WST体外及体内药效学研究
摘要
背景和目的：近年来，以血管内皮生长因子(VEGF)及其受体(VEGFR)为靶点进行的抗血管生成治疗已成为肿瘤研究的新热点。WST是由人血管内皮生长因子受体VEGFR-1(Flt-1)和VEGFR-2(KDR)中与VEGF结合的结构域以及人免疫球蛋白IgG中的Fc片断组成。本课题即对WST的抗肿瘤作用及其作用机制进行了探讨和研究。
方法：体外部分，采用ELISA法考察WST与VEGF的结合能力，采用CCK-8法考察WST对人脐静脉血管内皮细胞(HUVEC)生长的影响；体内部分，建立人结肠癌LoVo和人乳腺癌MCF-7裸鼠皮下移植瘤模型，考察WST在接种肿瘤细胞后及时给药和先成瘤后给药两种不同给药方式中，对肿瘤生长的影响。最后，对WST联合化疗药物进行抗肿瘤治疗的效果进行了初步研究。
结果：WST对VEGF具有高度的亲和能力(Kd=12.5 pM)，强于已上市的同类产品贝伐单抗Avastin(Kd=228.6 pM)；WST能够抑制HUVEC细胞的生长增殖。10 nM以上的WST对0.2 nM VEGF诱导的内皮细胞增殖有明显的抑制作用(≥50％)，40 nM的WST对0.2 nM VEGF诱导的内皮细胞增殖抑制效应＞90％，而40 nM Avastin对0.2 nM VEGF诱导的内皮细胞增殖抑制效应为60％，表明在相同浓度下WST具有更好的抑制HUVEC细胞生长的作用；WST对人结肠癌LoVo和人乳腺癌MCF-7裸鼠皮下移植瘤模型中肿瘤的生长均具有较好的抑制作用，并呈现良好剂量效应关系。接种细胞及时给药与先成瘤后给药两种方式中，WST都显示出了较好的抑瘤效果，且与贝伐单抗Avastin相比，抑瘤效果更佳。病理组织学检查结果尚显示，WST的抗肿瘤作用与抑制肿瘤内血管新生及血管内皮细胞的生长有关。WST单用的抗肿瘤效果明显好于单用化疗药，而二者合用的抗肿瘤效果最佳，WST单用及与化疗药合用对动物体重均无影响。
结论：WST具有较为广泛的抗肿瘤活性，对人结肠癌LoVo和人乳腺癌MCF-7肿瘤均有较为明显的抑制作用，其抗肿瘤效果与剂量之间呈正相关；接种细胞及时给药与先成瘤后给药两种方式，WST均显示出较好的抑瘤效果；其与化疗药合用的抑瘤效果最佳。其抑瘤作用可能与其对VEGF的高度亲和有关，故而能够有效抑制血管内皮细胞的生长增殖，导致肿瘤内血管新生受到抑制而最终抗肿瘤。其体内、体外生物学活性均强于已上市的同类产品贝伐单抗Avastin。
Background and Aim: Vascular endothelial growth factor (VEGF) or its receptor (VEGFR) has been a new hot spot in cancer therapy study recently which is considered to be an important therapeutic target to angiogenesis. WST is made up of Fc fragment of IgG and the structure of VEGFR-1/Flt-1 and VEGFR-2/KDR which can combine with VEGF. In this research, its effect on tumors and mechanism was studied.
Methods: In vitro, the ability of connecting to VEGF was studied with ELISA method, and the effect on HUVEC was studied with CCK-8 method. In vivo, on the foundation of transplantated tumor model including human colon carcinoma (LoVo) and breast cancer (MCF-7) on the nude mice, the effect on tumors were studied with different ways in which some mice were administrated after inoculating tumor cells immediately, others were administrated after the tumors had grown up. Finally, the effect on tumors was studied after WST and chemotherapeutics were administered alliance.
Results: WST has a high binding ability to VEGF (Kd is 12.5 PM). This is more powerful than Avastin (Kd is 228.6 PM). WST could inhibit the growth of HUVEC. When its concentration exceed 10nM, its inhibition is obvious (>50%).when its concentration exceed 40nM, its inhibition exceeds 90%. But the inhibition of Avastin is 60% at same concention. So, WST has more excellent effect on HUVEC than Avastin. WST have obvious inhibition to human colon carcinoma (LoVo) and breast cancer (MCF-7). The inhibition relatived to its dose. WST has satisfactory inhibition to tumors not only being administered immediately after inoculating tumor cells, but also after the tumors had grown up. WST has more obvious inhibition than Avastin. The results of histopathologic examination indicated that the effect was relatived to the inhibition of tumor angiogensis and the growth of vascular endothelial cells. The inhibition of WST is stronger than chemotherapeutics, but the inhibition is strongest after they are administered alliance. WST has not effect on the body weigh of animal. Conclusion: WST have obvious effect on tumors. It can obviously inhibit the growth of human colon carcinoma (LoVo) and breast cancer (MCF-7). The inhibition relative with its dose.WST has satisfactory inhibition to tumor not only by administration immediately after inoculating tumor cells, but also by administration after the tumor had grown up. The inhibition.is strongest after WST and chemotherapeutics are co-administration. The effect could relatived with its ability of connecting to VEGF. So, it can inhibit the growth of vascular endothelial cells, angiogensis and tumors. Its biology activity is stronger than Avastin in vitro or in vivo.
