王　鲁
 
论文题目：α干扰素及其它制剂干预肝癌转移复发和肿瘤生长的实验研究
 
作者简介：王　鲁，男，1968年10月出生，1997年08月师从于复旦大学汤钊猷教授，于2000年07月获博士学位。
 
 

摘 要

原发性肝癌是亚、非洲最常见的恶性肿瘤之一，年平均发病约为30万人^[1]。手术切除是重要的治疗手段，由于术后较高的复发率和大多数肝癌病人诊断时已不能手术，肝癌的预后差。肝癌对化疗不敏感，目前对肝癌转移复发有抑制作用的实验性干预药物难以上临床。肝癌转移是多步骤的过程，针对肝癌转移复发分子机理的生物治疗药物是干预的重要方向，肝癌是典型的多血管肿瘤，其复发与肿瘤血管形成有关^[2]，抗肿瘤血管形成治疗对肝癌转移复发的干预有特殊意义。寻找有效的抗肝癌转移复发的药物，对一些已在临床应用的药物探索其潜在的抗转移作用，筛选出具有实际应用价值者为临床直接应用，将极大地改善肝癌病人的预后。并通过对其作用机理的研究推动肝癌复发机理的研究。

第一部分：干预不同环节药物对肝癌转移复发和肿瘤生长作用的初步探索

一 标化实验动物模型(LCI-D20)

裸鼠人肝癌转移模型LCI-D20是我所首次建立的原位移植人肝癌高转移模型，该瘤株在宿主体内展示100％的移植生长和转移，能在人体外模拟人肝癌转移的自然过程^[3]，是一株可供研究实验性干预人肝癌转移复发的理想模型。在此基础上，观察分别于原位移植LCI-D20肿瘤后不同时间根治性切除移植瘤后及未切肿瘤的荷瘤裸鼠的转移复发率和生存时间。结果发现：裸鼠种植LCI-D20肿瘤组织后第3周可出现肝内播散，肺转移率达70%，平均生存期45±4天。于移植后7-14天切除肝移植瘤，术后处死时间延至5周，肿瘤转移复发率为100％，移植后11天切除肝移植瘤裸鼠平均生存期67±4天。从而确定本研究采用的动物模型一般分两组，一组为接种后24小时即用药，观测药物对肝移植瘤生长的抑制，另一组为移植后7－14天切除肝移植瘤，24小时后给药，用药4～5周，观察其对转移复发的疗效。

二 干预不同环节的药物对肝癌转移复发和肿瘤生长作用的初步探索

肝癌的转移与肿瘤及癌周组织表达雌雄激素受体有关^[4]，但临床肝癌的激素治疗效果不肯定^{[5, 6, 7]}。肝癌是典型的多血管肿瘤，肝癌组织中微血管密度与肝癌术后的转移复发有关。Droloxifene和2-羟基氟他胺分别阻断雌、雄激素受体与相应配体结合。维甲酸通过影响肿瘤细胞的增殖、分化和凋亡而起抗肿瘤作用，也有抑制肿瘤血管形成的作用^[8-12]。a干扰素具有影响细胞增殖、分化、免疫功能的作用，也具有抗肿瘤血管形成的活性^{[13, 14]}。本实验通过裸鼠人肝癌转移模型LCI-D20进行体内抑制肿瘤转移复发及肿瘤生长实验，以探索有潜在阻断肝癌转移可能的已在临床应用的Droloxifene、2-羟基氟他胺(futamine)、全反式维甲酸(all-trans retinoic acid, ATRA)、a干扰素(interferon a，IFN-a-1b，商品名Sinogen) 对肝癌转移复发和肿瘤生长的作用。发现Droloxifene, 2-羟基氟他胺，ATRA对根治性切除术后肝癌的转移复发及肿瘤生长无抑制作用。在早期（7天）切除后大剂量IFN-a(3×10⁷ U/kg/天)治疗组肝内复发率、肝内复发瘤大小、肝内播散灶、肺转移率、肺转移灶数目分别为16.7% ，3±2 mm3，0个，0%和0个；而对照组分别为100%，3224±1297 mm3，4±1个，100％，及6+2 个。两者比较，差别有统计学意义(P<0.01)。结果初步提示早期应用大剂量IFN-a对肝癌转移复发有抑制作用。

