
Three immunosuppressive agents effects on phenotypic and cytoskeleton changes, proliferation, apoptosis, cell cycles, migration, adhesion, and invasiveness on MHCC97H cell line were also studied in vitro by scanning electron microscopy, F-actin polymerization, MTT, flow cytometry, Boyden chamber methods, and gelatin zymograph, respectively. And the expression of molecules (E-cadherin, ICAM-1, and MMP-2) implicated in tumor progression were analyzed by real-time PCR, western blot analysis in vitro, and immunohistochemistry, ELISA in vivo, respectively.
The results showed that, in vivo, rapamycin inhibited tumor growth and lung metastasis compared with control in nude mice (0.76±0.38 g vs. 2.09±0.75 g, P=0.001, 3/7 vs. 7/7, P=0.007). The number of metastatic nodules in lung was increased in cyclosporine group as compared with control group (6±2 vs. 4±1, P=0.046). There was no significant difference between FK506 and control in lung metastasis rate (5/7 vs. 7/7, P=0.078). Rapamycin but not FK506 or cyclosporine inhibited tumor cell proliferation, induced apoptosis, and decreased tumor angiogenesis in LCI-D20 model.
In vitro, treatment of MHCC97H resulted in striking morphological alterations in cyclosporine group, including numerous pseudopodial protrusions, increased cell motility, and metastasis abilities. Confocal laser scan microscopy of MHCC97H cells stimulated in cyclosporine showed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. Cyclosporine could also evidently increase the secretion of MMP-2 by MHCC97H and tumor cells in LCI-D20 model. Rapamycin blocked proliferation of MHCC97H and induced cell cycle arrest at the G1 checkpoint, but did not induce apoptosis. FK506 and rapamycin inhibited the ability of migration, adhesion, and invasiveness of MHCC97H. They also resulted in a significant decrease in the levels of expression of ICAM-1 and MMP-2.
Our findings suggest that cyclosporine can promote hepatocellular carcinoma cell progression by a direct cellular effect, FK506 may not promote hepatocellular carcinoma cell invasiveness, and rapamycin seems to inhibit the growth, motility and invasiveness of hepatocellular carcinoma cell.
Section II. Preliminary study on the molecular mechanisms of antiangiogenic effects of rapamycin in hepatocellular carcinoma
The first section demonstrated that rapamycin inhibited tumor growth and recurrence in the LCI-D20 xenograft model in nude mice by suppressing tumor angiogenesis. However, the underlying molecular mechanism was not fully elucidated. In this study, a cDNA microarray analysis followed by Real-time PCR, Western blot, and Immunohistochemistry analysis revealed that VEGF, Hypoxia-inducible factor 1 a (HIF-1a) and Angiopoietin-2 might be inhibited by rapamycin in vivo. Then we demonstrated that rapamycin could also downregulate VEGF expression in protein levels in a time-dependent manner, as well as downregulating HIF-1a expression in MHCC97H cells treated with the hypoxia mimetic agent, CoCl_2, in vitro. While HIF-1a is a well characterized regulator of VEGF expression, we may lead to the initial conclusion that rapamycin could suppress VEGF synthesis and secretion by downregulating HIF-1a expression. Our results also suggest that the anti-angiogenesis treatment of rapamycin may be associated with suppression of VEGF and Angiopoietin-2.
In LCI-D20 nude mice model, when comparison was made among control, rapamycin 2mg/kg/day, interferon alpha (IFNα) 1×10~6 U/kg/day, and combined treatment of rapamycin and IFNa for treated groups; tumor volume was 4.10cm~3±0.58cm~3, 1.82cm~3±0.90cm~3, 0.70cm~3±0.39cm~3, and 0.20cm~3±31cm~3, and incidence of lung metastasis being 100%, 83.3%, 50%, and 0%, respectively. The combined treatment did not increase the inhibition effect on the growth of tumor compared with rapamycin group (P=0.128). However, the combined therapy significantly improved the effect on lung metastasis with single treatment with either rapamycin (P=0.023) or IFNa (P=0.000). Considering the fact that IFNa is used in liver transplant patients with chronic hepatitis C and rapamycin acts as a primary immune suppressant, combination therapy of rapamycin plus IFNa has the potential clinical implication, particularly for the prevention of recurrence and metastasis after liver transplantation for hepatocellular carcinoma.
Section HI: Experimental studies on the effects of immunosuppression on tumor growth, recurrence and metastasis in rats
The pro- and anti- tumor effects of three key immunosuppressive drugs (FK506, rapamycin, and cyclosporine) had been discussed in severe combined
immunodeficient BALB/c mice in section I and II. Here we investigated the effects of rapamycin, FK506, and cyclosporine on established tumors in rats simultaneously bearing a liver allograft.
In one tumor-transplant model, SD rats received subcutaneous syngenic walker-256 carcinosarcoma cells 3 days after orthotopic liver transplantation (Wistar →SD) with character of acute rejection. Rapamycin (1 mg/kg/d), FK506 (1 mg/kg/d) or CsA (20 mg/kg/d) was initiated with transplantation. The controls received a similar volume of saline. The clinical characters, the liver function, the transplantated liver pathologic character and the cytotoxicity of spleen mononuclear from recipient against the Yac-1 cells were observed and measured on tumor postimplantative day 14. The treatment and control groups were evaluated for tumor volume thrice every one week.
In a second model system, a transplanted hepatoma model was made in rats by implantation of histologically intact walker-256 tumor fragment into the left lateral lobe of the liver. On the 14th days after implantation, the hepatic tubercle was excised to be examined by histological method to confirm the successful establishment of primary carcinoma of the livers in rats. Then the orthotopic liver transplantation (SD →SD) with character of no acute rejection was performed in different groups. Intrahepatic recurrence and lung metastasis were recorded.
In the first model system, results showed that the function of graft was improved, the cytotoxicity against the Yac-1 cells decreased, and the rejection reaction inhibited in the immunosuppressive treatment groups compared with control groups. The rapamycin treatment group showed a significantly decreased tumor volume (1.41±0.87cm3 vs. 3.65±0.87cm3, P=0.047), and CsA or FK506 group increased tumor size versus controls (9.56±2.81cm3 vs. 3.65±0.87cm3, P=0.000; 8.11±1.69cm3 vs. 3.65±0.87cm3, P=0.001) at the end of treatment.
In the second model system, when comparison was made among control, FK506, rapamycin, and cyclosporine, incidence of recurrent tumor was 0%, 20%, 16.7% and 0%; incidence of lung metastasis being 42.9%, 60.0%, 33.3% and 0%, respectively. The incidence of lung metastasis was decreased in the rapamycin-treated animals, and tumor recurrence in liver was seen only in the cyclosporine-treated and FK506-treated animals.
In conclusion, this study demonstrates that rapamycin simultaneously protects allografts from rejection and attacks tumors in a complex transplant-tumor situation.
Notably, CsA or FK506 protects allografts from rejection, but cancer progression is promoted in transplant recipients.
Section IV: Retrospective analysis of 134 cases of liver transplantation for hepatocellular carcinoma at the Liver cancer institute of Fudan University, china
A total of 134 patients received orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) from January 2004 to October 2005 in authors' institute were enrolled in this study to evaluate the clinical value and prognostic factors of OLT for HCC. Their clinicopathological characteristics [including age, gender, the history of hepatitis, Child-Pugh class, α-fetal protein (AFP), blood transfusion, tumor size, number, capsule, micro-vascular invasion, tumor differentiation, pTNM stage, Milan criteria, and immunosuppressive protocol], and follow-up data [including tumor recurrence, overall (OS) and disease-free survival (DFS) of patients] were retrospectively analyzed.
