人肝细胞癌血管内皮生长因子及受体基因表达和其反义RNA体内外抑瘤作用的研究
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摘要
原发性肝癌是常见的消化系统恶性肿瘤，约占消化系统肿瘤的20％以上，其中肝细胞肝癌约占90％，其生物学特征为易复发、转移、预后差、死亡率高。尽管目前原发性肝癌的治疗方案(手术切除、介入治疗、生物治疗、激光及微波治疗等)日趋完善，但仍难以取得令人满意的疗效，患者生存期极短。因此研究肝癌的发病机理以及探讨新的可行的治疗方案具有极其重要的临床意义。
随着肿瘤学领域中基础研究的不断深入，人们发现在肿瘤组织中存在一些与肿瘤血管生成有关的生长因子及其受体过表达的现象；并且提出了肿瘤生长的血管依赖性学说，为探讨肿瘤的发生机制奠定了基础。血管内皮生长因子(VEGF)是一种重要的促血管生长因子，它参与肿瘤细胞的生长、分化、转移、增殖等多方面的过程。肝癌是富含血管的实体性肿瘤，因此VEGF可能与肝癌的发生、发展有重要的相关性联系。
目前，国内外针对VEGF及其受体基因表达与肝细胞癌分化程度、生长方式、临床特点及转移活性的关系尚未进行深入系统的研究。针对肝癌的血管内皮生成因子(VEGF)反义基因治疗尚无报道。本课题从三个角度进行了系统研究：(1)VEGF基因表达与肝癌恶性程度、临床特点、转移活性、微血管生成及其受体之间的相关性。(2)采用脂质体体外转染VEGF反义RNA，观察转染后肿瘤细胞在体外的生长情况及VEGF表达情况。(3)VEGF反义RNA转染后肿瘤细胞体内生长情况观
天津医科大学博士学位论文
察。结果将为探讨以VEGF为优选靶进行基因治疗，阻止肿瘤的发生
和发展，提高生存率，延长生存期奠定理论基础。
第一部分
人肝细胞癌VEGF及其受体基因表达与肝癌转移活性
和血管形成的相关性研究
本研究对 60例肝细胞癌（HCC）、10例正常肝组织采用逆转录－
聚合酶链反应、免疫组化法分别检测VEGFmRNA表达及其与相应受
体＊hi lflnl蛋白表达情况；采用＂m23评价肝细胞癌的转移活性；采
用Vlll因子相关抗原兔疫染色确定肿瘤微血管密度，从而分析肝细胞癌
VEGF基因表达与肿瘤血管生成、转移之间的关系。
1．人肝细胞癌VEGFmRNA表达的定量检测
通过RT－PCR方法检测60例人肝细胞癌和10例正常肝脏组织
VEGFmRNA 表达水平：发现正常肝脏组织和肝细胞肝癌的
VEGFmRNA阳性表达率分别为 30％和 78．3％，后者显著高于前者（P
＜0刀 1）：以 p．actin为内参照检测正常肝组织 VEGFmRNA表达水平明
显低于肝癌组织：W级肝细胞癌＊＊＊＊m＊＊A的表达水平为1．36土0．19，
而＊、11级肝细胞癌VEGFmRNA的表达水平分别为1．1210．25、0．79
士0．26，三者之间有明显差异（Pwto刀5人 因此提示，VEGFmRNA表
达水平与肿瘤病理分级呈正相关。此外发现：瘤栓组VEGFmRNA表
达水平为 1．31士0．13，无瘤栓组为 0．89土0．23；无包膜组 VEGFmRNA
表达水平为 1．28士 0．15，有包膜组为 0．96上 0．18，相应的两组之间有显
著性差异。而VEGFmRNA表达水平在不同大小肿瘤之间差别则无显
著性（P＞0．05）。此结果表明＊＊＊FmR＊A表达水平增高与临床上肿瘤
的侵袭特牲有关。
2
天津医科人学博土学位论文
2．VEGF及其受体 fit－llflk－l的蛋白表达
采用免疫组化法检测VEGF蛋白表达。正常肝组织和肝癌组织的
VEGF阳性表达率分别为40％、sl．3％，后者明显高于前者（Prto刀1）。
而不同分化程度的肝细胞肝癌的VEGF蛋白表达强度有显著差异（P
t 0．05）。表明：肝癌组织 VEGF蛋白表达水平与肝癌的病理分级呈正
相关。比较不同形态特征的肿瘤之间VEGF蛋白表达水平发现：瘤栓
组VEGF蛋白表达强度大于无瘤栓组，无包膜组VEGF蛋白表达强度
大于有包膜组，对应的两组之间有显著性差异。而不同直径的肿瘤之
间＊＊＊F蛋白表达水平无显著性差异（P＞0刀5）。
采用免疫组化法检测fi t－llflkl 蛋白表达。正常肝组织和肝癌组
织的月卜1什！卜1阳性表达率分别为40％历0％、80畅侣8．3用，后者明显高
于前者（P＜0刀5）。而不同分化程度的肝细胞肝癌的门卜lin卜1表达强
度也有显著差异，并与肝癌的病理分级呈正相关（P t 0刀5）。比较不
同形态肿瘤之间＊－llflkJ蛋白表达水平发现：fltllflki蛋白表达水
平也与瘤栓、包膜有关、与肿瘤直径无关。瘤栓组n卜1用卜1蛋白表达
强度大于无瘤栓组，无包膜组＊卜1厅！卜1蛋白表达强度大于有包膜组，
对应的两组之间有显著性差异。（P＜0乃5）
3．肝癌转移?
Liver cancer is one of the common types of gastrointestinal tumors with poor prognosis % metastasis and high mortality , in which hepatocellular carcinoma (HCC) accounts for about 95% . Although the different available multi-modalities of therapies , including radical surgical operation % intervention therapy > laser therapy et al, have currently been used in the treatment for liver cancer , the prognosis of liver cancer has not been markedly improved. Therefore, it has an important clinic significance to study on the mechanism of the development and progression of liver cancer and the new strategies for the treatment of liver cancer with the molecular biology. With the development of the basic research on oncological fields, it was found that many tumors have the overexpression in growth factors & their receptors, and progression of tumors depends on angiogenesis. The vascular endothelial growth factor ( VEGF) is an important factor which elicits the angiogensis in tumors. It takes an important role in the
growth, differentiation , metastasis, proliferation of tumors. The HCC is well known to be vascular rich tumors and its growth is highly depended on angiogenesis, so VEGF is considered to have key roles in the development of HCC. By now , the systemic researches on the
relationship between the expression of VEGF and histopathologic type, metastasis activity > angiogenesis of HCC have not been reported. And there are also no reports on gene therapy against angiogeneis of liver cancer. This study contains three parts of work: The first, the relationships between the expression of VEGF gene & its receptors ( flk-1 , flt-1 ) in HCC and the tumor grade , tumor metastasis , angiogenesis of liver cancer. The secondi the inhibitory effect of VEGF antisense RNA on the growth of 7721 hepatocellular cell line (7721 cancer hepatocytes)in vivo. The third, the inhibitory effect of VEGF antisense RNA on 7721 cell in nude mouse.