方法：体外部分，采用ELISA法考察WST与VEGF的结合能力，采用CCK-8法考察WST对人脐静脉血管内皮细胞(HUVEC)生长的影响；体内部分，建立人结肠癌LoVo和人乳腺癌MCF-7裸鼠皮下移植瘤模型，考察WST在接种肿瘤细胞后及时给药和先成瘤后给药两种不同给药方式中，对肿瘤生长的影响。最后，对WST联合化疗药物进行抗肿瘤治疗的效果进行了初步研究。
结果：WST对VEGF具有高度的亲和能力(Kd=12.5 pM)，强于已上市的同类产品贝伐单抗Avastin(Kd=228.6 pM)；WST能够抑制HUVEC细胞的生长增殖。10 nM以上的WST对0.2 nM VEGF诱导的内皮细胞增殖有明显的抑制作用(≥50％)，40 nM的WST对0.2 nM VEGF诱导的内皮细胞增殖抑制效应＞90％，而40 nM Avastin对0.2 nM VEGF诱导的内皮细胞增殖抑制效应为60％，表明在相同浓度下WST具有更好的抑制HUVEC细胞生长的作用；WST对人结肠癌LoVo和人乳腺癌MCF-7裸鼠皮下移植瘤模型中肿瘤的生长均具有较好的抑制作用，并呈现良好剂量效应关系。接种细胞及时给药与先成瘤后给药两种方式中，WST都显示出了较好的抑瘤效果，且与贝伐单抗Avastin相比，抑瘤效果更佳。病理组织学检查结果尚显示，WST的抗肿瘤作用与抑制肿瘤内血管新生及血管内皮细胞的生长有关。WST单用的抗肿瘤效果明显好于单用化疗药，而二者合用的抗肿瘤效果最佳，WST单用及与化疗药合用对动物体重均无影响。
结论：WST具有较为广泛的抗肿瘤活性，对人结肠癌LoVo和人乳腺癌MCF-7肿瘤均有较为明显的抑制作用，其抗肿瘤效果与剂量之间呈正相关；接种细胞及时给药与先成瘤后给药两种方式，WST均显示出较好的抑瘤效果；其与化疗药合用的抑瘤效果最佳。其抑瘤作用可能与其对VEGF的高度亲和有关，故而能够有效抑制血管内皮细胞的生长增殖，导致肿瘤内血管新生受到抑制而最终抗肿瘤。其体内、体外生物学活性均强于已上市的同类产品贝伐单抗Avastin。
Background and Aim: Vascular endothelial growth factor (VEGF) or its receptor (VEGFR) has been a new hot spot in cancer therapy study recently which is considered to be an important therapeutic target to angiogenesis. WST is made up of Fc fragment of IgG and the structure of VEGFR-1/Flt-1 and VEGFR-2/KDR which can combine with VEGF. In this research, its effect on tumors and mechanism was studied.
Methods: In vitro, the ability of connecting to VEGF was studied with ELISA method, and the effect on HUVEC was studied with CCK-8 method. In vivo, on the foundation of transplantated tumor model including human colon carcinoma (LoVo) and breast cancer (MCF-7) on the nude mice, the effect on tumors were studied with different ways in which some mice were administrated after inoculating tumor cells immediately, others were administrated after the tumors had grown up. Finally, the effect on tumors was studied after WST and chemotherapeutics were administered alliance.
Results: WST has a high binding ability to VEGF (Kd is 12.5 PM). This is more powerful than Avastin (Kd is 228.6 PM). WST could inhibit the growth of HUVEC. When its concentration exceed 10nM, its inhibition is obvious (>50%).when its concentration exceed 40nM, its inhibition exceeds 90%. But the inhibition of Avastin is 60% at same concention. So, WST has more excellent effect on HUVEC than Avastin. WST have obvious inhibition to human colon carcinoma (LoVo) and breast cancer (MCF-7). The inhibition relatived to its dose. WST has satisfactory inhibition to tumors not only being administered immediately after inoculating tumor cells, but also after the tumors had grown up. WST has more obvious inhibition than Avastin. The results of histopathologic examination indicated that the effect was relatived to the inhibition of tumor angiogensis and the growth of vascular endothelial cells. The inhibition of WST is stronger than chemotherapeutics, but the inhibition is strongest after they are administered alliance. WST has not effect on the body weigh of animal. Conclusion: WST have obvious effect on tumors. It can obviously inhibit the growth of human colon carcinoma (LoVo) and breast cancer (MCF-7). The inhibition relative with its dose.WST has satisfactory inhibition to tumor not only by administration immediately after inoculating tumor cells, but also by administration after the tumor had grown up. The inhibition.is strongest after WST and chemotherapeutics are co-administration. The effect could relatived with its ability of connecting to VEGF. So, it can inhibit the growth of vascular endothelial cells, angiogensis and tumors. Its biology activity is stronger than Avastin in vitro or in vivo.