The 1- and 2-year OS rates of the 134 patients were 82.75%±3.47% and 68.80%±7.07% and the DFS rates were 69.13%±4.42% and 41.33%±17.20%, respectively. Univariate analysis revealed Child-Pugh class (P=0.000), tumor size (P=0.032), micro-vascular invasion (P=0.019), blood transfusion (P=0.037), Milan criteria (P=0.007) were statistically significant factors affecting OS; and Child-Pugh class (P=0.009) , tumor size (P=0.002), tumor number (P=0.041), micro-vascular invasion (P=0.000), Milan criteria (P=0.000), pTNM stage (P=0.001) were statistically significant factors affecting DFS. Multivariate analysis using Cox proportional hazards regression model demonstrated the Child-Pugh class (P=0.000) and Milan criteria (P=0.001) were the independent and statistically significant factors affecting OS; and the Child-Pugh class (P=0.009), Milan criteria (P=0.004), and micro-vascular invasion (P=0.004) were independent and statistically significant factors affecting DFS.
Taking the Milan criteria as a categorized variable, the 134 cases were classified into two subgroups: within the Milan criteria group (n= 61) and beyond group (n=73). In the Milan group, with the Cox regression, only Child-Pugh class (P=0.043) was identified as the independent predictors for OS, only micro-vascular invasion (P=0.014) had independent predictive significance for DFS.
In the beyond Milan criteria group, the patients with the sirolimus-based protocol (n= 27) were significantly associated with a better OS (P=0.011) as well as DFS (P=0.037), and later posttransplant recurrence time (263.0± 100.0 天 vs. 140.1± 123.5 天, P=0.035), compared with the FK506-based patients (n=46). Immunosuppressive protocol after OLT was one of the leading independent prognostic factors for both OS (P=0.015) and DFS (P=0.015) in multivariate Cox models analyses. Acute rejection (n=6), thrombocytopenia (n=3), anemia (n=12), and oral aphthous ulcers (n=7) were found in beyond Milan cohort and were easily manageable.
We can draw the following theoretical considerations based on our results that The sirolimus based immunosuppression protocol appears to have beneficial effects on tumor recurrence and survival with an acceptable rate of rejection and toxicity in beyond Milan criteria group. The risk of recurrence and metastasis after OLT for HCC may be predicted preoperatively using imaging-based criteria (presence of macroscopic vascular invasion and size/number of tumors, which are surrogates for microscopic invasion), and the predictive model may be refined based on information gained on pathologic study of the explanted livers (e.g. microscopic invasion). And the prognosis of these patients depends not only on the stage of the tumor at the time of diagnosis, but also on the degree of deterioration in liver function.
引文
1. Parkin DM, Bray F, Ferlay J, et al. Estimating the world cancer burden: Globocan 2000. Int J Cancer, 2001, 94(2): 153-156
2. Tang ZY, Ye SL, Liu YK, et al. A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol, 2004, 130(4): 191-195
3．李常海，陈孝平，黄志勇，等．肝移植治疗原发性肝癌的现状．中华实验外科杂志，2005，22(5)：636-638
4. Ringe B. Transplantation for liver tumors: current status. Liver, 2002, 22(1):1-7
5. Kelly DM, Emre S, Guy SR, et al. Liver transplant recipients are not at increased risk for nonlymphoid solid organ tumors. Cancer, 1998,83(6):1237-1243
6. Oruc MT, Soran A, Jain AK, et al. De novo breast cancer in patients with liver transplantation: University of Pittsburgh's experience and review of the literature. Liver Transpl, 2004,10(1): 1-6
7. Torbenson M, Grover D, Boitnott J, et al. De Novo Hepatocellular Carcinoma in a Liver Allograft Associated with Recurrent Hepatitis B. Transplant Proc,2005,37(5):2205-2206
8. Majno P, Giostra E, Mentha G, et al. Is there a customised immunosuppressive regimen for patients transplanted with hepatocellular carcinoma? J Hepatol,2005,43 (4):577-584
9. Lin HL, Lui WY, Liu TY, et al. Reversal of Taxol resistance in hepatoma by cyclosporin A: involvement of the PI-3 kinase-AKT 1 pathway. Br J Cancer,2003, 88(6):973-980
10. Sakai M, Miyake H, Tashiro S, et al. Inhibitory effect of FK506 and cyclosporine A on the growth and invasion of human liver cancer cells. J Med Invest,2004, 51(1-2):63-69
11. Mousses S, Wagner U, Chen Y, et al. Failure of hormone therapy in prostate cancer involves systematic restoration of androgen responsive genes and activation of rapamycin sensitive signaling. Oncogene, 2001, 20(46):6718-6723
12. Kneteman NM, Oberholzer J, Al Saghier M, et al. Sirolimus-based immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma. Live Transpl, 2004, 10(10): 1301-1311
13. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibitsprimary and metastatic tumor growth by antiangiogenesis: involvementof vascular endothelial growth factor. Nat Med,2002,8(2):128-135
14. Hojo M, Morimoto T, Maluccio M, et al. Cyclosporine induces cancerprogression by a cell-autonomous mechanism. Nature,1999,397(6719):530-534
15. Vivarelli M, Bellusci R, Cucchetti A, et al. Low recurrence rate of hepatocellul carcarcinoma after liver transplantation: better patient selection or lower immunosuppression? Transplantation, 2002,74(12): 1746-1751
16. Maluccio M, Sharma V, Lagman M, et al. Tacrolimus enhances transforming growth factor-betal expression and promotes tumor progression. Transplantation,2003,76(3): 597-602
17. Wang L, Tang ZY, Qin LX, et al. High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential. Hepatology,2000,32(1):43-48
1. Majno P, Giostra E, Mentha G, et al. Is there a customised immunosuppressive regimen for patients transplanted with hepatocellular carcinoma? J Hepatol,2005,43(4):577-584
2. Hojo M, Morimoto T, Maluccio M, et al. Cyclosporine induces cancer progression by a cell-autonomous mechanism. Nature,1999,397(6719):530-534
3. Lin HL, Lui WY, Liu TY, et al. Reversal of Taxol resistance in hepatoma by cyclosporin A: involvement of the PI-3 kinase-AKT 1 pathway. Br J Cancer,2003,88(6):973-980
4. Schumacher G, Oidtmann M, Rosewicz S, et al. Sirolimus inhibits growth of human hepatoma cells in contrast to tacrolimus which promotes cell growth. Transplant Proc,2002,34(5): 1392-1393
5. Maluccio M, Sharma V, Lagman M, et al. Tacrolimus enhances transforming growth factor-betal expression and promotes tumor progression. Transplantation,2003,76(3):597-602
6. Sakai M, Miyake H, Tashiro S, et al. Inhibitory effect of FK506 and cyclosporine A on the growth and invasion of human liver cancer cells. J Med Invest,2004, 51(1-2):63-69