Part I : Study on the expression of VEGF and its receptors and its relationship to metastasis activity and angiogenesis of human HCC
1. Expression of VEGF gene in human HCC and normal liver tissue The gene expression of VEGF in 60 human HCCs and 10 normal
liver were studied by RT-PCR method. The expressive rates of HCCs and normal tissue were 78.3% and 30% respectively. The levels of VEGFmRNA (VEGFmRNA/P -actin mRNA) were 0.79 + 0.26 in grade IK 1.12 + 0.25 in grade IIK 1.36?.19 in grade IV, respectively . The expression levels of VEGFmRNA were positively correlated to pathologic grades^ embolus and envelops of tumors.
2. The protein expression of VEGF and its specific receptor in human HCC and normal liver tissue
The protein expression of VEGF in 60 human HCCs and 10 normal liver were studied by immunohistochemistry .The expression rates of HCCs and normal tissue were 81.3% and 40% respectively. The level of VEGF protein was also significantly correlated to pathologic grades>
embolus and capsules of the tumors.
The protein expression of flk-l/flt-1 in 60 human HCCs and 10 normal liver were studied by immunohistochemical method. The expression rates of flk-1 in HCCs and normal tissue were 88.3% and 50% respectively. The expression rates of flt-1 in HCCs and normal tissue were 80% and 40% respectively. The levels of fik-l/fit-1 were correlated to pathologic grades^ embolus and capsules of the tumors.
The overexpressions of VEGFmRNA -. VEGF protein and its fll-l/flk-1 were found in HCC. It suggested that the VEGF gene play an important role in the malignant progression of cancers. The overexpression of VEGF and the correlation between VEGF expression and tumor grade provided a useful parameter for evaluating the degree of malignancy at molecular level and for selecting the target gene in gene therapy.
Part II The inhibitory effect of VEGF antisense RNA on the growth of HCC cells in vitro
Because there was no effective treatments on liver cancers, it is very urgent and important to find out new methods to solve this problem. According to the results in part I , w
随着肿瘤学领域中基础研究的不断深入，人们发现在肿瘤组织中存在一些与肿瘤血管生成有关的生长因子及其受体过表达的现象；并且提出了肿瘤生长的血管依赖性学说，为探讨肿瘤的发生机制奠定了基础。血管内皮生长因子(VEGF)是一种重要的促血管生长因子，它参与肿瘤细胞的生长、分化、转移、增殖等多方面的过程。肝癌是富含血管的实体性肿瘤，因此VEGF可能与肝癌的发生、发展有重要的相关性联系。
目前，国内外针对VEGF及其受体基因表达与肝细胞癌分化程度、生长方式、临床特点及转移活性的关系尚未进行深入系统的研究。针对肝癌的血管内皮生成因子(VEGF)反义基因治疗尚无报道。本课题从三个角度进行了系统研究：(1)VEGF基因表达与肝癌恶性程度、临床特点、转移活性、微血管生成及其受体之间的相关性。(2)采用脂质体体外转染VEGF反义RNA，观察转染后肿瘤细胞在体外的生长情况及VEGF表达情况。(3)VEGF反义RNA转染后肿瘤细胞体内生长情况观
天津医科大学博士学位论文
察。结果将为探讨以VEGF为优选靶进行基因治疗，阻止肿瘤的发生
和发展，提高生存率，延长生存期奠定理论基础。
第一部分
人肝细胞癌VEGF及其受体基因表达与肝癌转移活性
和血管形成的相关性研究
本研究对 60例肝细胞癌（HCC）、10例正常肝组织采用逆转录－
聚合酶链反应、免疫组化法分别检测VEGFmRNA表达及其与相应受
体＊hi lflnl蛋白表达情况；采用＂m23评价肝细胞癌的转移活性；采
用Vlll因子相关抗原兔疫染色确定肿瘤微血管密度，从而分析肝细胞癌
VEGF基因表达与肿瘤血管生成、转移之间的关系。
1．人肝细胞癌VEGFmRNA表达的定量检测
通过RT－PCR方法检测60例人肝细胞癌和10例正常肝脏组织
VEGFmRNA 表达水平：发现正常肝脏组织和肝细胞肝癌的
VEGFmRNA阳性表达率分别为 30％和 78．3％，后者显著高于前者（P
＜0刀 1）：以 p．actin为内参照检测正常肝组织 VEGFmRNA表达水平明
显低于肝癌组织：W级肝细胞癌＊＊＊＊m＊＊A的表达水平为1．36土0．19，
而＊、11级肝细胞癌VEGFmRNA的表达水平分别为1．1210．25、0．79
士0．26，三者之间有明显差异（Pwto刀5人 因此提示，VEGFmRNA表
达水平与肿瘤病理分级呈正相关。此外发现：瘤栓组VEGFmRNA表
达水平为 1．31士0．13，无瘤栓组为 0．89土0．23；无包膜组 VEGFmRNA
表达水平为 1．28士 0．15，有包膜组为 0．96上 0．18，相应的两组之间有显
著性差异。而VEGFmRNA表达水平在不同大小肿瘤之间差别则无显
著性（P＞0．05）。此结果表明＊＊＊FmR＊A表达水平增高与临床上肿瘤
的侵袭特牲有关。
2
天津医科人学博土学位论文
2．VEGF及其受体 fit－llflk－l的蛋白表达
采用免疫组化法检测VEGF蛋白表达。正常肝组织和肝癌组织的
VEGF阳性表达率分别为40％、sl．3％，后者明显高于前者（Prto刀1）。
而不同分化程度的肝细胞肝癌的VEGF蛋白表达强度有显著差异（P
t 0．05）。表明：肝癌组织 VEGF蛋白表达水平与肝癌的病理分级呈正
相关。比较不同形态特征的肿瘤之间VEGF蛋白表达水平发现：瘤栓
组VEGF蛋白表达强度大于无瘤栓组，无包膜组VEGF蛋白表达强度
大于有包膜组，对应的两组之间有显著性差异。而不同直径的肿瘤之
间＊＊＊F蛋白表达水平无显著性差异（P＞0刀5）。
采用免疫组化法检测fi t－llflkl 蛋白表达。正常肝组织和肝癌组
织的月卜1什！卜1阳性表达率分别为40％历0％、80畅侣8．3用，后者明显高
于前者（P＜0刀5）。而不同分化程度的肝细胞肝癌的门卜lin卜1表达强
度也有显著差异，并与肝癌的病理分级呈正相关（P t 0刀5）。比较不
同形态肿瘤之间＊－llflkJ蛋白表达水平发现：fltllflki蛋白表达水
平也与瘤栓、包膜有关、与肿瘤直径无关。瘤栓组n卜1用卜1蛋白表达
强度大于无瘤栓组，无包膜组＊卜1厅！卜1蛋白表达强度大于有包膜组，
对应的两组之间有显著性差异。（P＜0乃5）
3．肝癌转移?