第二部分 IFN-a预防肝癌切除后转移复发和抑制肿瘤生长的作用

一 LCI-D20裸鼠体内应用IFN-a对肝癌转移复发和肿瘤生长的抑制

为探索IFN-a对肝癌生长、复发的作用及其剂量效应关系。采用不同剂量IFN-a通过裸鼠人肝癌转移模型LCI-D20进行体内实验。共种植105只LCI-D20模型，其中64只裸鼠作为预防组于种植后第11天根治性切除移植瘤。于切除后第2天开始皮下注射不同剂量的IFN-a，连续用药35天，于37天处死裸鼠，检测IFN-a对肝癌转移复发的预防作用。剩余的41只荷瘤裸鼠于种植后第2天开始给予不同剂量IFN-a治疗。治疗35天，于37天后每组剩余5只裸鼠继续治疗至死亡以观察IFN-a对生存期的影响，其余裸鼠处死，检测IFN-a对较大肿瘤负荷的治疗作用。结果裸鼠肝内移植瘤大小对照组为8475±2636 mm³，IFN-a1.5×10⁷U/kg治疗组为769±287 mm³，与对照组比较差别有统计学意义(P<0.001)；3×10⁷U/kgIFN-a治疗组为13±9 mm³，与对照组比较差别有统计学意义(P<0.001)。裸鼠肺转移率对照组为100％(5/5)， IFN-a3×10⁷U/kg治疗组为0%，与对照组比较差别有统计学意义(P<0.05)。IFN-a可延长荷瘤裸鼠生存时间。对照组平均生存期为45±4天，而IFN-a治疗1.5×10⁷U/kg剂量组和3×10⁷U/kg剂量组裸鼠平均生存期分别为81±6天、105±24天，与对照组比较差别有统计学意义(P<0.05)。IFN-a对根治性切除后肝癌转移复发有抑制作用。裸鼠肺转移率、肝内复发率、复发瘤大小对照组分别为100％、100％、1786±538 mm³。IFN-a1.5×10⁷U/kg治疗组分别为0、62.5%、11±4 mm³，与对照组比较差别有统计学意义(P<0.05)； IFNa3×10⁷U/kg治疗组0、12.5%、0.5 mm³，与对照组比较差别有统计学意义(P<0.001)。与对照组相比IFN-a1×10⁶U/kg 可明显抑制肝内复发瘤生长(1786±538mm³ v 260±57mm³, P<0.001)。按体表面积计算相当于人5.5MU，每周3次。为临床应用提供了参考。我们的结果显示长期大剂量应用IFN-a可以抑制肝癌术后转移复发和肿瘤生长，并呈剂量效应关系，为临床特别是肝癌术后方法复发的抑制提供了依据。

二 IFN-a对皮下移植肝癌生长的抑制

为了直接观察IFN-a对肿瘤生长的作用，我们将LCI-D20肿瘤组织植入裸鼠背部皮下。于移植后第2天开始皮下注射IFN-a(1.5×10⁷ U/kg/天)，连续用药35天，对照组皮下注射相同体积生理盐水，每5天观察肿瘤大小。对照组肿瘤在第35天肿瘤平均体积为6053mm³。应用1.5×10⁷ U/kg/天剂量的IFN-a对肿瘤生长有明显抑制，在第35天肿瘤平均体积为3308mm³，与对照组相比差别有统计学意义(P<0.05)。

三 ATRA对IFN-a抑制肝癌转移复发和肿瘤生长的协同效应

本实验通过裸鼠人肝癌转移模型LCI-D20进行体内实验，联用不同剂量IFN-a和ATRA并和单独应用IFN-a的疗效相比较，验证ATRA对IFN-a是否有疗效的协同效应。结果显示在荷瘤裸鼠应用ATRA与不同剂量的IFN-a，治疗组裸鼠肿瘤大小分别为8097±2531 mm³、839±304 mm³和15±7mm³；肺转移率分别为80％、40％和0％。而单独应用上述剂量IFN-a肿瘤大小分别为7963±3214 mm³、769±287 mm³和13±9 mm³；肺转移率分别为80％、40％和0％。结果与单独应用IFN-a比较，差别无统计学意义 (P>0.05)。在根治性切除移植瘤的裸鼠应用ATRA与不同剂量的IFN-a治疗组裸鼠肝内复发瘤大小为1067±756、16±9和1 mm³；肺转移率分别为90％、0％和0％。单独应用上述剂量的IFN-a肝内复发瘤大小分别为1345±559 mm³、11±4 mm³、1mm³；肺转移率为75％、0％和0％。二者比较差别无统计学意义 (P>0.05)。提示ATRA对IFN-a抑制肝癌转移复发和肿瘤生长无协同作用。