7. Yang R, Rescorla FJ, Reilly CR, et al. A reproducible rat liver cancer model for experimental therapy: introducing a technique of intrahepatic tumor implantation. J Surg Res,1992,52(3):193-198
8. Uehara H, Kim SJ, Karashima T, et al. Effects of blocking platelet-derived growth factor-receptor signaling in a mouse model of experimental prostate cancer bone metastases. J Natl Cancer Inst,2003,95(6):458-470
9. Patterson MK Jr: Measurement of growth and viability of cells in culture. In: Jakoby WB, Pastin IH, eds. Methods in Enzymology. Volume 58. New York: Academic Press Inc, 1979,141-152
10. Romanov VI, Durand DB, Petrenko VA. Phage display selection of peptides that affect prostate carcinoma cells attachment and invasion. Prostate,2001,47(4): 239-251
11. Sun FX, Tang ZY, Lui KD, et al. Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues. Int J Cancer, 1996,66(2):239-43
12. Maluccio M, Sharma V, Lagman M, et al. Tacrolimus enhances transforming growth factor-beta1 expression and promotes tumor progression. Transplantation,2003,76(3):597-602
13.Luan FL, Hojo M, Maluccio M, et al. Rapamycin blocks tumor progression: unlinking immunosuppression from antitumor efficacy. Transplantation,2002, 73(10):1565-1572
14. Li Y, Tang ZY, Ye SL, et al. Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol,2001,7(5):630-636
15. Grewe M, Gansauge F, Schmid RM, et al. Regulation of cell growth and cyclin D1 expression by the constitutively active FRAP-p70s6K pathway in human pancreatic cancer cells. Cancer Res,1999,59(15):3581-3587
16. Nourse J, Firpo E, Flanagan WM, et al. Interleukin-2-mediated elimination of the p27Kipl cyclin-dependent kinase inhibitor prevented by rapamycin. Nature, 1994(6506), 372:570-573
17. Hosoi H, DiLling MB, Shikata T, et al. Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells. Cancer Res, 1999,59(4):886-894
18. Mahalati K, Kahan BD. Clinical pharmacokinetics of sirolimus. Clin Pharmacokinet, 2001,40(8):573-585
19. Van Haastert PJ, Devreotes PN. Chemotaxis: signalling the way forward. Nat Rev Mol Cell Biol, 2004,5(8):626-634
20. Lambrechts A, Van Troys M, Ampe C. The actin cytoskeleton in normal and pathological cell motility. Int J Biochem Cell Biol, 2004,36( 10): 1890-1909
21.Upadhyaya A, van Oudenaarden A. Actin polymerization: forcing flat faces forward. Curr Biol, 2004,14(12):467-469
22. Sun JJ, Zhou XD, Liu YK, et al. Invasion and metastasis of liver cancer: expression of intercellular adhesion molecule 1. J Cancer Res Clin Oncol, 1999,125(1):28-34
23. Sun JJ, Zhou XD, Zhou G, et al. Expression of intercellular adhesive molecule-1 in liver cancer tissues andliver cancer metastasis. World J Gastroenterol, 1998,4(3):202-205
24. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med, 2002, 8(2): 128-135
1. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med, 2002, 8(2): 128-135
2. Luan FL, Ding R, Sharma VK, et al. Rapamycin is an effective inhibitor of human renal cancer metastasis. Kidney Int, 2003, 63(3): 917-926
3. Mise M, Arii S, Higashituji H, et al. Clinical significance of vascular endothenial growth factor and basic fibroblast growth factor gene expression in liver tumor. Hepatology, 1996, 23 (3):455-464
4. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med, 2003, 9(6): 677-684
5. Talks KL, Turley H. The expression and distribution of the hypoxia - inducible factors HIF- lalpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol, 2000, 157 (2):411-421
6．丁磊，陈孝平，王海平。缺氧诱导因子-1α蛋白在肝癌组织中的表达及临床意义。中华肝脏病杂志，2004，12(11)：656-659
7. Gao N, Nester RA., Sarkar MA. 4-Hydroxy estradiol but not 2-hydroxy estradiol induces expression of hypoxia-inducible factor la and vascular endothelial growth factor A through phosphatidylinositol 3-kinase/Akt/FRAP pathway in OVCAR-3 and A2780-CP70 human ovarian carcinoma cells. Toxicology and Applied Pharmacology,2004,196(1): 124-135
8. Jiang BH, Jiang G., Zheng JZ, Lu Z, et al. Phosphatidylinositol 3-kinase signaling mediates the induction of hypoxia- inducible factor 1 by insulin or epidermal growth factor. Cell Growth Differ, 2001, 12(7):363-369
9. Richard DE, Berra E, Gothie E, et al. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor lalpha (HIF-lalpha) and enhance the transcriptional activity of HIF-1. J Biol Chem, 1999,274(46):32631- 32637
10. Laughner E, Taghavi P, Chiles K, et al. HER2 (neu) signaling increases the rate of hypoxia-inducible factor lalpha (HIF-lalpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol, 2001,21(12):3995- 4004
11. Hudson CC, Liu M, Chiang GG, et al. Regulation of Hypoxia-Inducible Factor 1αExpression and Function by the Mammalian Target of Rapamycin. Mol Cell Biol, 2002, 22(20):7004-7014
12. Makino Y, Cao R, Svensson K, et al. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature,2001,414(6863):550-554
13. Blancher C, Moore JW, Robertson N, et al. Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1 alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway. Cancer Res,2001,61(19): 7349-7355
14. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev,1997,18(1):4-25
15. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol,2001,61(3):253-270
16. Asahara T, Chen D, Takahashi T, et al. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ Res, 1998,83(3):233-40
17. Tanaka S, Mori M, Sakamoto Y, et al. Biologic significance of angiopoietin-2 expression in human hepatocellular carcinoma. J Clin Invest, 1999,103(3): 341-345
18. Mitsuhashi N, Shimizu H, Ohtsuka M, et al. Angiopoietins and Tie-2 expression in angiogenesis and proliferation of human hepatocellular carcinoma. Hepatology 2003,37(5):1105-1113
19. Torimura T, Ueno T, Kin M, et al. Overexpression of angiopoietin-1 and angiopoietin-2 in hepatocellular carcinoma. J Hepatol 2004,40(5):799-807
20. Moon WS, Rhyu KH, Kang MJ, et al. Overexpression of VEGF and angiopoietin 2: a key to high vascularity of hepatocellular carcinoma? Mod Pathol 2003,16(6):552-557
21. Peng L, Sun J, Wang WD, et al. Biological effect of ectopic expression of angiopoietin-1 and -2 in hepatocellular carcinoma cell line. Hepatobiliary Pancreat Dis Int,2003 ,2(1):94-97
22. Ahmad SA, Liu W, Jung YD, et al. Differential expression of angiopoietin-1 and angiopoietin-2 in colon carcinoma. A possible mechanism for the initiation of angiogenesis. Cancer, 2001,92(5):1138-1143
23. Yoshiji H, Kuriyama S, Noguchi R, et al. Angiopoietin 2 displays a vascular endothelial growth factor dependent synergistic effect in hepatocellular carcinoma development in mice. Gut, 2005,54(12): 1768-1775
24. Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin- proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redoxinduced changes. J Biol Chem,1997,272(36):22642-22647
25. Mathew P, Thall PF, Jones D, et al. Platelet-derived growth factor receptor inhibitor imatinib mesylate and docetaxel: a modular phase I trial in androgen-independent prostate cancer. J Clin Oncol, 2004,22(16):3323-3329
26. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer, 2002,2(10):727-739
27. Wang L, Tang ZY, Qin LX, et al. High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential. Hepatology,2000,32(1):43-48
28. Shakil AO, McGuire B, Crippin J, et al. A pilot study of interferon alfa and ribavirin combination in liver transplant recipients with recurrent hepatitis C. Hepatology,2002,36(5): 1253-1258
29. Samuel D, Bizollon T, Feray C, et al. Interferon alfa 2b plus ribavirin in patients with recurrent HCV infection after liver transplantation: a randomized study. Gastroenterology,2003,124(3):642-650
30. Yedibela S, Schuppan D, Muller V, et al. Successful treatment of hepatitis C reinfection with interferon-a2b and ribavirin after liver transplantation. Liver International,2005,25(4):717-722
31. Kayler LK, Merion RM, Arenas JD, et al. Epithelioid hemangioendothelioma of the liver disseminated to the peritoneum treated with liver transplantation and interferon alpha-2B. Tansplantation,2002,74(1): 128-130
1. An international panel. Banff schema for grading liver allograft rejection: international consensus document. Hepatology, 1997, 25:658-663
2. Yano K, Yasuhiko F, Dohi K, et al. Development of a rat model for orthotopic liver transplantation for hepatocellular carcinoma. Surgery, 1995, 118(3):539-546
3. Matsuzaki T, Murase N, Yagihashi A, et al. Liver transplantation for diethylnitrosamine-indueed hepatocellular carcinoma in rats. Transplant Proc, 1992, 24(2):748-751
4．曾兆林，韩德恩，韩丽姝，等．三氧化二砷预防大鼠肝癌肝移植后肿瘤复发的研究．中华实验外科杂志，2005，22(2)：169-170
5. He YC, Chen JW, Cao J, et al. Toxicities and therapeutic effect of 5-fluorouracil controlled release implant on tumor-bearing rats. World J Gastroenterol, 2003, 9(8): 1795-1798
6．彭永海，李琦，刘庆，等．大鼠肝包膜下接种Walker-256瘤株肺转移的观察．第二军医大学学报，2002，23(10)：1084-1122
7．顾伟，沈婕，瞿笑枫．大鼠移植性肝癌模型生物学行为与分期的初步探讨．中西医结合学报，2005，3(2)：136-138
8．智星，严律南，杨培，等．CTLA4-Ig抗大鼠肝移植排斥反应及诱导免疫耐受的实验研究．中国普外基础与临床杂志，2005，12(6)：600-604
9. Vivarelli M, Cucchetti A, Piscaglia F, et al. Analysis of risk factors for tumor recurrence after liver transplantation for hepatocellular carcinoma: key role of immunosuppression. Liver Transpl, 2005, 11(5):497-503
10. Hallett WH, Murphy WJ. Natural killer cells: biology and clinical use in cancer therapy. Cell Mol Immunol, 2004, 1(1): 12-21
11. Moretta L, Bottino C, Pende D, et al. Human natural killer cells: Molecular mechanisms controlling NK cell activation and tumor cell lysis. Immunol Lett. 2005, 100(1):7-13
12. Koehl GE, Andrassy J, Guba M, et al. Rapamycin protects allografts from rejection while simultaneously attacking tumors in immunosuppressed mice. Transplantation, 2004, 77(9): 1319-1326
13. Stippel DL, Kasper HU, Schleimer K, et al. Successful use of sirolimus in a patient with bulky ovarian metastasis of hepatocellular carcinoma after liver transplantation. Transplant Proc, 2005, 37(5):2185-2187
14. Elsharkawi M, Staib L, Henne-Bruns D, et al. Complete remission of postransplant lung metastases from hepatocellular carcinoma under therapy with sirolimus and mycophenolate mofetil. Transplantation,2005,79(7):854-857
15. Stepkowski SM. Preclinical Results of Sirolimus Treatment in Transplant Models. Transplant Proc,2003,35(3 Suppl):219S-226S
1. Tran TT, Nissen N, Poordad FF, et al. Advances in liver transplantation. New strategies and current care expand access, enhance survival. Postgrad Med,2004,115(5):73-76
2. LH Sobin, Ch. Wittekind. TNM Classification of Malignant Tumours, 6th Edition. Wiley-Liss, New York, 2002
3. Plessier A, Codes L, Consigny Y, et al. Underestimation of the influence of satellite nodules as a risk factor for post-transplantation recurrence in patients with small hepatocellular carcinoma. Liver Transplantation,2004,10(2 Suppl 1):86-90
4. Leung JY, Zhu AX, Gordon FD, et al. Liver transplantation outcomes for early-stage hepatocellular carcinoma: results of a multicenter study. Liver Transpl,2004,10(11): 1343-1354
5. Marsh JW, Dvorchik I, Bonham CA, et al. Is the pathologic TNM staging system for patients with hepatoma predictive of outcome? Cancer,2000,88(3):538-543
6. Zavaglia C, De Carlis L, Alberti AB, et al. Predictors of long-term survival after liver transplantation for hepatocellular carcinoma. Am J Gastroenterol, 2005,100(12):2708-2716
7. Herold C, Reck T, Fischler P, et al. Prognosis of a large cohort of patients with hepatocellular carcinoma in a single European centre. Liver,2002,22 (1):23-28
8. Mazzaferro V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med,1996,334(ll):693-699
9. Margarit C, Charco R, Hidalgo E, et al. Liver transplantation for malignant diseases: selection and pattern of recurrence. World J Surg,2002,26(2):257-263
10. Khakhar A, Solano E, Steel D, et al. Survival after liver transplantation for hepatocellular carcinoma. Transplant Proc,2003,35(7):2438-2441
11. Merli M, Nicolini G, Gentili F, et al. Predictive factors of outcome after liver transplantation in patients with cirrhosis and hepatocellular carcinoma. Transplant Proc,2005,37(6):2535-2540
12. Martinez Ares D, Suarez Lopez FJ, Souto Ruzo J, et al. Liver transplantation in patients with hepatocellular carcinoma: factors implicated in tumor relapse. Rev Esp Enferm Dig,2004,96 (1):22-31
13. Del Gaudio M, Grazi GL, Principe A, et al. Influence of prognostic factors on the outcome of liver transplantation for hepatocellular carcinoma on cirrhosis: a univariate and multivariate analysis. Hepatogastroenterology,2004,51(56):510 -514
14. Kneteman NM, Oberholzer J, Al Saghier M, et al. Sirolimus-based immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma. Live Transpl,2004,10(10):1301-1311
15. Watson CJ, Friend PJ, Jamieson NV, et al. Sirolimus: a potent new immimosuppressant for liver transplantation. Transplantation, 1999,67(4): 505-509
16. McAlister VC, Peltekain KM, Malatjalian DA, et al. Orthotopic liver transplantation using low-dose tacrolimus and sirolimus. Liver Transpl,2001,7(8): 701-708
17. Dunkelberg JC, Trotter JF, Wachs M, et al. Sirolimus as primary immunosuppression in liver transplantation is not associated with hepatic artery or wound complications. Liver Transpl,2003,9(5):463-468