Liver cancer is one of the common types of gastrointestinal tumors with poor prognosis % metastasis and high mortality , in which hepatocellular carcinoma (HCC) accounts for about 95% . Although the different available multi-modalities of therapies , including radical surgical operation % intervention therapy > laser therapy et al, have currently been used in the treatment for liver cancer , the prognosis of liver cancer has not been markedly improved. Therefore, it has an important clinic significance to study on the mechanism of the development and progression of liver cancer and the new strategies for the treatment of liver cancer with the molecular biology. With the development of the basic research on oncological fields, it was found that many tumors have the overexpression in growth factors & their receptors, and progression of tumors depends on angiogenesis. The vascular endothelial growth factor ( VEGF) is an important factor which elicits the angiogensis in tumors. It takes an important role in the
growth, differentiation , metastasis, proliferation of tumors. The HCC is well known to be vascular rich tumors and its growth is highly depended on angiogenesis, so VEGF is considered to have key roles in the development of HCC. By now , the systemic researches on the
relationship between the expression of VEGF and histopathologic type, metastasis activity > angiogenesis of HCC have not been reported. And there are also no reports on gene therapy against angiogeneis of liver cancer. This study contains three parts of work: The first, the relationships between the expression of VEGF gene & its receptors ( flk-1 , flt-1 ) in HCC and the tumor grade , tumor metastasis , angiogenesis of liver cancer. The secondi the inhibitory effect of VEGF antisense RNA on the growth of 7721 hepatocellular cell line (7721 cancer hepatocytes)in vivo. The third, the inhibitory effect of VEGF antisense RNA on 7721 cell in nude mouse.
Part I : Study on the expression of VEGF and its receptors and its relationship to metastasis activity and angiogenesis of human HCC
1. Expression of VEGF gene in human HCC and normal liver tissue The gene expression of VEGF in 60 human HCCs and 10 normal
liver were studied by RT-PCR method. The expressive rates of HCCs and normal tissue were 78.3% and 30% respectively. The levels of VEGFmRNA (VEGFmRNA/P -actin mRNA) were 0.79 + 0.26 in grade IK 1.12 + 0.25 in grade IIK 1.36?.19 in grade IV, respectively . The expression levels of VEGFmRNA were positively correlated to pathologic grades^ embolus and envelops of tumors.
2. The protein expression of VEGF and its specific receptor in human HCC and normal liver tissue
The protein expression of VEGF in 60 human HCCs and 10 normal liver were studied by immunohistochemistry .The expression rates of HCCs and normal tissue were 81.3% and 40% respectively. The level of VEGF protein was also significantly correlated to pathologic grades>
embolus and capsules of the tumors.
The protein expression of flk-l/flt-1 in 60 human HCCs and 10 normal liver were studied by immunohistochemical method. The expression rates of flk-1 in HCCs and normal tissue were 88.3% and 50% respectively. The expression rates of flt-1 in HCCs and normal tissue were 80% and 40% respectively. The levels of fik-l/fit-1 were correlated to pathologic grades^ embolus and capsules of the tumors.
The overexpressions of VEGFmRNA -. VEGF protein and its fll-l/flk-1 were found in HCC. It suggested that the VEGF gene play an important role in the malignant progression of cancers. The overexpression of VEGF and the correlation between VEGF expression and tumor grade provided a useful parameter for evaluating the degree of malignancy at molecular level and for selecting the target gene in gene therapy.
Part II The inhibitory effect of VEGF antisense RNA on the growth of HCC cells in vitro
Because there was no effective treatments on liver cancers, it is very urgent and important to find out new methods to solve this problem. According to the results in part I , w
引文
1．张天泽，徐光炜主编．肿瘤学．天津：天津科学技术出版社．1997：1548-49
2．王庆生，郝希山，于世北．天津市1993年-1997年恶性肿瘤死亡率分析．中国肿瘤临床．2001；28：71-2
3．汤钊猷主编．原发型肝癌．上海：上海科学技术出版社．1981：1
4. Ott M; Schmidt HH; Emerging therapies in hepatology: liver-directed gene transfer and hepatocyte transplantation. Cells Tissues Organs 2000; 167(2-3):81-7.
5. Fevery J .Non-surgical treatment of hepatocellular carcinoma (HCC). Acta Gastroenterol Belg 2000 Apr-Jun;63(2):233-5.
6. Ruiz J; Qian C Gene therapy of viral hepatitis and hepatocellular carcinoma. J Viral Hepat 1999 Jan;6(1):17-34.
7. Usatoff V; Habib N .Hepatic malignancy: challenges and opportunities for the surgeon. J R Coll Surg Edinb 2000 Apr;45(2):99-109.
8. Mitry RR; Mansour MR .Gene therapy for liver tumours. Adv Exp Med Biol 2000;465(7): 193-205.
9. Leung DW, Cachlanes G,et al. Vascular endothelial growth factor is a secreted angiogenic mitogen .Science 1989;246:1306-1309
10. Folkman J,Klagsbrun M.Angiogenic factors. Science 1987;235:442-447
11. Deroanne CF, et al .Angiogenesis by fibroblast growth factor 4 is mediated through an autcrin up-regulation of vascular endothelial growth factor expression.Cancer Res, 1999,57:5590
12. Poltorak Z,Cohen T,et al. VEGF_(145), a secreted vascular endothelial growth factor isoform that binds to extracellular matrix .J Biol Chem, 1997 ;272:7151
13. Bowen W. The effects of surgical interference with the blood supply on the growth of transplanted carcinomata and sarcomata. Sci Rep Imperial Cancer Res Fund 1908; 3:146-158
14. Tanock IF. The relationship between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Br J Cancer 1968;22:258-273
15. Folkman J. Anti-angiogenesis: new concept for therapy of solid tumours. Ann Surg 1972;175:409-416
16. Gimbrone M, Leapman S,et,al.Tumour dormancy in vivo by prevention of neo-vascularisation .J Exp Med 1972; 136:261-276
17. Folkman JD. Clinical application of research on angiogenesis.N Engl J Med 1998;333(26): 1757-1763
18. Furcht LT. Critical factors controlling angiogenesis :cell products, cell matrix and growth factors. Lab Invest 1986;55(5);505-509
19. Hobson B. Denekamp J.Endothelial proliferation in tumours and normal tissues continous labelling studies. Br J Cancer 1984:49:405-413
20. Liotta L. Kleinerman J. Quantitative relationship of intravascular tumout cells, tumour vssels and pulmonary metastasis following tumour implantation . Cancer Res 1974:34:997-1004
21. Alguire GH. The transparent chamber technique as a tool in experiment tumour therapy .In Symposia : approaches to tumour chemotherapy .Washington DC: American Association for the Advancement of Science .1947:13-26
22． Alguire GH. Vascular reactions of normal and malignant tumours in vivol. Vascular reaction of mice to wounds and to normal and to neoplastic . J Nat) Cancer Inst 1945;6;73-85
23． Folkman J. Tumor angiogenesis : therapeutic implications. N Engl J Med 1971 ;285:1182-1186