四 体内外检测IFN-a抑制肝癌血管形成的作用

体外检测IFN-a对肝癌细胞株增殖的作用以及对人脐静脉血管内皮细胞(HUVECs)增殖和移动的影响。通过裸鼠角膜微囊移植模型检测IFN-a对裸鼠角膜形成新生血管的影响，以探讨IFN-a作用机理。结果表明IFN-a在浓度为1000U/ml对BEL-7402细胞增殖的抑制率>50%，而其它肝癌细胞株包括MHCC97的增殖几乎不受IFN-a的影响。IFN-a可显著抑制HUVECs增殖，其ED₅₀为50U/ml，并呈剂量效应关系。IFN-a 浓度达100U/ml即显著抑制HUVECs的移动。通过肝癌裸鼠角膜微囊移植模型直观发现应用IFN-a使裸鼠角膜形成新生血管被明显抑制，在第21天，治疗组角膜新生血管积分为16±4，而对照组为27±6。

第三部分 IFN-a抑制肝癌血管形成的机理

一 IFN-a治疗后裸鼠血清血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)水平的变化

检测切除移植瘤后应用IFN-a (1.5×10⁷U/kg/天和3×10⁷U/kg/天)治疗裸鼠血清血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)和碱性成纤维细胞生长因子(basic fibroflast growth factor, bFGF)水平，并与单纯切除移植瘤裸鼠及未切瘤的荷瘤裸鼠血清中VEGF和bFGF的水平比较。发现荷瘤者、单纯切瘤者、肿瘤切除后应用IFN-a 1.5×10⁷U/kg/天和 3×10⁷U/kg/天裸鼠血清VEGF含量分别为197.5±67.8pg/mL，265±154.7pg/mL，53.3±9.9pg/mL和65.2±17.9pg/mL。切除肿瘤后经IFN-a治疗者，其VEGF水平显著低于切除而未经治疗者，也低于未切除的对照组(P<0.05)。而IFN-a治疗对bFGF水平则无明显影响，治疗与非治疗者相仿。证实IFN-a可抑制肝癌细胞VEGF的产生。

二 IFN-a对血管内皮生长因子(VEGF)和诱导型一氧化氮合酶

(iNOS) mRNA表达的影响

一氧化氮合酶活性的增高与VEGF及肿瘤血管形成关系密切，一氧化氮在VEGF诱导新生血管形成过程的每一步骤均起重要作用^[15]。应用RT-PCR方法分析根治性切除肝移植瘤后IFN-a治疗组(1.5×10⁷U/kg)和对照组复发瘤组织血管内皮生长因子(vascular endothelial growth factor, VEGF)和诱导型一氧化氮合酶 (inducible nitric oxide synthase, iNOS)表达水平结果显示，对照组肿瘤组织VEGF表达水平高于治疗组(P<0.05)。iNOSmRNA经PT-PCR扩增后，对照组肿瘤组织iNOSmRNA表达水平高于治疗组(P<0.05)。

三 IFN-a 治疗后肝癌组织VEGF、iNOS表达及微血管密度(MVD) 的变化

应用免疫组化方法分别检测到不同剂量IFN-a(1.5×10⁷U/kg/天，n=9；3×10⁷U/kg/天，n＝10)连续35治疗及给予相同体积生理盐水的LCI-D20裸鼠肿瘤组织标本中iNOS和VEGF均为细胞浆着色，在19例治疗组肝癌组织中分别有36.8%(7/19)表达iNOS和VEGF；在13例对照组肝癌组织中分别有69.2%(9/13) 表达iNOS，76.9%(10/13)表达VEGF，治疗前后VEGF表达差异有统计意义(P<0.05)。1.5×10⁷U/kg/天、3×10⁷U/kg/天治疗组肝癌组织肿瘤微血管密度(microvessel density, MVD)平均分别为66±5/每高倍视野，21±9 /每高倍视野，而对照组肿瘤微血管密度平均为114±18/每高倍视野(P<0.01)。结果提示IFN-a可以抑制VEGF的产生进而阻断肝癌血管形成。应用IFN-a可抑制VEGF及iNOS 的核酸表达。初步表明IFN-a主要通过抑制VEGF、iNOS进而阻断肝癌形成新生血管。也提示VEGF及 iNOS在促进肝癌的肿瘤血管形成中起一定的作用。