1 Ramsay HM, Fryer AA, Hawley CM, et al. Br J Dermatol, 2002, 147:950-956.
2 Euvrard S, Kanitakis J, Claudy A. N Engl J Med, 2003, 348:1681-1691.
3 Hunt SA. Transplant Proc, 2002, 34:1874-1876.
4 Jensen P, Hansen S, Moller B, et al. J Am Acad Dermatol, 1999, 40:177-186.
5 Vivarelli M, Bellusci R, Cucchetti A, et al. Transplantation, 2002, 74:1746-1751.
6 Hojo M, Morimoto T, Maluccio M, et al. Nature, 1999, 397: 530-534.
7 Guba M, von Breitenbuch P, Steinbauer M, et al. Nat Med, 2002, 8:128-135.
8 Shihab FS, Bennett WM, Isaac J, et al. Kidney Int, 2003, 63: 522-533.
9 Herman M, Weinstein T, Korzets A, et al. J Lab Clin Med, 2001, 137:14-20.
10 Dantal J, Hourmant M, Cantarovich D, et al. Lancet, 1998, 351:623-628.
11 Tremblay F, Femandes M, Habbab F, et al. Ann Surg Oncol, 2002, 9:785-788.
12 Freise CE, Ferrell L, Liu T, et al. Transplantation, 1999, 67:510-513.
13 Lin HL, Lui WY, Liu TY, et al. Br J Cancer, 2003, 88:973-980.
14 Malueeio M, Sharma V, Lagman M, et al. Transplantation, 2003, 76:597-602.
15 Jonas S, Rayes N, Neumann U, et al. Cancer, 1997, 80:1141-1150.
16 Sakai M, Miyake H, Taahiro S, et al. J Med Invest, 2004, 51:63-69.
17 Luan FL, Hojo M, Maheeio M, et al. Transplantation, 2002, 73:1565-1572.
18 Huang S, Houghton PJ. Curr Opin Pharmacol, 2003, 3:371-377.
19 Chen H, Lee JM, Zong Y, et al. J Virol, 2001, 75: 2929-2937.
20 Luan FL, Ding R, Sharma VK, et al. Kidney Int, 2003, 63:917-926.
21 Kahan BD, Knight R, Sehoenberg L, et al. Transplant Proc, 2003, 35:S25-S34.
22 Kneteman NM, Oberholzer J, Al Saghier M, et al. Liver Transpl, 2004,10:1301-1311.
23 Elsharkawi M, Staib L, Henne-Bruns D, et al. Transplantation, 2005,79:855-857.
24 Cai J, Zheng T, Lotz M, et al. Blood, 1997, 89:1491-1500.
25 Mazzaferro V, Rondinara GF, Rossi G, et al. Transplant Proc, 1994,26:3557-3560.
26 Tressler RJ, Garvin LJ, Slate DL. lnt J Cancer, 1994, 57:568-573.
27 Yokoyama I, Hayashi S, Kobayashi T, et al. Transpl lnt , 1995, 8:251-255.
28 Hussein MM, Mooij JM, Roujouleh HM. Nephrol Dial Transplant, 2000,15:1103-1104.
29 Eberhard OK, Kliem V, Brunkhorst R. Transplantation, 1999, 67:180-184.
1 Sehgal SN. Clin Biochem, 1998, 31:335-340.
2 Hackstein H, Tarter T, Logar AJL, et al. Blood,2002,100:1084-1087.
3 Monti P, Mercalli A, Leone BE, et al. Transplantation,2003,75(1):137-145.
4 Baboolal K. Tnumsplantation,2003,75(8): 1404-1408.
5 Kalan BD. Transplantation, 2001,71(11): SS52-57.
6 Me Alister Vc,Gao Z, Peltekian K, et al. Lancet, 2000, 355:375-376.
7 Qi S, Xu D, Peng J, et al. Transplantation, 2000, 69(7):1275-1283.
8 Johannes P, Squifflet JP, Wlodarczyk Z, et al. Transplantation, 2003,75(12):1934-1939.
9 Kreis H, Cisterne J, Land W, et al. Transplantation, 2000, 69:1252-1260.
10 Morales JM, Wramner L, Kreis H, et al. Am J Transplant, 2002,2:436-442.
11 Nair S, Eason J, Loss G. Liver Transpl, 2003, 9(2): 126-129.
12 Hong JC, Kahan BD. Transplantation, 2001, 71:1579-1584.
13 Sindhi R, Webber S, Venkataramanan R, et al. Transplantation, 2001,72:851-855.
14 Oliveira J, Xavier P, Sampaio S, et al. Transplantation, 2002,73:915-919.
15 Zhu J, Wu J, Frizell E, et el. Gastroenterology, 1999, 117(5):1198-1204.
16 Hosoi H, Diling MB, Shikata T, et al. Cancer Res, 1999, 59:886-894.
17 Testa JR, Bellacosa A. PNAS, 2001,98: 10983-10985.
18 Huang S, Liu LN, Hosoi H, et al. Cancer Res, 2001,61:3373-3381.
19 Luan FL, Hojo M, Maluccio M, et al. Transplantation, 2002, 73:1565-1572.
20 Luan FL, Ding R, Sharma VK, et al. Kidney Int, 2003, 63:917-926.
21 Maluccio M, Sharma V, Lagman Mila, et al. Transplantation, 2003,76(3):597-602.
22 Guba M, Breitenbuch PV, Steinbauer M, et al. Nature Medicine,2002,8:128-135.
23 Koehl G, Guba M, Seeliger H, et al. Transplantation Proceedings,2003,35:2135-2136.
24 Pescovit M, Govani M. Am J Kidney Dis, 2001,38(2):S16-21.
25 Kahan BD, Pedbielski J, Napoli KL, et al.Transplantation,1998, 66(8):1040-1046.
26. Kreis H, Cistern JM, Land W, et al. Transplantation, 2000,69(7):1252-1260.
27 Morales JM, Wramner H, Kreis D, et al. X VⅢ International Congress of the Transplantation Soceity,2000,428:140.
28 Stallone G, Paolo SD, Schena A, et al. J Am Soc Nephrol, 2004,15:228-233
2. Tang ZY, Ye SL, Liu YK, et al. A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol, 2004, 130(4): 191-195
3．李常海，陈孝平，黄志勇，等．肝移植治疗原发性肝癌的现状．中华实验外科杂志，2005，22(5)：636-638
4. Ringe B. Transplantation for liver tumors: current status. Liver, 2002, 22(1):1-7
5. Kelly DM, Emre S, Guy SR, et al. Liver transplant recipients are not at increased risk for nonlymphoid solid organ tumors. Cancer, 1998,83(6):1237-1243
6. Oruc MT, Soran A, Jain AK, et al. De novo breast cancer in patients with liver transplantation: University of Pittsburgh's experience and review of the literature. Liver Transpl, 2004,10(1): 1-6
7. Torbenson M, Grover D, Boitnott J, et al. De Novo Hepatocellular Carcinoma in a Liver Allograft Associated with Recurrent Hepatitis B. Transplant Proc,2005,37(5):2205-2206
8. Majno P, Giostra E, Mentha G, et al. Is there a customised immunosuppressive regimen for patients transplanted with hepatocellular carcinoma? J Hepatol,2005,43 (4):577-584
9. Lin HL, Lui WY, Liu TY, et al. Reversal of Taxol resistance in hepatoma by cyclosporin A: involvement of the PI-3 kinase-AKT 1 pathway. Br J Cancer,2003, 88(6):973-980
10. Sakai M, Miyake H, Tashiro S, et al. Inhibitory effect of FK506 and cyclosporine A on the growth and invasion of human liver cancer cells. J Med Invest,2004, 51(1-2):63-69
11. Mousses S, Wagner U, Chen Y, et al. Failure of hormone therapy in prostate cancer involves systematic restoration of androgen responsive genes and activation of rapamycin sensitive signaling. Oncogene, 2001, 20(46):6718-6723
12. Kneteman NM, Oberholzer J, Al Saghier M, et al. Sirolimus-based immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma. Live Transpl, 2004, 10(10): 1301-1311
13. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibitsprimary and metastatic tumor growth by antiangiogenesis: involvementof vascular endothelial growth factor. Nat Med,2002,8(2):128-135
14. Hojo M, Morimoto T, Maluccio M, et al. Cyclosporine induces cancerprogression by a cell-autonomous mechanism. Nature,1999,397(6719):530-534
15. Vivarelli M, Bellusci R, Cucchetti A, et al. Low recurrence rate of hepatocellul carcarcinoma after liver transplantation: better patient selection or lower immunosuppression? Transplantation, 2002,74(12): 1746-1751
16. Maluccio M, Sharma V, Lagman M, et al. Tacrolimus enhances transforming growth factor-betal expression and promotes tumor progression. Transplantation,2003,76(3): 597-602
17. Wang L, Tang ZY, Qin LX, et al. High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential. Hepatology,2000,32(1):43-48
1. Majno P, Giostra E, Mentha G, et al. Is there a customised immunosuppressive regimen for patients transplanted with hepatocellular carcinoma? J Hepatol,2005,43(4):577-584