24． Folkman J. What ie the evidence that tumours are angiogenesis dependent? J Natl Cancer Inst 1990:82:4-7
25． Folkman J. Watson K,et,al. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989;339:58-61
26． Weidner N.Carroll PR.et al. Tumour angiogenesis correlates with metastasis in invasive prostate carcinoma .Am J Pathol 1993:143:401-409
27． Hanahan D, Folkman J .Patterns and emerging mechanism of the angiogenic switch during tumorigenesis .Cell 1996;86;353-364
28． Gospodarowicz D. Lau K. Pituitary follicular cells secret both vascular epidermal growth factor and follistatin . Biochem Biophys Res Commun 1989:165:292-298
29． Dvorak HF. Broun LF. Detmar M,et al . Vascular permeability factor /vascular endothelial growth factor ,microvascular hyperpermeability and angiogenesis .Am J Pathol 1999;146:1029-1039
30． Gimbrone M. Leapman S. Cotran R. Folkman J. Tumor dormancy in vivo by prevention of neo-vascularisation J Exp Med 1972 :136:261-276
31． Klagbrum M. Amore PA. Regulators of angiogenesis . Annu Rev Physiol 1994;53;217-239
32． Plate KH, et al. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo .Natuer ,1992． 359:845
33． Samoto K.et al. Expression of vascular growth factor and its possible relation with neovascularization in human brain tumors. Cancer Res;1995;55:1189
34． Lichtor T, Dohrmann GJ, Gurney ME, et al .Cytokine gene expression by human gliomas. Neurosurgery 1990; 26;788-793
35． Takanami I, et al. Vascular endothelial growth factor and its receptor correlate with angiogenesis and survival in pulmonary adenocarcinoma. Anti Cancer Res ,1997:17;2811
36． Mattern J. Coexpression of VEGF and bFGF in human epidermoid lung carcinoma is associated with increased vessel density . Anti Cancer Res ,1997;17:2242
37． Maeda K, et al. Prognostic of VEGF endothelial growth factor expression in gastric carcinoma .Cancer ,1996,77:858
38． 陶厚权, 林言箴等. 血管内皮生长因子表达在胃癌浸润转移中的作用. 中华消化杂志 1998, 10,18(5) : 287
39． Zhang HT, et al. Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells. J Natl Cancer Inst ,1995,87:213
40． Fox SB, Gatter KC,Bicknell B, et al. Relationship of endothelial cell proliferation to tumor vascularity in human breast cancer . Cancer Res,1993 :53:4161-4163
41． Boocock CA, et al . Expression of vascular endothelial growth factor and its receptors Fit and KDR in ovarian carcinoma. J Natl Cancer Inst , 1999,87:506-509
42． Senger DR. Gvorak AM, et al. Tumor cells secrete a vascular permeability
factor that promotes accumulation ascites fluid. Science 1983:219(4587):983-988
43. UICC. TNM classification of malignant tumours .4th Ed. Berlin : Springer-Verlag, 1987:53
44．卢圣栋，主编。现代分子生物学技术。中国协和医科大学出版社。
45．金冬雁，黎孟枫等译。J．萨姆布鲁斯．分子克隆 北京：科学出版社．1992
46．李晓明、汤钊猷等。血管内皮细胞生长因子的表达与肝细胞癌侵袭和转移的关系。中华肿瘤杂志，1998；20：(1)12-14
47. Maeda K, Chung KS, Takatsuka S, et al. Tumor angiogenesis as a predictor of recurrence in gastric carcinoma. J Clin Oncol, 1995:13:477
48. Ferrara N. Henzal WJ. Pituitary follicular cells secrets a novel heparinbinding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989:161:851-858
49. Muller YA. Christinger HW. Keyt BA. Et al. The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 resulution : multiple copy flexidility and receptor binding . Structer, 1995;5(10):1325
50. Siemeister G. Marm D. Marting BG. The alpha-helical domain near the amino terminus is essential for dimerization of vscular endothelial growth factor. J Biol Chem ,1998:273 (18):11115
51. Soker S. Gollamudi S. Fikler H et al. Inhibition of vascular endothelial growth factor-induced endothelial cell proliferation by a peptide corresponding to the Exon-encoded domain of VEGF_(165). J Biol Chem. 1997; 272;31582
52. Brow LF. Lanir N. McDonagh J, Et al . Fibroblast migration in fibrin gel matrices. Am J Pathol ,1998:142(1):273
53. Kohn E. Liotta L. Molecular insights into cancer invasion :strategies for prevention and intervention. Cancer Res ,1995:55:1865
54. Folkman J. Angiogenesis in cancer , vascular , rheumatoid and other diseases. Nature Med , 1997:1:27
55. Senger D. Van De Walter L. et al . Vascular permeability factor (VPF/VEGF) in tumor biology .Cancer Metastasis Rev 1993:12:303-305
56. Collins PD. Connolly DT. Williams TJ. Characterization of the increase in vascular permeability induced by vascular permeability factor in vivo. Br J Pharamacol 1999: 109:195-199
57. Miura H. Miyazaki T et al . Increased expression of vascular endothelial growth factor in human hepatocellular carcinoma. J Hepatol 1997:27(5):854-61
58. Li XM et al. Serum vascular endothelial growth factor is a predictor of invasion and metastasis in hepatocellular carcinoma J Exp Clin Cancer Res 1999:18(4)511-7
59. Torimura T. Sata M et al . Increased expression of vascular endothelial growth factor is associated with tumour progression in hepatocellular carcinoma . Hum Pathol 1999:29(9):986-91
60. Suzuki K. Hayashi N et al . Expression of Vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma. Cancer Res 1999:56(13):3004-9
61. Mise M . Arii S et al. Clinical significance of vascular endothelial growth factor and basic fibroblast growth factor gene expression in liver cancer. Hepatology 1996;23(3);455-64
62．蒋扬富、杨治华等。血管内皮生长因子和基质金属蛋白酶-9 在原发性肝癌中的表达及其临床意义。癌症 2000：19(1)17-19
63．纪子钊、王宝胜等。VEGF p53和p21ras在原发性肝细胞癌中的表达及其临床病理意义。中国现代医学杂志 1999；9(4)：6-8
64. Yamaguchi R. Yano H et al. Expression and localization of vascular endothelial growth factor receptors in human hepatocellular carcinoma and non-HCC tissues. Oncol Rep 2000:7(4):725-9
65. Suzuki H. Seto K et al . Paracrine upregulation of VEGF receptor mRNA in endothelial cells by hypoxia-exposed hep G_2 cells. Am J Physiol 1999:276(1):G92-7
66．李江、王文亮、刘斌。人类肝细胞肝癌的细胞凋亡和癌组织内血管形成的调控。华人消化杂志 1998：6(12)：1057-1060。
67. Steeg PS. Bevilacqua G. Kopper L, et al. Evidence for a novel gene associated with low tumor metastasis potential . J Natl Cancer Inst. 1988: 80:200-204
68. Bevilaequa G. Sobel ME. Liotta LA, et al . Association of low nm23 RNA levels in human primary infiltrating ductal breast caricinoma with lymph node involvment and other histopathological indicators of high metastasis potential . Cancer Res . 1989:49:5185-5190
69. Yamaguchi A. Urano T. Fushida S, et al. Inverse association of nm23-H_1 expression by colorectal cancer with liver metastasis .Br J Cancer 1993, 68:1020-1024
70. Wang L. Patel U. Ghosh L, et al . Mutation in the nm23 gene is association with metastasis in colorectal cancer. Cancer Res, 1993:53:3652
71. Lizuka N. Masski O. Name T, et al . nm23-H_1 and nm23-H_2 messanger RNA abundance in HCC. Cancer Res ,1999:55:652
72. Igawa M. Rukstalis DB. Tanabe T, et al. High level of nm23 expression are related to cell proliferation in human prostate cancer .Cancer Res 1994;54: 1313
73. Howlett AR. Petersen OW. Steeg PS, et al . A novel fuction for the nm23 H_1 gene :overexpression in human breast carcinoma cells lead to the formation of basement membrane and growth arrest. J Natl Cancer Inst ,1999;86:1838
74．郑晓勇、林芷英等。原位微环境与nm23-H_1、H-rasmRNA量及肝癌肝内转移的关系。中华肿瘤杂志。1998；20(1)15-17