引文
1．国家药品监督管理局，《药品非临床研究质量管理规范》，2003年8月
2．卫生部药政管理局，《新药(西药)临床前研究指导原则汇编》，1993年。
3．徐叔云，卞如濂，陈修主编．《药理试验方法学》第三版，人民卫生出版社，2005年5月
4．贺师鹏，胡雅儿，夏宗勤主编．《受体研究技术》，北京大学医学出版社，2004年
5．杨宝峰主编，《药理学》第6版，人民卫生出版社，2003年
6. Kim Eugene S, et al., Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. PNAS 99(17): 11399-11404, 2002
7. Mitsuharu and Murray. Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic Cancer Cell Lines. Clinical Cancer Research 10: 3327-3332, 2004
8. Napoleone F. Vascular endothelial growth factor as a target for anticancer therapy. The Oncologist 9 (suppl 1): 2-10, 2004
9. Yoko H, et al., Soluble Flt-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Research 62: 2019-2023, 2002
10. Jocelyn H, Sam D, Nick P, et al., VEGF-Trap: A VEGF blocker with potent antitumor effects. PNAS 99 (17): 11393-11398, 2002
11. Neufeld, Cohen 1, Stela G, et al. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J, 1999,18(1)30～32
12. Lymboussaki A, Achen MG, Stacker SA, et al. Growth factors regulating lymphatic vessels[J]. Curr Top Microbiol Immunol, 2000, 25(1):75～82
13. Baillie R, Harada K, Bradley NJ, et al. Expression of vascular endothelial growth factor in normal and tumor oral tissues with different antibodies[J]. The Histochemical Journal, 2001, 33(2): 287～294.
14．陈珊，金伟，闵平等．血管内皮生长因子家族及其受体与肿瘤血管生成研究进展[J]．生命科学，2004，16(1)：19～23
1．国家药品监督管理局，《药品非临床研究质量管理规范》，2003年8月
2．卫生部药政管理局，《新药(西药)临床前研究指导原则汇编》，1993年。
3．徐叔云，卞如濂，陈修主编．《药理试验方法学》第三版，人民卫生出版社，2005年5月
4．贺师鹏，胡雅儿，夏宗勤主编．《受体研究技术》，北京大学医学出版社，2004年
5．司徒镇强主编，《细胞培养》第一版，世界图书出版公司西安分公司出版，2004年
6．杨宝峰主编，《药理学》第6版，人民卫生出版社，2003年
7. Jocelyn H, et al., VEGF-Trap: A VEGF blocker with potent antitumor effects. PNAS 99:11393-11398, 2002
8. Monica A, et al., Role of PIGF in the intra-and intermolecμlar cross talk between the VEGF receptors Fltl and Flk1. Nature Science 9(7): 936-943, 2003
9. Jacques G, et al., Preclinical pharmacokinetics of ranibizumab (rhuFabV2) after a single intravitreal administration. Investigative Ophthalmology & Visual Science 46(2): 726-733, 2005
10. Deeba H, et al., Safety and Efficacy of Intravitreal Injection of Ranibizμmab in Combination With Verteporfin PDT on Experimental Choroidal Neovascularization in the monkey. Arch Ophthalmol 123:509-516, 2005
11. Dvorak HF, Brown LF, Dermar M , et al. Vascular permeability factor, vascular endothelial growth factor, Micro vascular hyper permeability factor and angiogenesis[J]. A.J. Path 1995, 146:1029.