2. Hojo M, Morimoto T, Maluccio M, et al. Cyclosporine induces cancer progression by a cell-autonomous mechanism. Nature,1999,397(6719):530-534
3. Lin HL, Lui WY, Liu TY, et al. Reversal of Taxol resistance in hepatoma by cyclosporin A: involvement of the PI-3 kinase-AKT 1 pathway. Br J Cancer,2003,88(6):973-980
4. Schumacher G, Oidtmann M, Rosewicz S, et al. Sirolimus inhibits growth of human hepatoma cells in contrast to tacrolimus which promotes cell growth. Transplant Proc,2002,34(5): 1392-1393
5. Maluccio M, Sharma V, Lagman M, et al. Tacrolimus enhances transforming growth factor-betal expression and promotes tumor progression. Transplantation,2003,76(3):597-602
6. Sakai M, Miyake H, Tashiro S, et al. Inhibitory effect of FK506 and cyclosporine A on the growth and invasion of human liver cancer cells. J Med Invest,2004, 51(1-2):63-69
7. Yang R, Rescorla FJ, Reilly CR, et al. A reproducible rat liver cancer model for experimental therapy: introducing a technique of intrahepatic tumor implantation. J Surg Res,1992,52(3):193-198
8. Uehara H, Kim SJ, Karashima T, et al. Effects of blocking platelet-derived growth factor-receptor signaling in a mouse model of experimental prostate cancer bone metastases. J Natl Cancer Inst,2003,95(6):458-470
9. Patterson MK Jr: Measurement of growth and viability of cells in culture. In: Jakoby WB, Pastin IH, eds. Methods in Enzymology. Volume 58. New York: Academic Press Inc, 1979,141-152
10. Romanov VI, Durand DB, Petrenko VA. Phage display selection of peptides that affect prostate carcinoma cells attachment and invasion. Prostate,2001,47(4): 239-251
11. Sun FX, Tang ZY, Lui KD, et al. Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues. Int J Cancer, 1996,66(2):239-43
12. Maluccio M, Sharma V, Lagman M, et al. Tacrolimus enhances transforming growth factor-beta1 expression and promotes tumor progression. Transplantation,2003,76(3):597-602
13.Luan FL, Hojo M, Maluccio M, et al. Rapamycin blocks tumor progression: unlinking immunosuppression from antitumor efficacy. Transplantation,2002, 73(10):1565-1572
14. Li Y, Tang ZY, Ye SL, et al. Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol,2001,7(5):630-636
15. Grewe M, Gansauge F, Schmid RM, et al. Regulation of cell growth and cyclin D1 expression by the constitutively active FRAP-p70s6K pathway in human pancreatic cancer cells. Cancer Res,1999,59(15):3581-3587
16. Nourse J, Firpo E, Flanagan WM, et al. Interleukin-2-mediated elimination of the p27Kipl cyclin-dependent kinase inhibitor prevented by rapamycin. Nature, 1994(6506), 372:570-573
17. Hosoi H, DiLling MB, Shikata T, et al. Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells. Cancer Res, 1999,59(4):886-894
18. Mahalati K, Kahan BD. Clinical pharmacokinetics of sirolimus. Clin Pharmacokinet, 2001,40(8):573-585
19. Van Haastert PJ, Devreotes PN. Chemotaxis: signalling the way forward. Nat Rev Mol Cell Biol, 2004,5(8):626-634
20. Lambrechts A, Van Troys M, Ampe C. The actin cytoskeleton in normal and pathological cell motility. Int J Biochem Cell Biol, 2004,36( 10): 1890-1909
21.Upadhyaya A, van Oudenaarden A. Actin polymerization: forcing flat faces forward. Curr Biol, 2004,14(12):467-469
22. Sun JJ, Zhou XD, Liu YK, et al. Invasion and metastasis of liver cancer: expression of intercellular adhesion molecule 1. J Cancer Res Clin Oncol, 1999,125(1):28-34
23. Sun JJ, Zhou XD, Zhou G, et al. Expression of intercellular adhesive molecule-1 in liver cancer tissues andliver cancer metastasis. World J Gastroenterol, 1998,4(3):202-205
24. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med, 2002, 8(2): 128-135
1. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med, 2002, 8(2): 128-135
2. Luan FL, Ding R, Sharma VK, et al. Rapamycin is an effective inhibitor of human renal cancer metastasis. Kidney Int, 2003, 63(3): 917-926
3. Mise M, Arii S, Higashituji H, et al. Clinical significance of vascular endothenial growth factor and basic fibroblast growth factor gene expression in liver tumor. Hepatology, 1996, 23 (3):455-464
4. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med, 2003, 9(6): 677-684
5. Talks KL, Turley H. The expression and distribution of the hypoxia - inducible factors HIF- lalpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol, 2000, 157 (2):411-421
6．丁磊，陈孝平，王海平。缺氧诱导因子-1α蛋白在肝癌组织中的表达及临床意义。中华肝脏病杂志，2004，12(11)：656-659
7. Gao N, Nester RA., Sarkar MA. 4-Hydroxy estradiol but not 2-hydroxy estradiol induces expression of hypoxia-inducible factor la and vascular endothelial growth factor A through phosphatidylinositol 3-kinase/Akt/FRAP pathway in OVCAR-3 and A2780-CP70 human ovarian carcinoma cells. Toxicology and Applied Pharmacology,2004,196(1): 124-135
8. Jiang BH, Jiang G., Zheng JZ, Lu Z, et al. Phosphatidylinositol 3-kinase signaling mediates the induction of hypoxia- inducible factor 1 by insulin or epidermal growth factor. Cell Growth Differ, 2001, 12(7):363-369
9. Richard DE, Berra E, Gothie E, et al. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor lalpha (HIF-lalpha) and enhance the transcriptional activity of HIF-1. J Biol Chem, 1999,274(46):32631- 32637
10. Laughner E, Taghavi P, Chiles K, et al. HER2 (neu) signaling increases the rate of hypoxia-inducible factor lalpha (HIF-lalpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol, 2001,21(12):3995- 4004
11. Hudson CC, Liu M, Chiang GG, et al. Regulation of Hypoxia-Inducible Factor 1αExpression and Function by the Mammalian Target of Rapamycin. Mol Cell Biol, 2002, 22(20):7004-7014
12. Makino Y, Cao R, Svensson K, et al. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature,2001,414(6863):550-554
13. Blancher C, Moore JW, Robertson N, et al. Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1 alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway. Cancer Res,2001,61(19): 7349-7355
14. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev,1997,18(1):4-25
15. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol,2001,61(3):253-270
16. Asahara T, Chen D, Takahashi T, et al. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ Res, 1998,83(3):233-40
17. Tanaka S, Mori M, Sakamoto Y, et al. Biologic significance of angiopoietin-2 expression in human hepatocellular carcinoma. J Clin Invest, 1999,103(3): 341-345
18. Mitsuhashi N, Shimizu H, Ohtsuka M, et al. Angiopoietins and Tie-2 expression in angiogenesis and proliferation of human hepatocellular carcinoma. Hepatology 2003,37(5):1105-1113
19. Torimura T, Ueno T, Kin M, et al. Overexpression of angiopoietin-1 and angiopoietin-2 in hepatocellular carcinoma. J Hepatol 2004,40(5):799-807
20. Moon WS, Rhyu KH, Kang MJ, et al. Overexpression of VEGF and angiopoietin 2: a key to high vascularity of hepatocellular carcinoma? Mod Pathol 2003,16(6):552-557
21. Peng L, Sun J, Wang WD, et al. Biological effect of ectopic expression of angiopoietin-1 and -2 in hepatocellular carcinoma cell line. Hepatobiliary Pancreat Dis Int,2003 ,2(1):94-97