结 论

1. 本实验证实拮抗雌雄激素受体药物Droloxifene、2-羟基氟他胺以及分化诱导剂全反式维甲酸对肝癌肿瘤生长及根治性切除术后转移复发无抑制作用。

2. 大剂量长疗程应用IFN-a治疗能够抑制肝癌的转移复发和肿瘤生长，其抑制术后复发的作用较抑制未经切除的移植瘤的作用更为明显，其效应呈剂量依赖性。证实应用1×10⁶U/kg/天剂量IFN-a可显著抑制根治性术后肝内复发瘤的生长，相当于人5.5MU，每周3次。

3. IFN-a的作用机理为阻断肝癌的肿瘤血管形成。

4. IFN-a阻断肝癌肿瘤血管形成主要通过抑制VEGF而非bFGF的产生。一氧化氮在介导肝癌血管形成中与VEGF有协同作用。

ABSTRACT

Hepatocellular carcinoma is one of the most common malignacies in Asia and Africa, in China it is the second major cause of cancer death in males and the third in females. It was estimated to 315,000 cases worldwide in the year of 1985. Although the surgical resection is the first treatment of choice, unfortunately, the survival rate for HCC after resection remains low because of the high recurrent rate, only 9-27% of patients with HCC are suitable for resection. HCC is generally not sensitive to chemotherapy. Although there were a lot of drugs reported could inhibit recurrene and metastasis of HCC, however, most of them have not yet applied to patients. The metastasis of HCC is a multistep process, biotherapy that targeted to molecular mechanism of recurrence is one of the important approach. HCC is a typical hypervascular tumor, its recurrence was associated with tumor angiogenesis. Therefore, anti-angiogenesis may play important role in inhibiting HCC recurrence. It is necessary to establish effective adjuvant treatment to prevent postoperative recurrence and improve prognosis. This study is to evaluate whether drugs that currently used patients in clinic could inhibit tumor growth and recurrence after resection, and studies on the mechanism will also be reported.

Section I：Preliminary studies on the effect of pharmaceuticals with different mechanism on tumor growth and recurrence of hepatocelluar carcinoma

I. Standardizing experimental animal model (LCI-D20)

Metastatic model of human hepatocellular carcinoma in nude mice (LCI-D20) was established via orthotopic implantation, all mice with transplanted tumors exhibited 100% transplantability and metastasis, and is a patient-like nude mice model. Therefore, this model is an ideal model for the study of experimental intervention of HCC recurrence. To this end, we investigate the tumor growth and metastasis with or without curative resection of primary tumor in LCI-D20 model to verify incidence of intrahepatic recurrence, incidence of lung metastasis and life-span. The results showed that LCI-D20 model represented intrahepatic dissemination and lung metastasis (the incidence was 70%) in three weeks, the life-span was 45±4 days. Curative resection of tumor on postimplantative day 7 to 14, incidence of intrahepatic recurrence and lung metastasis were 100% on postoperative day 35, the life-span was 67±4 days. We employed two groups with or without resection to test the effects of drugs on prevention with small tumor burden and treatment with well established tumor burden. Therapy began on postimplantative or postoperative day 2, the period of treatment was 4 to 5 weeks.

II. Preliminary studies on the effects of parmaceuticals with

different mechanism on recurrence and tumor growth of

hepatocellular carcinoma

Expression of androgen and oestrogen receptors in hepatocellular carcinoma and surrounding liver parenchyma were associated with intrahepatic recurrence after hepatic resection, however, the results of hormonetherapy are controversal. HCC is typical hypervascular tumor, its recurrence was associated with tumor angiogenesis. Droloxifene and futamine prevent the banding of androgen and oestrogen receptors to their ligand resepectively. The effect of retinoic acid correlates with their ability to modulate growth tumor cells by influencing proliferation, differentiation, and apoptosis. Retinoic acids are also effective antiangiogenic agents. IFN-a is multifaceted agent long known for their ability to influencing proliferation, differentiation, and the immune system. In addition, IFN-a also posses antiangiogenic activity. In this study, we identify the effects of droloxifene, futamine, all-trans retinoic acid (ATRA), and interferon a (IFN-a-1b) on prevention of HCC tumor growth and recurrent tumor after curative resection in nude mice bearing HCC xenograft with high metastatic potential (LCI-D20 model). We found that droloxifene, futamine，and ATRA can not inhibit recurrence and lung metastasis after curative resection, as well as tumor growth in tumor bearing mice. But in mice with resection on postimplantative day 7, when comparison was made between control and IFN-a (IFN-a-1b) 1.5×10⁷ U/kg/day treated group, the incidence of intrahepatic recurrence rate was 16.7% versus 100%; recurrent tumor volume being 3224±1297 mm³ vs. 3±2 mm³; lesions of intrahepatic dissemination 4±1 vs. 0; incidence of lung metastasis being 100% vs. 0%; lesions of lung metastasis being 6+2 vs. 0 (P<0.01). This results showed that IFN-a inhibited recurrence after resection of HCC.