12. Hewett RW, Murry JC. Co-expression of Flt-1,Flt-4 and KDR in freshly isolated and cultured human endothelial cells [J]. Biochem Biophys Res Common, 1996, 221(3): 697～ 702
13. Makinen T, Jussila L, Veikkola T, et al. Inhibition of lymphangiogenesis with resulting lympbedema in transgenic mice expressing soluble VEGF receptor-3[J]. Nat Med, 2001, 7(2): 199～205
14. Lymboussaki A, Achen MG, Stacker SA, et al. Growth factors regulating lymphatic vessels[J]. Curr Top Microbiol Immunol, 2000, 25(1):75～82
1．国家药品监督管理局，《药品非临床研究质量管理规范》，2003年8月
2．卫生部药政管理局，《新药(西药)临床前研究指导原则汇编》，1993年。
3．徐叔云，卞如濂，陈修主编．《药理试验方法学》第三版，人民卫生出版社，2005年5月
4．贺师鹏，胡雅儿，夏宗勤主编．《受体研究技术》，北京大学医学出版社，2004年
5．司徒镇强主编，《细胞培养》第一版，世界图书出版公司西安分公司出版，2004年
6．杨宝峰主编，《药理学》第6版，人民卫生出版社，2003年
7．国家药品监督管理局，《细胞毒类抗肿瘤药物非临床研究技术指导原则》第二稿，2006年
8．国家卫生部，《抗肿瘤药物药效学指导原则》，1993年
9．国家食品药品监督管理局，《治疗用生物制品非临床安全性技术审评一般原则》(第二稿)，2005年
10. Gasparini G, Harris A. Clinical importance of the determination of tumor angiogenesis in breast carcinima: much more than a new prognostic tool[J]. J Clin Oncol, 1995, 13(3):765～782
11. Folkman J.What is the evidence that tumors are angio-genesis-dependent[J]. J Nat ICancer Inst, 1990,82(1):4～6
12. JainRK. Vascular and interstitial barriers to delivery of therapeutic agents in tumors [J] .Cancer Metastasis Rev, 1990,9:253～266
13．祝浩杰，李运曼，刘国卿．肿瘤血管生成抑制剂[J]．药学进展，2001，25(1)：17～21
14．王鹏，陈震，黄雯霞．抗VEGF单抗Avastin(bevacizumab)在肿瘤治疗中的应用研究进展[J]．国外医学肿瘤学分册，2004，12(31)：1～4
15. Kim Eugene S, et al., Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. PNAS, 2002, 99(17): 11399～11404
16. Mitsuharu and Murray. Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic Cancer Cell Lines. Clinical Cancer Research 2004,10:3327～3332
17. Napoleone F. Vascular endothelial growth factor as a target for anticancer therapy. The Oncologist, 2004, 9 (1): 2～10
18. Yoko H, et al., Soluble Flt-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Research , 2002, 6(2): 2019～2023
19. Jocelyn H, Sam D, Nick P, et al., VEGF-Trap: A VEGF blocker with potent antitumor effects. PNAS ,2002, 99 (17): 11393-11398
20. Monica A, et al., Role of PIGF in the intra-and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nature Science, 2003, 9(7): 936—943
21. Jacques G, et al., Preclinical pharmacokinetics of ranibizumab (rhuFabV2) after a single intravitreal administration. Investigative Ophthalmology & Visual Science, 2005, 46(2): 726-733,
22. Deeba H, et al., Safety and Efficacy of Intravitreal Injection of Ranibizumab in Combination With Verteporfin PDT on Experimental Choroidal Neovascularization in the monkey. Arch Ophthalmol ,2005,12(3): 509-516
23. Hans-Peter Gerber and Napoleone Ferrara. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cacer Res 2005,65(3): 671—680
24. Annette TB, Leorah R,Joceyln H, et al., Vascular endothelial growth factor-trap decreases tumor burden, inhibits ascites, and causes dramatic vascular remodeling in an ovarian cancer model. Clinical Cancer Research, 2003, 9:5721—5728,
25. Gerber HP, Napoleone F. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cacer Res 2005, 65(3):671-680
26. Limin H, Judith H, Jocelyn H, et al, Vascular endothelial growth factor trap combined with paclitaxel strikingly inhibits tumor and ascites, prolonging survival in a human ovarian cancer model. Clin Cancer Res, 2005, 11(19):6966—6971
27. Jacquemier J M P, Mathoulin—Portier R, Valtola E, et al. Prognosis of breast carcinoma lymphangiogenesis evaluated by immunohistochemical investigation of vascularendothelial growth factor receptor 3. Int Cancer, 2000, 89: 69—73
1．国家药品监督管理局，《药品非临床研究质量管理规范》，2003年8月
2．卫生部药政管理局，《新药(西药)临床前研究指导原则汇编》，1993年。
3．徐叔云，卞如濂，陈修主编．《药理试验方法学》第三版，人民卫生出版社，2005年5月
4．贺师鹏，胡雅儿，夏宗勤主编．《受体研究技术》，北京大学医学出版社，2004年
5．司徒镇强主编，《细胞培养》第一版，世界图书出版公司西安分公司出版，2004年
6．杨宝峰主编，《药理学》第6版，人民卫生出版社，2003年
7．国家药品监督管理局，《细胞毒类抗肿瘤药物非临床研究技术指导原则》第二稿，2006年
8．国家卫生部，《抗肿瘤药物药效学指导原则》，1993年
9．国家食品药品监督管理局，《治疗用生物制品非临床安全性技术审评一般原则》(第二稿)，2005年
10. Kim Eugene S, et al., Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. PNAS 99(17): 11399-11404, 2002
11. Mitsuharu and Murray. Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic Cancer Cell Lines. Clinical Cancer Research 10: 3327-3332, 2004
12. Napoleone F. Vascular endothelial growth factor as a target for anticancer therapy. The Oncologist 9 (suppl 1): 2-10, 2004
13. Yoko H, et al., Soluble Fit-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Research 62: 2019-2023, 2002
14. Jocelyn H, Sam D, Nick P, et al., VEGF-Trap: A VEGF blocker with potent antitumor effects. PNAS 99 (17): 11393-11398, 2002
15. Monica A, et al., Role of PIGF in the intra-and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nature Science 9(7): 936-943, 2003
16. Jacques G, et al., Preclinical pharmacokinetics of ranibizumab (rhuFabV2) after a single intravitreal administration. Investigative Ophthalmology & Visual Science, 46(2): 726-733, 2005
17. Deeba H, et al., Safety and Efficacy of Intravitreal Injection of Ranibizμmab in Combination With Verteporfin PDT on Experimental Choroidal Neovascularization in the monkey. Arch Ophthalmol 123: 509-516,2005
18. Hans-Peter Gerber and Napoleone Ferrara. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cacer Res 65(3): 671-680,2005
19. Annette TB, Leorah R,Joceyln H. et al., Vascular endothelial growth factor-trap decreases tumor burden, inhibits ascites, and causes dramatic vascular remodeling in an ovarian cancer model. Clinical Cancer Research 9:5721-5728,2003
20. Gerber HP, Napoleone F. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cacer Res 65(3):671-680, 2005
21. Limin H, Judith H, Jocelyn H, et al, Vascular endothelial growth factor trap combined with paclitaxel strikingly inhibits tumor and ascites, prolonging survival in a human ovarian cancer model. Clin Cancer Res 11(19):6966-6971, 2005
[1] David W Leung, George Cachianes, Wun-Jing Kuang, et al. Vascular endothelial growth factor is a secreted angiogenicmitogen. Science, 1989, 246(4 935): 1306～1310.