75. Weidner N. Semple JP. Welch WR, et al. Tumor angiogenesis and metastasis :correlation in invasive breast carcinoma .N Engl J Med ,1991:324:1
76. Weinder N. Carroll PR, Flax J, et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Path ,1993:143:401
77. Graham CH. Rivers J. Kerbel RS, et al. Extent of vascularization as a prognostic indicator in thin malignant melanomas. Am J Pathol 1994:145:510
78. Macchiarini P. Fontanini G. Hardin MJ, et al. Relation of neovascularization to metastasis of non-small-cell lung cancer. Lancet, 1996:340:145
79. Liotta L. Kleinerman J. Saldel G, et al. Quantitative relationship of intravascular tumor cells , tumor vessels, and pulmonary metstasis following tumor implantation .Cancer Res ,1974:34:997
80. Folkman J. How is blood vessel growth regulated in normal and neoplastic tissue? Cancer Res ,1986:46:467
81. Maeda K. Chung KS. Takatsuka S, et al. Tumor angiogenesis as a predictor
of recurrence in gastric carcinoma. J Clin Oncol, 1998:13:477
82． 徐杰等. 肝癌组织中VEGF、MVD的表达与肿瘤转移、复发之间的相关性. 1998:20(6) 440-442
83． El Assal ON. Yamanoi A, et al. Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver. Hepatology 1998:27(6) 1554-62
84． 夏景林、杨秉辉等. 原发性肝癌肿瘤血管密度及其表达的临床病理学意义. 中华肿瘤杂志, 1998, 20(6) :400-402
85． 俞民澎. 医学分子遗传学. 第一版. 北京: 科学出版社, 1990;491
86． Green P J, Pines O, Jnouye M . The role of antisense RNA in gene regulation . Ann Rev Biochem, 1986;55:569-97
87． Eguchi Y. Itoh T. Tomizawa J. Antisense RNA. Ann Rev Biochem .1991; 60:631-652
88． Denhart DT. Mechanism of action of antisense RNA. Ann N Y Acad Sci. 1992:660:70-76
89． Carter G. Lemorise N R. Antisense technology for cancer therapy : does it make sense? Br J Cancer ; 1993:67; 869-876
90． Kopper L. Kovalszkym I. Antisense tumor therapy ( a dream under construction ) In vivo .1994; 8:781-786
91． Zhang Wei Wei , Roth J A. Anti-oncogene and tumor suppressor gene therapy . In vivo ,1994: 8; 755-770
92． Rosenberg SA. Aebersold P. Cornetta K, et al . Gene transfer into human immunotherapy of patients with advanced medanoma, using tumor infiltrating lymphocytes modified by retroviral gene transdution . N Engl J Med. 1990, 323: 570
93． Jie Mu . Yoshiko Abe. Tateki Tsutsui, et al. Inhibition of growth and metastasis of ovarian carcinoma by administering a drug capable of interfering with vascular endothelial growth factor activity. Cancer Res , 1996; 87: 963-971
94． Ellis LM. Wenbiao L. Wilson M. Down-regulation of vascular endothelial growth factor in human colon carcinoma cell lines by antisense transfection decreases endothelial cell proliferation . Surgery. 1999; 120:871-878
95． Borgstrom. Per Kennetch J. Et al . Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody : novel concepts of angiostatic therapy from intravital videmicroscopy , Cancer Res , 1999:56:4032-4039
96． Zhu Z. Witte LSR. Inhibition of tumor growth and metastasis by targeting tumor-associated angiogenesis with antagonists to the receptors of vascular endothelial growth factor. Invest New Drug , 1999; 17(3) :195-212
97． Ke LD. Fueyo J, et al. A novel approach to glioma gene therapy: down-rugulation of the vascular endothelial growth factor in glioma cells using ribozymes. Int J Oncol ,1998; 12(6) :1391-1396
98． Rendall RL. Thomas KA. et al. Inhibition of vascular endithelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci 1993; 90;10705-10709
99． Wedge SR. Ogilvie DJ. Dukes M et al. ZD4190: an orally active inhibitor of vascular endothelial growth factor signaling with broad spectrum antitumor efficacy. Cancer Res .2000: 60: (4) :970-975
100. Ramakrishnan S. Olson TA. Bautch V, et al. Vascular endothelial growth factor-toxin conjuate specifically inhibits KDR/flk-1 positive endothelial cell proliferaton in vitro and angiogenesis in vivo. Cancer Res.1996: 56:1324-1330
101. Zamecnic PC, Stephenson ML. Inhibition of RSV replication and cell transplantation by a specific oligdexynucleotide. Proc Natl Acad Sci USA 1978: 75:280
102. Dachs GU. Doupherty GJ. Stratford IJ, et al. Targeting gene therapy to cancer: a review. Oncol Res 1997; 9:313-25
103. Kopper L. Alsaky IK. Antisense tumor therapy (a dream under construction). In vivo .1994: 8:781-6
104. Helene C. Control of oncogene expression by antisense nucleic acids. Eur J Cancer 1994; 30A;1721-6
105. Feng M. Cabrera G. Deshane J,et al . Neoplastic reversion accomplished by high efficiency adenoviral-mediated delivery of an anti-ras Ribozyme. Cancer Res 1995; 55:2024-8
106. Gibson I. Antisense approaches to the gene therapy of cancer .Cancer and Metastasis Rev. 1996:15:287-99
107. Philipl .Feigner . Lipofection : a highly efficient , lipid-mediated DNA transfection procedure .Proc Natl Acad Sci USA, 1987, 84; 7413
108. Xiao HZ. Leaf H,et al. DNA transfection mediated by cationic liposomes containing lipolysine : charactrization and mechanism of action .Biochem Biophys Acta, 1995, 135:1189
109．王彪、林其谁。核酸转染技术和阳离子脂质体。细胞生物学杂志，1998：20：21
110．张灵芝，温进坤。影响外源性基因在体内表达的因素。国外医学分子生物学分册。1993，15；124
111．姜泊。分子生物学常见实验方法。人民军医出版社，1996，20
112. Millauer B, Shawver LK, Plate KH,et al . Giloblastoma growth inhibited in vivo by a dominant-negative flk-1mutant. Nature, 1994:367:576-599
113. Saleh M. Stacker SA. Wilks AF. Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res 1996; 56:393-401
114. Wyllie AH. Kerr JFR. Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980, 68:251-306
115. Wyllie AH. The biology of cell death in tumors. Anticancer Res .1985,5;131-142
116. Wright SC. Zhong J. Larrick JW. Inhibition of apoptosis as a mechanism of tumor promotion. FASEB J. 1994,8:654-60
117. Kamohara Y , Kanematsu T . Treatment of liver cancer: current status and future prospectives. Gan To Kagaku Ryoho 2000 Jul; 27(7):987-92
118. Fevery J. Non-surgical treatment of hepatocellular carcinoma (HCC) . Acta Gastroenterol Belg 2000; 63(2):233-5
119. Badvie S. Hepatocellular carcinoma. Postgrad Med J 2000 Jan;76(891):4-11
120. Fried MW. Treatment of hepatocellular carcinoma: medical options. Liver Transpl Surg 1998;4(5 Suppl 1):S92-7
121. Ruiz J, Qian C, Prieto J. Gene therapy of liver tumors: principles and applications. Digestion 1998 Jul;59 Suppl 2:92-6
122. Carroll NM, Tanabe KK. Gene therapy for liver tumors. Surg Oncol Clin N Am 1998 Jul;7(3):617-26
123. Havlik R, Kral V. Gene therapy of liver tumors . Rozhl Chir 2000 Jun;79(6):235-8
124. Mitry RR, Mansour MR, Havlik R, Habib NA. Gene therapy for liver tumours. Adv Exp Med Biol 2000;465:193-205
125. Ott M, Schmidt HH, Cichon G, Manns MP. Emerging therapies in hepatology:liver-directed gene transfer and hepatocyte transplantation. Cells Tissues Organs 2000; 167(2-3):81-7
126．彭黎明．六种细胞凋亡检测方法的比较．中华病理学杂志，1999，28 (1)：55-57
127．李晓明，汤钊猷等．突变型flk-1基因转染抑制肝癌在裸鼠体内血管生成、生长和转移。中国肿瘤生物治疗杂志，1998，09 30：5(3)：160-162
128. Li H; Griscelli F et al . Lindenmeyer F Systemic delivery of antiangiogenic adenovirus AdmATF induces liver resistance to metastasis and prolongs survival of mice. Hum Gene Ther 1999 Dec 10;10(18):3045-53.