22. Ahmad SA, Liu W, Jung YD, et al. Differential expression of angiopoietin-1 and angiopoietin-2 in colon carcinoma. A possible mechanism for the initiation of angiogenesis. Cancer, 2001,92(5):1138-1143
23. Yoshiji H, Kuriyama S, Noguchi R, et al. Angiopoietin 2 displays a vascular endothelial growth factor dependent synergistic effect in hepatocellular carcinoma development in mice. Gut, 2005,54(12): 1768-1775
24. Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin- proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redoxinduced changes. J Biol Chem,1997,272(36):22642-22647
25. Mathew P, Thall PF, Jones D, et al. Platelet-derived growth factor receptor inhibitor imatinib mesylate and docetaxel: a modular phase I trial in androgen-independent prostate cancer. J Clin Oncol, 2004,22(16):3323-3329
26. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer, 2002,2(10):727-739
27. Wang L, Tang ZY, Qin LX, et al. High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential. Hepatology,2000,32(1):43-48
28. Shakil AO, McGuire B, Crippin J, et al. A pilot study of interferon alfa and ribavirin combination in liver transplant recipients with recurrent hepatitis C. Hepatology,2002,36(5): 1253-1258
29. Samuel D, Bizollon T, Feray C, et al. Interferon alfa 2b plus ribavirin in patients with recurrent HCV infection after liver transplantation: a randomized study. Gastroenterology,2003,124(3):642-650
30. Yedibela S, Schuppan D, Muller V, et al. Successful treatment of hepatitis C reinfection with interferon-a2b and ribavirin after liver transplantation. Liver International,2005,25(4):717-722
31. Kayler LK, Merion RM, Arenas JD, et al. Epithelioid hemangioendothelioma of the liver disseminated to the peritoneum treated with liver transplantation and interferon alpha-2B. Tansplantation,2002,74(1): 128-130
1. An international panel. Banff schema for grading liver allograft rejection: international consensus document. Hepatology, 1997, 25:658-663
2. Yano K, Yasuhiko F, Dohi K, et al. Development of a rat model for orthotopic liver transplantation for hepatocellular carcinoma. Surgery, 1995, 118(3):539-546
3. Matsuzaki T, Murase N, Yagihashi A, et al. Liver transplantation for diethylnitrosamine-indueed hepatocellular carcinoma in rats. Transplant Proc, 1992, 24(2):748-751
4．曾兆林，韩德恩，韩丽姝，等．三氧化二砷预防大鼠肝癌肝移植后肿瘤复发的研究．中华实验外科杂志，2005，22(2)：169-170
5. He YC, Chen JW, Cao J, et al. Toxicities and therapeutic effect of 5-fluorouracil controlled release implant on tumor-bearing rats. World J Gastroenterol, 2003, 9(8): 1795-1798
6．彭永海，李琦，刘庆，等．大鼠肝包膜下接种Walker-256瘤株肺转移的观察．第二军医大学学报，2002，23(10)：1084-1122
7．顾伟，沈婕，瞿笑枫．大鼠移植性肝癌模型生物学行为与分期的初步探讨．中西医结合学报，2005，3(2)：136-138
8．智星，严律南，杨培，等．CTLA4-Ig抗大鼠肝移植排斥反应及诱导免疫耐受的实验研究．中国普外基础与临床杂志，2005，12(6)：600-604
9. Vivarelli M, Cucchetti A, Piscaglia F, et al. Analysis of risk factors for tumor recurrence after liver transplantation for hepatocellular carcinoma: key role of immunosuppression. Liver Transpl, 2005, 11(5):497-503
10. Hallett WH, Murphy WJ. Natural killer cells: biology and clinical use in cancer therapy. Cell Mol Immunol, 2004, 1(1): 12-21
11. Moretta L, Bottino C, Pende D, et al. Human natural killer cells: Molecular mechanisms controlling NK cell activation and tumor cell lysis. Immunol Lett. 2005, 100(1):7-13
12. Koehl GE, Andrassy J, Guba M, et al. Rapamycin protects allografts from rejection while simultaneously attacking tumors in immunosuppressed mice. Transplantation, 2004, 77(9): 1319-1326
13. Stippel DL, Kasper HU, Schleimer K, et al. Successful use of sirolimus in a patient with bulky ovarian metastasis of hepatocellular carcinoma after liver transplantation. Transplant Proc, 2005, 37(5):2185-2187
14. Elsharkawi M, Staib L, Henne-Bruns D, et al. Complete remission of postransplant lung metastases from hepatocellular carcinoma under therapy with sirolimus and mycophenolate mofetil. Transplantation,2005,79(7):854-857
15. Stepkowski SM. Preclinical Results of Sirolimus Treatment in Transplant Models. Transplant Proc,2003,35(3 Suppl):219S-226S
1. Tran TT, Nissen N, Poordad FF, et al. Advances in liver transplantation. New strategies and current care expand access, enhance survival. Postgrad Med,2004,115(5):73-76
2. LH Sobin, Ch. Wittekind. TNM Classification of Malignant Tumours, 6th Edition. Wiley-Liss, New York, 2002
3. Plessier A, Codes L, Consigny Y, et al. Underestimation of the influence of satellite nodules as a risk factor for post-transplantation recurrence in patients with small hepatocellular carcinoma. Liver Transplantation,2004,10(2 Suppl 1):86-90
4. Leung JY, Zhu AX, Gordon FD, et al. Liver transplantation outcomes for early-stage hepatocellular carcinoma: results of a multicenter study. Liver Transpl,2004,10(11): 1343-1354
5. Marsh JW, Dvorchik I, Bonham CA, et al. Is the pathologic TNM staging system for patients with hepatoma predictive of outcome? Cancer,2000,88(3):538-543
6. Zavaglia C, De Carlis L, Alberti AB, et al. Predictors of long-term survival after liver transplantation for hepatocellular carcinoma. Am J Gastroenterol, 2005,100(12):2708-2716
7. Herold C, Reck T, Fischler P, et al. Prognosis of a large cohort of patients with hepatocellular carcinoma in a single European centre. Liver,2002,22 (1):23-28
8. Mazzaferro V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med,1996,334(ll):693-699
9. Margarit C, Charco R, Hidalgo E, et al. Liver transplantation for malignant diseases: selection and pattern of recurrence. World J Surg,2002,26(2):257-263
10. Khakhar A, Solano E, Steel D, et al. Survival after liver transplantation for hepatocellular carcinoma. Transplant Proc,2003,35(7):2438-2441
11. Merli M, Nicolini G, Gentili F, et al. Predictive factors of outcome after liver transplantation in patients with cirrhosis and hepatocellular carcinoma. Transplant Proc,2005,37(6):2535-2540
12. Martinez Ares D, Suarez Lopez FJ, Souto Ruzo J, et al. Liver transplantation in patients with hepatocellular carcinoma: factors implicated in tumor relapse. Rev Esp Enferm Dig,2004,96 (1):22-31
13. Del Gaudio M, Grazi GL, Principe A, et al. Influence of prognostic factors on the outcome of liver transplantation for hepatocellular carcinoma on cirrhosis: a univariate and multivariate analysis. Hepatogastroenterology,2004,51(56):510 -514
14. Kneteman NM, Oberholzer J, Al Saghier M, et al. Sirolimus-based immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma. Live Transpl,2004,10(10):1301-1311
15. Watson CJ, Friend PJ, Jamieson NV, et al. Sirolimus: a potent new immimosuppressant for liver transplantation. Transplantation, 1999,67(4): 505-509
16. McAlister VC, Peltekain KM, Malatjalian DA, et al. Orthotopic liver transplantation using low-dose tacrolimus and sirolimus. Liver Transpl,2001,7(8): 701-708
17. Dunkelberg JC, Trotter JF, Wachs M, et al. Sirolimus as primary immunosuppression in liver transplantation is not associated with hepatic artery or wound complications. Liver Transpl,2003,9(5):463-468