[2] 姜恒，郭光金．VEGF及其受体的研究进展[J]．局解手术学杂志，2004，13(2)：126～127．
[3] Neufeld, Cohen 1, Stela G, et al. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J, 1999,18(1)30～32
[4] Tischer E, Mitchell R, Hartman H, et al. The human gene for vascular endothelial growth factor [J]. J.Biol Chem, 1991, 266(18): 11947.
[5] Toi M, Matsumoto T, Bando H. Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications [J]. Lancet Oncol, 2001, 2(11): 667～673.
[6] Brekken RA, Thorpe PE. Vascular endothelial growth factor and vascular targeting of solid tumors [J]. Anticancer Res, 2001, 21 (6B): 4221～4229.
[7] Dvorak HF, Brown LF, Dermar M , et al. Vascular permeability factor, vascular endothelial growth factor, Micro vascular hyper permeability factor and angiogenesis[J]. A.J. Path 1995, 146:1029.
[8] Hewett RW, Murry JC. Co-expression of Flt-1,Flt-4 and KDR in freshly isolated and cultured human endothelial cells [J]. Biochem Biophys Res Common, 1996, 221(3): 697～702
[9] 李永东．血管内皮生长因子的研究进展[J]．中国误诊学杂志，2002，2(2)：200～203
[10] 姜恒，郭光金．VEGF及其受体的研究进展[J]．局解手术学杂志，2004，13(2)：126-127
[11] Jiang h, Guo gj. Advances in the studies of VEGF and its receptors [J]. J Regional Anat & Operative Surg, 2004, 13(2): 126～127
[12] Olofsson B, Jeltsch M, Eriksson U, et al. Current biology of VEGF-B and VEGF-C Curr Opin Biotechnol, 1999, 10(6): 528～535
[13] 周泉．血管内皮生长因子的生物学特性及临床研究进展[J]．实用心脑肺血管病杂志，2001，9(1)：51～54．
[14] He Y, Kozaki K, Karpanen T, et al. Suppression of tumor lymph angiogen- esis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling [J]. J Nath Cancer Inst, 2002, 94(11): 819～825
[15] 毛华，赵敏芳，袁爱力，等．血管内皮生长因子对肝癌细胞侵袭能力和同质性粘附作用影响[J]．肿瘤，2002，22(3)：197～199
[16] 陈治，杜钰．血管内皮细胞特异性受体在肿瘤血管靶向治疗中的应用[J]．世界华人消化杂志，2001，9(6)：702～705
[17] Yokoyama Y, Sato S, Futagami M, et al. Prognostic significance of vascular endothelial growth factor and its receptors in endometrial carcinoma [J].GynecolOncol, 2000, 77: 413～418
[18] 胥文春，陈新敏，罗春丽．血管内皮生长因子受体与恶性肿瘤[j]．国外医学临床生物化学与检验学分册，2003，24(3)：158～159
[19] SachieH, Yoshiro M, Akiko O, et al. Involvement of Flt-1 tyrosine kinase(vascular endothelial growth factor receptor-1) in pathological angiogenesis[J].Cancer Res, 2001, 61(3):1207～1213.