129. Sherczinger et al . Variables affecting antisense RNA inhibition of gene expression . Ann N Y Acad Sci , 1998; 660:45-56
130. Roninson I B,Gudkov A V, et al .Genetic suppressor elements:New tools for molecular oncology-thirteenth Cornelius P. Rhoads Memorial Award Lecture . Cancer Res ,1999:55:4023-4028
2．王庆生，郝希山，于世北．天津市1993年-1997年恶性肿瘤死亡率分析．中国肿瘤临床．2001；28：71-2
3．汤钊猷主编．原发型肝癌．上海：上海科学技术出版社．1981：1
4. Ott M; Schmidt HH; Emerging therapies in hepatology: liver-directed gene transfer and hepatocyte transplantation. Cells Tissues Organs 2000; 167(2-3):81-7.
5. Fevery J .Non-surgical treatment of hepatocellular carcinoma (HCC). Acta Gastroenterol Belg 2000 Apr-Jun;63(2):233-5.
6. Ruiz J; Qian C Gene therapy of viral hepatitis and hepatocellular carcinoma. J Viral Hepat 1999 Jan;6(1):17-34.
7. Usatoff V; Habib N .Hepatic malignancy: challenges and opportunities for the surgeon. J R Coll Surg Edinb 2000 Apr;45(2):99-109.
8. Mitry RR; Mansour MR .Gene therapy for liver tumours. Adv Exp Med Biol 2000;465(7): 193-205.
9. Leung DW, Cachlanes G,et al. Vascular endothelial growth factor is a secreted angiogenic mitogen .Science 1989;246:1306-1309
10. Folkman J,Klagsbrun M.Angiogenic factors. Science 1987;235:442-447
11. Deroanne CF, et al .Angiogenesis by fibroblast growth factor 4 is mediated through an autcrin up-regulation of vascular endothelial growth factor expression.Cancer Res, 1999,57:5590
12. Poltorak Z,Cohen T,et al. VEGF_(145), a secreted vascular endothelial growth factor isoform that binds to extracellular matrix .J Biol Chem, 1997 ;272:7151
13. Bowen W. The effects of surgical interference with the blood supply on the growth of transplanted carcinomata and sarcomata. Sci Rep Imperial Cancer Res Fund 1908; 3:146-158
14. Tanock IF. The relationship between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Br J Cancer 1968;22:258-273
15. Folkman J. Anti-angiogenesis: new concept for therapy of solid tumours. Ann Surg 1972;175:409-416
16. Gimbrone M, Leapman S,et,al.Tumour dormancy in vivo by prevention of neo-vascularisation .J Exp Med 1972; 136:261-276
17. Folkman JD. Clinical application of research on angiogenesis.N Engl J Med 1998;333(26): 1757-1763
18. Furcht LT. Critical factors controlling angiogenesis :cell products, cell matrix and growth factors. Lab Invest 1986;55(5);505-509
19. Hobson B. Denekamp J.Endothelial proliferation in tumours and normal tissues continous labelling studies. Br J Cancer 1984:49:405-413
20. Liotta L. Kleinerman J. Quantitative relationship of intravascular tumout cells, tumour vssels and pulmonary metastasis following tumour implantation . Cancer Res 1974:34:997-1004
21. Alguire GH. The transparent chamber technique as a tool in experiment tumour therapy .In Symposia : approaches to tumour chemotherapy .Washington DC: American Association for the Advancement of Science .1947:13-26
22． Alguire GH. Vascular reactions of normal and malignant tumours in vivol. Vascular reaction of mice to wounds and to normal and to neoplastic . J Nat) Cancer Inst 1945;6;73-85
23． Folkman J. Tumor angiogenesis : therapeutic implications. N Engl J Med 1971 ;285:1182-1186
24． Folkman J. What ie the evidence that tumours are angiogenesis dependent? J Natl Cancer Inst 1990:82:4-7
25． Folkman J. Watson K,et,al. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989;339:58-61
26． Weidner N.Carroll PR.et al. Tumour angiogenesis correlates with metastasis in invasive prostate carcinoma .Am J Pathol 1993:143:401-409
27． Hanahan D, Folkman J .Patterns and emerging mechanism of the angiogenic switch during tumorigenesis .Cell 1996;86;353-364
28． Gospodarowicz D. Lau K. Pituitary follicular cells secret both vascular epidermal growth factor and follistatin . Biochem Biophys Res Commun 1989:165:292-298
29． Dvorak HF. Broun LF. Detmar M,et al . Vascular permeability factor /vascular endothelial growth factor ,microvascular hyperpermeability and angiogenesis .Am J Pathol 1999;146:1029-1039
30． Gimbrone M. Leapman S. Cotran R. Folkman J. Tumor dormancy in vivo by prevention of neo-vascularisation J Exp Med 1972 :136:261-276
31． Klagbrum M. Amore PA. Regulators of angiogenesis . Annu Rev Physiol 1994;53;217-239
32． Plate KH, et al. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo .Natuer ,1992． 359:845
33． Samoto K.et al. Expression of vascular growth factor and its possible relation with neovascularization in human brain tumors. Cancer Res;1995;55:1189
34． Lichtor T, Dohrmann GJ, Gurney ME, et al .Cytokine gene expression by human gliomas. Neurosurgery 1990; 26;788-793
35． Takanami I, et al. Vascular endothelial growth factor and its receptor correlate with angiogenesis and survival in pulmonary adenocarcinoma. Anti Cancer Res ,1997:17;2811
36． Mattern J. Coexpression of VEGF and bFGF in human epidermoid lung carcinoma is associated with increased vessel density . Anti Cancer Res ,1997;17:2242
37． Maeda K, et al. Prognostic of VEGF endothelial growth factor expression in gastric carcinoma .Cancer ,1996,77:858
38． 陶厚权, 林言箴等. 血管内皮生长因子表达在胃癌浸润转移中的作用. 中华消化杂志 1998, 10,18(5) : 287
39． Zhang HT, et al. Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells. J Natl Cancer Inst ,1995,87:213
40． Fox SB, Gatter KC,Bicknell B, et al. Relationship of endothelial cell proliferation to tumor vascularity in human breast cancer . Cancer Res,1993 :53:4161-4163
41． Boocock CA, et al . Expression of vascular endothelial growth factor and its receptors Fit and KDR in ovarian carcinoma. J Natl Cancer Inst , 1999,87:506-509
42． Senger DR. Gvorak AM, et al. Tumor cells secrete a vascular permeability
factor that promotes accumulation ascites fluid. Science 1983:219(4587):983-988
43. UICC. TNM classification of malignant tumours .4th Ed. Berlin : Springer-Verlag, 1987:53
44．卢圣栋，主编。现代分子生物学技术。中国协和医科大学出版社。
45．金冬雁，黎孟枫等译。J．萨姆布鲁斯．分子克隆 北京：科学出版社．1992