Section II. The inhibitory effect of IFN-a on tumor growth and recurrence after curative resection

Therapy with IFN-a inhibits tmor growth and recurrence in LCI-D20 nude mice model

This study was to investigate the effects of interferon-a-1b (IFN-a-1b) on recurrent tumor and metastasis after curative resection in nude mice bearing HCC xenograft with high metastatic potential. Tumor tissues from LCI-D20 were orthotopically implanted in 105 nude mice. Eleven days later, 64 mice underwent curative resection of liver tumors. IFN-a at different doses was administered subcutaneously (s.c.) to mice with or without resection for 35 consecutive days. In mice without resection, when comparison was made among control, IFN-a 7.5×10⁶ , 1.5×10⁷ and 3×10⁷ U/kg/day treated groups, tumor volume was 8475±2636 mm³, 7963±3214 mm³, 769±287 mm³ and 13±9 mm³; incidence of lung metastasis being 100%, 80%, 40% and 0%; life span being 45±4 days, 53±8 days, 81±6 days and 105±24 days respectively. In mice with curative resection, when comparison was made among control, IFN 5×10⁵, 1×10⁶, 4×10⁶, 7.5×10⁶, 1.5×10⁷ and 3×10⁷ U/kg/day, incidence of recurrent tumor was 100%, 100%, 87.5%, 100%, 87.5%, 62.5% and 12.5%; incidence of lung metastasis being 100%, 75%, 87.5%, 50%, 62.5%, 0% and 0% respectively. The minimal effective dose which has significant preventive effect on recurrent tumor is 1×10⁶ U/kg/day in nude mice, according fo formula S=K×W^2/3, which generally equaled to 5.5 MU tiw in human. In conclusion, high-dose and long-term therapy with IFN-a inhibits tumor growth and recurrence after resection of HCC in a dose-dependent manner. These results provide potential clinical implication particularly for the prevention of recurrence after curative resection of HCC.

IFN-a inhibits tumor growth of subcutaneous hepatocellular carcinoma xenograft

To investigate the inhibitory effect of IFN-a on tumor growth, we implanted a piece of LCI-D20 tumor tissue subcutaneously in nude mice. Treatment began on postimplantative day 2, IFN-a (1.5×10⁷ U/kg/day) was administered sc and continued for 35 days. The controls received a similar volume of saline. The treatment and control groups were evaluated for tumor volume every 5 days. The IFN-a treatment group showed a significantly decreased tumor volume compared with control group at the end of treatment (3308mm³ v 6053mm³; P<0.05).

I. The combination of ATRA and IFN-a has no synergistic effect on recurrece and tumor growth of hepatocellular carcinoma

The aim of this study was to elucidate if there are synergistic effect on recurrent tumor and metastasis after curative resection by the combination of ATRA and IFN-a in nude mice bearing HCC xenograft with high metastatic potential. In tumor bearing mice, when comparison was made among IFN-a 7.5×10⁶, IFN-a 1.5×10⁷, IFN-a 7.5×10⁶ U/kg/day and IFN-a at different doses plus ATRA (20mg/kg/day) treated groups, tumor volume was 8097±2531mm³, 839±304 mm³, 15±7mm³, 8475±2636 mm³, 769±287 mm³ and 13±9 mm³; incidence of lung metastasis being 80%, 40%, 0%, 80%, 40%, and 0% respectively. In mice with curative resection, when comparison was made among IFN-a at different doses and the combination treatment group, recurrent tumor volume was 1067±756 mm³, 1 mm³, 16±9 mm³, 1345±559 mm³ 11±4 mm³, and 0.5 mm³; incidence of lung metastasis being 90％, 0%, 0%, 75%, 0%, and 0% respectively. Therefore, synergistic effect was not found when ATRA was added to IFN-a treatment.