[20] Lam PM, Briton-Jones C, Cheuntg CK et al. In vivo regulation of mRNA expression of Vascular endothelial growth factor receptors(KDR and fit-1)in the human oviduct. Fertil Steril, 2004, 81(2): 416～423
[21] Suhandja A, Hoffman H. Role of growth factors and their receptors in proliferation of microvascular endothelial cell. Microsc Res Tech, 2003, 60: 70～75
[22] 尼玛卓玛，李亚里，朱运峰．血管内皮生长因子受体研究进展[J]．武警医学，2005，16(7)：542～544
[23] Makinen T, Jussila L, Veikkola T, et al. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3 [J]. Nat Med, 2001, 7(2): 199～205
[24] Lymboussaki A, Achen MG, Stacker SA, et al. Growth factors regulating lymphatic vessels[J]. Curr Top Microbiol Immunol, 2000, 25(1):75～82
[25] Toster L, Clapp L, Lrickson M, et al. Botulinum toxin A and chronic low back pain: a randomized, double-blind study [J]. Neurology.2001, 56(10): 1290～1293
[26] Baillie R, Harada K, Bradley NJ, et al. Expression of vascular endothelial growth factor in normal and tumor oral tissues with different antibodies[J]. The Histochemical Journal, 2001，33(2)：287～294．
[27] 朱新峰，林坚．血管内皮生长因子与肿瘤关系研究进展[J]．广西医科大学学报，2006，23(2)：333～335
[28] Padro T, Bieker R, Ruiz S, et al. Overexpression of vascular endothelial growth factor(VEGF) and its cellular receptor KDR(VEGFR-2) in the bone marrow of patients with acute myeloid leukemia[J]. Leukemia, 2002, 16(7): 1302～1310.
[29] Alessandra F, Taghi M,Zeev E, et al. High levels of vascular endothelial growth factor receptor-2 correlate with shortened survival in chronic lymphocytic leukemia[J]. ClinC ancer Res, 2001, 7(4):795～799.
[30] Masood R, Cm I, Zheng T, et al. Vascular endothelial growth factor vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma [J]. Pro NatlAcad Sci USA, 1997, 94(3): 979～984
[31] 刘丽军，肖畅，朱迅．VEGF及其受体抗肿瘤血管治疗应用新进展[J]．国外医学肿瘤学分册，2001，28(2)：113～115
[32] Rapisarda A, Uranchimeg B, Scudicro DA, et al. Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway [J]. Cancer Res, 2002, 62:4316～4324
[33] Asano M, Yukita A, Suzuki H. Wide spectrum of anti-tumor activity of neutralizing monoclonal antibody to human vascular endothelial growth factor[J]. Jpn J Cancer Res, 1999, 90(1),93～100
[34] Galdmn CK, Kendall RL, Cabrera G, et al. Panacrine expression of a native soluble vascular endothelial growth factor receptor inhibits turner growth, metastasis, and mortality rate [J]. Proc Nail Acad Sei USA, 1998, 95:879～880
[35] Zhai Y, Yu J, Iruela-Arispel., et al. Inhibition of angiogenesis and breast cancer engraft tumor growth by VEGI, a novel cytokine of the NF superfamily [J]. Int J Cancer, 1999, 82(1): 131～136
[36] Reilly MS, Holmgren L, Shing Y, et al. Angionstatin a novel angiogensis inhibitor that mediates the suooression of metastases by a Lewis lung carcinoma [J]. Cell, 1994, 79(2): 315～328
[37] Xie CG, Wang XP. Effect of selective cyclooxygenase-2 inhibitor celebrex on expression of vascular endothelial growth factor in pancreatic carcinoma [J]. Chinese Journal of Cancer, 2003,22(10): 1042-1046.
[38] Ramakrishnan S, Olson TA, Bautch VI, et al. Vascular endothelial growth factor toxin conjugate specifically inhibits KDR/Flk-1 positive endothelial cell proliferation in vitro and angiogenesis in Vivo [J]. Cancer Res, 1996, 56(6): 1324～1330
2．卫生部药政管理局，《新药(西药)临床前研究指导原则汇编》，1993年。
3．徐叔云，卞如濂，陈修主编．《药理试验方法学》第三版，人民卫生出版社，2005年5月
4．贺师鹏，胡雅儿，夏宗勤主编．《受体研究技术》，北京大学医学出版社，2004年
5．杨宝峰主编，《药理学》第6版，人民卫生出版社，2003年
6. Kim Eugene S, et al., Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. PNAS 99(17): 11399-11404, 2002
7. Mitsuharu and Murray. Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic Cancer Cell Lines. Clinical Cancer Research 10: 3327-3332, 2004
8. Napoleone F. Vascular endothelial growth factor as a target for anticancer therapy. The Oncologist 9 (suppl 1): 2-10, 2004
9. Yoko H, et al., Soluble Flt-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Research 62: 2019-2023, 2002
10. Jocelyn H, Sam D, Nick P, et al., VEGF-Trap: A VEGF blocker with potent antitumor effects. PNAS 99 (17): 11393-11398, 2002
11. Neufeld, Cohen 1, Stela G, et al. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J, 1999,18(1)30～32
12. Lymboussaki A, Achen MG, Stacker SA, et al. Growth factors regulating lymphatic vessels[J]. Curr Top Microbiol Immunol, 2000, 25(1):75～82
13. Baillie R, Harada K, Bradley NJ, et al. Expression of vascular endothelial growth factor in normal and tumor oral tissues with different antibodies[J]. The Histochemical Journal, 2001, 33(2): 287～294.