46．李晓明、汤钊猷等。血管内皮细胞生长因子的表达与肝细胞癌侵袭和转移的关系。中华肿瘤杂志，1998；20：(1)12-14
47. Maeda K, Chung KS, Takatsuka S, et al. Tumor angiogenesis as a predictor of recurrence in gastric carcinoma. J Clin Oncol, 1995:13:477
48. Ferrara N. Henzal WJ. Pituitary follicular cells secrets a novel heparinbinding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989:161:851-858
49. Muller YA. Christinger HW. Keyt BA. Et al. The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 resulution : multiple copy flexidility and receptor binding . Structer, 1995;5(10):1325
50. Siemeister G. Marm D. Marting BG. The alpha-helical domain near the amino terminus is essential for dimerization of vscular endothelial growth factor. J Biol Chem ,1998:273 (18):11115
51. Soker S. Gollamudi S. Fikler H et al. Inhibition of vascular endothelial growth factor-induced endothelial cell proliferation by a peptide corresponding to the Exon-encoded domain of VEGF_(165). J Biol Chem. 1997; 272;31582
52. Brow LF. Lanir N. McDonagh J, Et al . Fibroblast migration in fibrin gel matrices. Am J Pathol ,1998:142(1):273
53. Kohn E. Liotta L. Molecular insights into cancer invasion :strategies for prevention and intervention. Cancer Res ,1995:55:1865
54. Folkman J. Angiogenesis in cancer , vascular , rheumatoid and other diseases. Nature Med , 1997:1:27
55. Senger D. Van De Walter L. et al . Vascular permeability factor (VPF/VEGF) in tumor biology .Cancer Metastasis Rev 1993:12:303-305
56. Collins PD. Connolly DT. Williams TJ. Characterization of the increase in vascular permeability induced by vascular permeability factor in vivo. Br J Pharamacol 1999: 109:195-199
57. Miura H. Miyazaki T et al . Increased expression of vascular endothelial growth factor in human hepatocellular carcinoma. J Hepatol 1997:27(5):854-61
58. Li XM et al. Serum vascular endothelial growth factor is a predictor of invasion and metastasis in hepatocellular carcinoma J Exp Clin Cancer Res 1999:18(4)511-7
59. Torimura T. Sata M et al . Increased expression of vascular endothelial growth factor is associated with tumour progression in hepatocellular carcinoma . Hum Pathol 1999:29(9):986-91
60. Suzuki K. Hayashi N et al . Expression of Vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma. Cancer Res 1999:56(13):3004-9
61. Mise M . Arii S et al. Clinical significance of vascular endothelial growth factor and basic fibroblast growth factor gene expression in liver cancer. Hepatology 1996;23(3);455-64
62．蒋扬富、杨治华等。血管内皮生长因子和基质金属蛋白酶-9 在原发性肝癌中的表达及其临床意义。癌症 2000：19(1)17-19
63．纪子钊、王宝胜等。VEGF p53和p21ras在原发性肝细胞癌中的表达及其临床病理意义。中国现代医学杂志 1999；9(4)：6-8
64. Yamaguchi R. Yano H et al. Expression and localization of vascular endothelial growth factor receptors in human hepatocellular carcinoma and non-HCC tissues. Oncol Rep 2000:7(4):725-9
65. Suzuki H. Seto K et al . Paracrine upregulation of VEGF receptor mRNA in endothelial cells by hypoxia-exposed hep G_2 cells. Am J Physiol 1999:276(1):G92-7
66．李江、王文亮、刘斌。人类肝细胞肝癌的细胞凋亡和癌组织内血管形成的调控。华人消化杂志 1998：6(12)：1057-1060。
67. Steeg PS. Bevilacqua G. Kopper L, et al. Evidence for a novel gene associated with low tumor metastasis potential . J Natl Cancer Inst. 1988: 80:200-204
68. Bevilaequa G. Sobel ME. Liotta LA, et al . Association of low nm23 RNA levels in human primary infiltrating ductal breast caricinoma with lymph node involvment and other histopathological indicators of high metastasis potential . Cancer Res . 1989:49:5185-5190
69. Yamaguchi A. Urano T. Fushida S, et al. Inverse association of nm23-H_1 expression by colorectal cancer with liver metastasis .Br J Cancer 1993, 68:1020-1024
70. Wang L. Patel U. Ghosh L, et al . Mutation in the nm23 gene is association with metastasis in colorectal cancer. Cancer Res, 1993:53:3652
71. Lizuka N. Masski O. Name T, et al . nm23-H_1 and nm23-H_2 messanger RNA abundance in HCC. Cancer Res ,1999:55:652
72. Igawa M. Rukstalis DB. Tanabe T, et al. High level of nm23 expression are related to cell proliferation in human prostate cancer .Cancer Res 1994;54: 1313
73. Howlett AR. Petersen OW. Steeg PS, et al . A novel fuction for the nm23 H_1 gene :overexpression in human breast carcinoma cells lead to the formation of basement membrane and growth arrest. J Natl Cancer Inst ,1999;86:1838
74．郑晓勇、林芷英等。原位微环境与nm23-H_1、H-rasmRNA量及肝癌肝内转移的关系。中华肿瘤杂志。1998；20(1)15-17
75. Weidner N. Semple JP. Welch WR, et al. Tumor angiogenesis and metastasis :correlation in invasive breast carcinoma .N Engl J Med ,1991:324:1
76. Weinder N. Carroll PR, Flax J, et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Path ,1993:143:401
77. Graham CH. Rivers J. Kerbel RS, et al. Extent of vascularization as a prognostic indicator in thin malignant melanomas. Am J Pathol 1994:145:510
78. Macchiarini P. Fontanini G. Hardin MJ, et al. Relation of neovascularization to metastasis of non-small-cell lung cancer. Lancet, 1996:340:145
79. Liotta L. Kleinerman J. Saldel G, et al. Quantitative relationship of intravascular tumor cells , tumor vessels, and pulmonary metstasis following tumor implantation .Cancer Res ,1974:34:997
80. Folkman J. How is blood vessel growth regulated in normal and neoplastic tissue? Cancer Res ,1986:46:467
81. Maeda K. Chung KS. Takatsuka S, et al. Tumor angiogenesis as a predictor
of recurrence in gastric carcinoma. J Clin Oncol, 1998:13:477
82． 徐杰等. 肝癌组织中VEGF、MVD的表达与肿瘤转移、复发之间的相关性. 1998:20(6) 440-442
83． El Assal ON. Yamanoi A, et al. Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver. Hepatology 1998:27(6) 1554-62
84． 夏景林、杨秉辉等. 原发性肝癌肿瘤血管密度及其表达的临床病理学意义. 中华肿瘤杂志, 1998, 20(6) :400-402
85． 俞民澎. 医学分子遗传学. 第一版. 北京: 科学出版社, 1990;491
86． Green P J, Pines O, Jnouye M . The role of antisense RNA in gene regulation . Ann Rev Biochem, 1986;55:569-97
87． Eguchi Y. Itoh T. Tomizawa J. Antisense RNA. Ann Rev Biochem .1991; 60:631-652