1 Ramsay HM, Fryer AA, Hawley CM, et al. Br J Dermatol, 2002, 147:950-956.
2 Euvrard S, Kanitakis J, Claudy A. N Engl J Med, 2003, 348:1681-1691.
3 Hunt SA. Transplant Proc, 2002, 34:1874-1876.
4 Jensen P, Hansen S, Moller B, et al. J Am Acad Dermatol, 1999, 40:177-186.
5 Vivarelli M, Bellusci R, Cucchetti A, et al. Transplantation, 2002, 74:1746-1751.
6 Hojo M, Morimoto T, Maluccio M, et al. Nature, 1999, 397: 530-534.
7 Guba M, von Breitenbuch P, Steinbauer M, et al. Nat Med, 2002, 8:128-135.
8 Shihab FS, Bennett WM, Isaac J, et al. Kidney Int, 2003, 63: 522-533.
9 Herman M, Weinstein T, Korzets A, et al. J Lab Clin Med, 2001, 137:14-20.
10 Dantal J, Hourmant M, Cantarovich D, et al. Lancet, 1998, 351:623-628.
11 Tremblay F, Femandes M, Habbab F, et al. Ann Surg Oncol, 2002, 9:785-788.
12 Freise CE, Ferrell L, Liu T, et al. Transplantation, 1999, 67:510-513.
13 Lin HL, Lui WY, Liu TY, et al. Br J Cancer, 2003, 88:973-980.
14 Malueeio M, Sharma V, Lagman M, et al. Transplantation, 2003, 76:597-602.
15 Jonas S, Rayes N, Neumann U, et al. Cancer, 1997, 80:1141-1150.
16 Sakai M, Miyake H, Taahiro S, et al. J Med Invest, 2004, 51:63-69.
17 Luan FL, Hojo M, Maheeio M, et al. Transplantation, 2002, 73:1565-1572.
18 Huang S, Houghton PJ. Curr Opin Pharmacol, 2003, 3:371-377.
19 Chen H, Lee JM, Zong Y, et al. J Virol, 2001, 75: 2929-2937.
20 Luan FL, Ding R, Sharma VK, et al. Kidney Int, 2003, 63:917-926.
21 Kahan BD, Knight R, Sehoenberg L, et al. Transplant Proc, 2003, 35:S25-S34.
22 Kneteman NM, Oberholzer J, Al Saghier M, et al. Liver Transpl, 2004,10:1301-1311.
23 Elsharkawi M, Staib L, Henne-Bruns D, et al. Transplantation, 2005,79:855-857.
24 Cai J, Zheng T, Lotz M, et al. Blood, 1997, 89:1491-1500.
25 Mazzaferro V, Rondinara GF, Rossi G, et al. Transplant Proc, 1994,26:3557-3560.
26 Tressler RJ, Garvin LJ, Slate DL. lnt J Cancer, 1994, 57:568-573.
27 Yokoyama I, Hayashi S, Kobayashi T, et al. Transpl lnt , 1995, 8:251-255.
28 Hussein MM, Mooij JM, Roujouleh HM. Nephrol Dial Transplant, 2000,15:1103-1104.
29 Eberhard OK, Kliem V, Brunkhorst R. Transplantation, 1999, 67:180-184.
1 Sehgal SN. Clin Biochem, 1998, 31:335-340.
2 Hackstein H, Tarter T, Logar AJL, et al. Blood,2002,100:1084-1087.
3 Monti P, Mercalli A, Leone BE, et al. Transplantation,2003,75(1):137-145.
4 Baboolal K. Tnumsplantation,2003,75(8): 1404-1408.
5 Kalan BD. Transplantation, 2001,71(11): SS52-57.
6 Me Alister Vc,Gao Z, Peltekian K, et al. Lancet, 2000, 355:375-376.
7 Qi S, Xu D, Peng J, et al. Transplantation, 2000, 69(7):1275-1283.
8 Johannes P, Squifflet JP, Wlodarczyk Z, et al. Transplantation, 2003,75(12):1934-1939.
9 Kreis H, Cisterne J, Land W, et al. Transplantation, 2000, 69:1252-1260.
10 Morales JM, Wramner L, Kreis H, et al. Am J Transplant, 2002,2:436-442.
11 Nair S, Eason J, Loss G. Liver Transpl, 2003, 9(2): 126-129.
12 Hong JC, Kahan BD. Transplantation, 2001, 71:1579-1584.
13 Sindhi R, Webber S, Venkataramanan R, et al. Transplantation, 2001,72:851-855.
14 Oliveira J, Xavier P, Sampaio S, et al. Transplantation, 2002,73:915-919.
15 Zhu J, Wu J, Frizell E, et el. Gastroenterology, 1999, 117(5):1198-1204.
16 Hosoi H, Diling MB, Shikata T, et al. Cancer Res, 1999, 59:886-894.
17 Testa JR, Bellacosa A. PNAS, 2001,98: 10983-10985.
18 Huang S, Liu LN, Hosoi H, et al. Cancer Res, 2001,61:3373-3381.
19 Luan FL, Hojo M, Maluccio M, et al. Transplantation, 2002, 73:1565-1572.
20 Luan FL, Ding R, Sharma VK, et al. Kidney Int, 2003, 63:917-926.
21 Maluccio M, Sharma V, Lagman Mila, et al. Transplantation, 2003,76(3):597-602.
22 Guba M, Breitenbuch PV, Steinbauer M, et al. Nature Medicine,2002,8:128-135.
23 Koehl G, Guba M, Seeliger H, et al. Transplantation Proceedings,2003,35:2135-2136.
24 Pescovit M, Govani M. Am J Kidney Dis, 2001,38(2):S16-21.
25 Kahan BD, Pedbielski J, Napoli KL, et al.Transplantation,1998, 66(8):1040-1046.
26. Kreis H, Cistern JM, Land W, et al. Transplantation, 2000,69(7):1252-1260.
27 Morales JM, Wramner H, Kreis D, et al. X VⅢ International Congress of the Transplantation Soceity,2000,428:140.
28 Stallone G, Paolo SD, Schena A, et al. J Am Soc Nephrol, 2004,15:228-233