14．陈珊，金伟，闵平等．血管内皮生长因子家族及其受体与肿瘤血管生成研究进展[J]．生命科学，2004，16(1)：19～23
1．国家药品监督管理局，《药品非临床研究质量管理规范》，2003年8月
2．卫生部药政管理局，《新药(西药)临床前研究指导原则汇编》，1993年。
3．徐叔云，卞如濂，陈修主编．《药理试验方法学》第三版，人民卫生出版社，2005年5月
4．贺师鹏，胡雅儿，夏宗勤主编．《受体研究技术》，北京大学医学出版社，2004年
5．司徒镇强主编，《细胞培养》第一版，世界图书出版公司西安分公司出版，2004年
6．杨宝峰主编，《药理学》第6版，人民卫生出版社，2003年
7. Jocelyn H, et al., VEGF-Trap: A VEGF blocker with potent antitumor effects. PNAS 99:11393-11398, 2002
8. Monica A, et al., Role of PIGF in the intra-and intermolecμlar cross talk between the VEGF receptors Fltl and Flk1. Nature Science 9(7): 936-943, 2003
9. Jacques G, et al., Preclinical pharmacokinetics of ranibizumab (rhuFabV2) after a single intravitreal administration. Investigative Ophthalmology & Visual Science 46(2): 726-733, 2005
10. Deeba H, et al., Safety and Efficacy of Intravitreal Injection of Ranibizμmab in Combination With Verteporfin PDT on Experimental Choroidal Neovascularization in the monkey. Arch Ophthalmol 123:509-516, 2005
11. Dvorak HF, Brown LF, Dermar M , et al. Vascular permeability factor, vascular endothelial growth factor, Micro vascular hyper permeability factor and angiogenesis[J]. A.J. Path 1995, 146:1029.
12. Hewett RW, Murry JC. Co-expression of Flt-1,Flt-4 and KDR in freshly isolated and cultured human endothelial cells [J]. Biochem Biophys Res Common, 1996, 221(3): 697～ 702
13. Makinen T, Jussila L, Veikkola T, et al. Inhibition of lymphangiogenesis with resulting lympbedema in transgenic mice expressing soluble VEGF receptor-3[J]. Nat Med, 2001, 7(2): 199～205
14. Lymboussaki A, Achen MG, Stacker SA, et al. Growth factors regulating lymphatic vessels[J]. Curr Top Microbiol Immunol, 2000, 25(1):75～82
1．国家药品监督管理局，《药品非临床研究质量管理规范》，2003年8月
2．卫生部药政管理局，《新药(西药)临床前研究指导原则汇编》，1993年。
3．徐叔云，卞如濂，陈修主编．《药理试验方法学》第三版，人民卫生出版社，2005年5月
4．贺师鹏，胡雅儿，夏宗勤主编．《受体研究技术》，北京大学医学出版社，2004年
5．司徒镇强主编，《细胞培养》第一版，世界图书出版公司西安分公司出版，2004年
6．杨宝峰主编，《药理学》第6版，人民卫生出版社，2003年
7．国家药品监督管理局，《细胞毒类抗肿瘤药物非临床研究技术指导原则》第二稿，2006年
8．国家卫生部，《抗肿瘤药物药效学指导原则》，1993年
9．国家食品药品监督管理局，《治疗用生物制品非临床安全性技术审评一般原则》(第二稿)，2005年
10. Gasparini G, Harris A. Clinical importance of the determination of tumor angiogenesis in breast carcinima: much more than a new prognostic tool[J]. J Clin Oncol, 1995, 13(3):765～782
11. Folkman J.What is the evidence that tumors are angio-genesis-dependent[J]. J Nat ICancer Inst, 1990,82(1):4～6
12. JainRK. Vascular and interstitial barriers to delivery of therapeutic agents in tumors [J] .Cancer Metastasis Rev, 1990,9:253～266
13．祝浩杰，李运曼，刘国卿．肿瘤血管生成抑制剂[J]．药学进展，2001，25(1)：17～21
14．王鹏，陈震，黄雯霞．抗VEGF单抗Avastin(bevacizumab)在肿瘤治疗中的应用研究进展[J]．国外医学肿瘤学分册，2004，12(31)：1～4
15. Kim Eugene S, et al., Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. PNAS, 2002, 99(17): 11399～11404
16. Mitsuharu and Murray. Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic Cancer Cell Lines. Clinical Cancer Research 2004,10:3327～3332
17. Napoleone F. Vascular endothelial growth factor as a target for anticancer therapy. The Oncologist, 2004, 9 (1): 2～10
18. Yoko H, et al., Soluble Flt-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Research , 2002, 6(2): 2019～2023
19. Jocelyn H, Sam D, Nick P, et al., VEGF-Trap: A VEGF blocker with potent antitumor effects. PNAS ,2002, 99 (17): 11393-11398
20. Monica A, et al., Role of PIGF in the intra-and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nature Science, 2003, 9(7): 936—943
21. Jacques G, et al., Preclinical pharmacokinetics of ranibizumab (rhuFabV2) after a single intravitreal administration. Investigative Ophthalmology & Visual Science, 2005, 46(2): 726-733,
22. Deeba H, et al., Safety and Efficacy of Intravitreal Injection of Ranibizumab in Combination With Verteporfin PDT on Experimental Choroidal Neovascularization in the monkey. Arch Ophthalmol ,2005,12(3): 509-516
23. Hans-Peter Gerber and Napoleone Ferrara. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cacer Res 2005,65(3): 671—680