88． Denhart DT. Mechanism of action of antisense RNA. Ann N Y Acad Sci. 1992:660:70-76
89． Carter G. Lemorise N R. Antisense technology for cancer therapy : does it make sense? Br J Cancer ; 1993:67; 869-876
90． Kopper L. Kovalszkym I. Antisense tumor therapy ( a dream under construction ) In vivo .1994; 8:781-786
91． Zhang Wei Wei , Roth J A. Anti-oncogene and tumor suppressor gene therapy . In vivo ,1994: 8; 755-770
92． Rosenberg SA. Aebersold P. Cornetta K, et al . Gene transfer into human immunotherapy of patients with advanced medanoma, using tumor infiltrating lymphocytes modified by retroviral gene transdution . N Engl J Med. 1990, 323: 570
93． Jie Mu . Yoshiko Abe. Tateki Tsutsui, et al. Inhibition of growth and metastasis of ovarian carcinoma by administering a drug capable of interfering with vascular endothelial growth factor activity. Cancer Res , 1996; 87: 963-971
94． Ellis LM. Wenbiao L. Wilson M. Down-regulation of vascular endothelial growth factor in human colon carcinoma cell lines by antisense transfection decreases endothelial cell proliferation . Surgery. 1999; 120:871-878
95． Borgstrom. Per Kennetch J. Et al . Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody : novel concepts of angiostatic therapy from intravital videmicroscopy , Cancer Res , 1999:56:4032-4039
96． Zhu Z. Witte LSR. Inhibition of tumor growth and metastasis by targeting tumor-associated angiogenesis with antagonists to the receptors of vascular endothelial growth factor. Invest New Drug , 1999; 17(3) :195-212
97． Ke LD. Fueyo J, et al. A novel approach to glioma gene therapy: down-rugulation of the vascular endothelial growth factor in glioma cells using ribozymes. Int J Oncol ,1998; 12(6) :1391-1396
98． Rendall RL. Thomas KA. et al. Inhibition of vascular endithelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci 1993; 90;10705-10709
99． Wedge SR. Ogilvie DJ. Dukes M et al. ZD4190: an orally active inhibitor of vascular endothelial growth factor signaling with broad spectrum antitumor efficacy. Cancer Res .2000: 60: (4) :970-975
100. Ramakrishnan S. Olson TA. Bautch V, et al. Vascular endothelial growth factor-toxin conjuate specifically inhibits KDR/flk-1 positive endothelial cell proliferaton in vitro and angiogenesis in vivo. Cancer Res.1996: 56:1324-1330
101. Zamecnic PC, Stephenson ML. Inhibition of RSV replication and cell transplantation by a specific oligdexynucleotide. Proc Natl Acad Sci USA 1978: 75:280
102. Dachs GU. Doupherty GJ. Stratford IJ, et al. Targeting gene therapy to cancer: a review. Oncol Res 1997; 9:313-25
103. Kopper L. Alsaky IK. Antisense tumor therapy (a dream under construction). In vivo .1994: 8:781-6
104. Helene C. Control of oncogene expression by antisense nucleic acids. Eur J Cancer 1994; 30A;1721-6
105. Feng M. Cabrera G. Deshane J,et al . Neoplastic reversion accomplished by high efficiency adenoviral-mediated delivery of an anti-ras Ribozyme. Cancer Res 1995; 55:2024-8
106. Gibson I. Antisense approaches to the gene therapy of cancer .Cancer and Metastasis Rev. 1996:15:287-99
107. Philipl .Feigner . Lipofection : a highly efficient , lipid-mediated DNA transfection procedure .Proc Natl Acad Sci USA, 1987, 84; 7413
108. Xiao HZ. Leaf H,et al. DNA transfection mediated by cationic liposomes containing lipolysine : charactrization and mechanism of action .Biochem Biophys Acta, 1995, 135:1189
109．王彪、林其谁。核酸转染技术和阳离子脂质体。细胞生物学杂志，1998：20：21
110．张灵芝，温进坤。影响外源性基因在体内表达的因素。国外医学分子生物学分册。1993，15；124
111．姜泊。分子生物学常见实验方法。人民军医出版社，1996，20
112. Millauer B, Shawver LK, Plate KH,et al . Giloblastoma growth inhibited in vivo by a dominant-negative flk-1mutant. Nature, 1994:367:576-599
113. Saleh M. Stacker SA. Wilks AF. Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res 1996; 56:393-401
114. Wyllie AH. Kerr JFR. Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980, 68:251-306
115. Wyllie AH. The biology of cell death in tumors. Anticancer Res .1985,5;131-142
116. Wright SC. Zhong J. Larrick JW. Inhibition of apoptosis as a mechanism of tumor promotion. FASEB J. 1994,8:654-60
117. Kamohara Y , Kanematsu T . Treatment of liver cancer: current status and future prospectives. Gan To Kagaku Ryoho 2000 Jul; 27(7):987-92
118. Fevery J. Non-surgical treatment of hepatocellular carcinoma (HCC) . Acta Gastroenterol Belg 2000; 63(2):233-5
119. Badvie S. Hepatocellular carcinoma. Postgrad Med J 2000 Jan;76(891):4-11
120. Fried MW. Treatment of hepatocellular carcinoma: medical options. Liver Transpl Surg 1998;4(5 Suppl 1):S92-7
121. Ruiz J, Qian C, Prieto J. Gene therapy of liver tumors: principles and applications. Digestion 1998 Jul;59 Suppl 2:92-6
122. Carroll NM, Tanabe KK. Gene therapy for liver tumors. Surg Oncol Clin N Am 1998 Jul;7(3):617-26
123. Havlik R, Kral V. Gene therapy of liver tumors . Rozhl Chir 2000 Jun;79(6):235-8
124. Mitry RR, Mansour MR, Havlik R, Habib NA. Gene therapy for liver tumours. Adv Exp Med Biol 2000;465:193-205
125. Ott M, Schmidt HH, Cichon G, Manns MP. Emerging therapies in hepatology:liver-directed gene transfer and hepatocyte transplantation. Cells Tissues Organs 2000; 167(2-3):81-7
126．彭黎明．六种细胞凋亡检测方法的比较．中华病理学杂志，1999，28 (1)：55-57
127．李晓明，汤钊猷等．突变型flk-1基因转染抑制肝癌在裸鼠体内血管生成、生长和转移。中国肿瘤生物治疗杂志，1998，09 30：5(3)：160-162
128. Li H; Griscelli F et al . Lindenmeyer F Systemic delivery of antiangiogenic adenovirus AdmATF induces liver resistance to metastasis and prolongs survival of mice. Hum Gene Ther 1999 Dec 10;10(18):3045-53.
129. Sherczinger et al . Variables affecting antisense RNA inhibition of gene expression . Ann N Y Acad Sci , 1998; 660:45-56
130. Roninson I B,Gudkov A V, et al .Genetic suppressor elements:New tools for molecular oncology-thirteenth Cornelius P. Rhoads Memorial Award Lecture . Cancer Res ,1999:55:4023-4028
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