II. The in vitro and in vivo effect of IFN-a on anti-angiogenesis

In this study, we investigated the effect of IFN-a on HCC cell and endothelial cells (human umbilical vein endothelial cell, HUVECs) growth as well as migration of HUVECs in vitro and the neovascularization induced by implanting LCI-D20 tumor tissue in mice corneas in vivo. IFN-a was effective on inhibition of proliferation in BEL-7402 only, but not in MHCC97 and other HCC cell lines, even at the concentration of 10,000 U/ml. The proliferation of HUVECs was inhibited by IFN-a in a dose-dependent manner with ED₅₀ of approximately 100U/ml. In addition, the chemotaxic migration of HUVECs was inhibited by IFN-a, which was effectively reduced at a dose of 100 U/ml. IFN-a inhibited neovascularization induced by LCI-D20 tumor specimens implanted into micropocket of the corneas of nude mice, the angiogenic score was 16±4 in IFN-a treatment group，and being 27±6 in the control group on postimplantative day 21.

Section III. The mechanism of IFN-a on anti-angiogenesis

I. Measurement of serum vascular endothelial growth factor

(VEGF) and basic fibroblast growthj factor (bFGF) in nude

mice after administration of IFN-a

We compared serum VEGF and bFGF concentration (ELISA) in nude mice among “HCC resection plus IFN-a treatment”, “HCC resection only” and the control (without resection and without IFN-a treatment) groups. The results showed that serum VEGF concentration was 197.5±67.8pg/mL in the control, being 265.7±154.7pg/mL in resected only group, and being 53.3±9.9pg/mL and 65.2±17.9pg/mL in IFN-a treated group with different dose (P<0.05). There were no significant difference among groups of bFGF concentration. The results showed that IFN-a prevent HCC angiogenesis mainly by inhibiting VEGF production .

II. IFN-a down-regulate expression of vascular endothelial growth

factor (VEGF) and inducible nitric oxide synthase (iNOS)

mRNA

Nitric oxide (NO) production is essential for tumor angiogenesis, and plays an important role in each step of VEGF inducing angiogenesis. We compared the levels of VEGF and iNOS mRNA expression (by RT-PCR) between the IFN-a (1.5×10⁷ U/kg) treatment group after resection of primary tumor and the control group. The results showed that the relative levels of VEGF and iNOS expression in treatment group were significantly lower than that in the control group. This results showed that IFN-a could down-regulate gene expression of VEGF and iNOS.

III. Observation of VEGF, iNOS and microvessel density (MVD)

using immunohistochemistry after IFN-a treatment

The VEGF, iNOS expression and MVD detection was carried out by immunohistochemistry. The tumor samples were collected from 19 LCI-D20 mice received IFN-a at different doses (1.5×10⁷U/kg/d，n=9；3×10⁷U/kg/d，n＝10) and received same volume saline (n=13) for 35 consecutive days. It was found that 36.8% (7/10) were positive for VEGF and iNOS in the treatment group, whereas in the control group, iNOS was expressed in 69.2% (9/13) and VEGF expressed in 76.9% (10/13) of samples. There was significant difference in VEGF expression after treatment with IFN-a (P<0.05). Using antibody against CD31, it was found that the number of blood vessels in tumor counted per field ×200 reduced from 114±18 in the control mice to 66±5 in the IFN-a 1.5×10⁷U/kg/d treated mice and to 21±9 in IFN-a3×10⁷U/kg/d treated mice. (P<0.01). Results demonstrated that IFN-a treatment decreased expression of VEGF, iNOS, and reduced microvessel density. The data also reconfirmed that VEGF is the main angiogenic factor in this study.

Conclusion

1. Anti-androgen receptor and anti-oestrogen receptor drugs, Droloxifene and futamin, as well as differentiation inducer all-trans retinoic acid were of no effect on tumor growth and recurrece after curative resection in this experiment.

2. High-dose and long-term therapy with IFN-a significantly inhibits tumor growth and recurrence after resection of HCC in a dose-dependent manner. The effect was more prominent for small tumor burden than that with well established tumor. The minimal effective with preventive effect on recurrent tumor is 1×10⁶ U/kg/day in nude mice, according to the formula S=K×W^2/3, which is generally equaled to 5.5 MU tiw in human.

3. The mechanism of the effectiveness of IFN-a treatment in this experiment is anti-angiogeneisis.

4. VEGF , but not bFGF, is involved in the anti-angiogenic effect induced by IFN-a treatment, and Nitric oxide might play a role in conjunction with VEGF.

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