24. Annette TB, Leorah R,Joceyln H, et al., Vascular endothelial growth factor-trap decreases tumor burden, inhibits ascites, and causes dramatic vascular remodeling in an ovarian cancer model. Clinical Cancer Research, 2003, 9:5721—5728,
25. Gerber HP, Napoleone F. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cacer Res 2005, 65(3):671-680
26. Limin H, Judith H, Jocelyn H, et al, Vascular endothelial growth factor trap combined with paclitaxel strikingly inhibits tumor and ascites, prolonging survival in a human ovarian cancer model. Clin Cancer Res, 2005, 11(19):6966—6971
27. Jacquemier J M P, Mathoulin—Portier R, Valtola E, et al. Prognosis of breast carcinoma lymphangiogenesis evaluated by immunohistochemical investigation of vascularendothelial growth factor receptor 3. Int Cancer, 2000, 89: 69—73
1．国家药品监督管理局，《药品非临床研究质量管理规范》，2003年8月
2．卫生部药政管理局，《新药(西药)临床前研究指导原则汇编》，1993年。
3．徐叔云，卞如濂，陈修主编．《药理试验方法学》第三版，人民卫生出版社，2005年5月
4．贺师鹏，胡雅儿，夏宗勤主编．《受体研究技术》，北京大学医学出版社，2004年
5．司徒镇强主编，《细胞培养》第一版，世界图书出版公司西安分公司出版，2004年
6．杨宝峰主编，《药理学》第6版，人民卫生出版社，2003年
7．国家药品监督管理局，《细胞毒类抗肿瘤药物非临床研究技术指导原则》第二稿，2006年
8．国家卫生部，《抗肿瘤药物药效学指导原则》，1993年
9．国家食品药品监督管理局，《治疗用生物制品非临床安全性技术审评一般原则》(第二稿)，2005年
10. Kim Eugene S, et al., Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. PNAS 99(17): 11399-11404, 2002
11. Mitsuharu and Murray. Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic Cancer Cell Lines. Clinical Cancer Research 10: 3327-3332, 2004
12. Napoleone F. Vascular endothelial growth factor as a target for anticancer therapy. The Oncologist 9 (suppl 1): 2-10, 2004
13. Yoko H, et al., Soluble Fit-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Research 62: 2019-2023, 2002
14. Jocelyn H, Sam D, Nick P, et al., VEGF-Trap: A VEGF blocker with potent antitumor effects. PNAS 99 (17): 11393-11398, 2002
15. Monica A, et al., Role of PIGF in the intra-and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nature Science 9(7): 936-943, 2003
16. Jacques G, et al., Preclinical pharmacokinetics of ranibizumab (rhuFabV2) after a single intravitreal administration. Investigative Ophthalmology & Visual Science, 46(2): 726-733, 2005
17. Deeba H, et al., Safety and Efficacy of Intravitreal Injection of Ranibizμmab in Combination With Verteporfin PDT on Experimental Choroidal Neovascularization in the monkey. Arch Ophthalmol 123: 509-516,2005
18. Hans-Peter Gerber and Napoleone Ferrara. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cacer Res 65(3): 671-680,2005
19. Annette TB, Leorah R,Joceyln H. et al., Vascular endothelial growth factor-trap decreases tumor burden, inhibits ascites, and causes dramatic vascular remodeling in an ovarian cancer model. Clinical Cancer Research 9:5721-5728,2003
20. Gerber HP, Napoleone F. Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cacer Res 65(3):671-680, 2005
21. Limin H, Judith H, Jocelyn H, et al, Vascular endothelial growth factor trap combined with paclitaxel strikingly inhibits tumor and ascites, prolonging survival in a human ovarian cancer model. Clin Cancer Res 11(19):6966-6971, 2005
[1] David W Leung, George Cachianes, Wun-Jing Kuang, et al. Vascular endothelial growth factor is a secreted angiogenicmitogen. Science, 1989, 246(4 935): 1306～1310.
[2] 姜恒，郭光金．VEGF及其受体的研究进展[J]．局解手术学杂志，2004，13(2)：126～127．
[3] Neufeld, Cohen 1, Stela G, et al. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J, 1999,18(1)30～32
[4] Tischer E, Mitchell R, Hartman H, et al. The human gene for vascular endothelial growth factor [J]. J.Biol Chem, 1991, 266(18): 11947.
[5] Toi M, Matsumoto T, Bando H. Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications [J]. Lancet Oncol, 2001, 2(11): 667～673.
[6] Brekken RA, Thorpe PE. Vascular endothelial growth factor and vascular targeting of solid tumors [J]. Anticancer Res, 2001, 21 (6B): 4221～4229.
[7] Dvorak HF, Brown LF, Dermar M , et al. Vascular permeability factor, vascular endothelial growth factor, Micro vascular hyper permeability factor and angiogenesis[J]. A.J. Path 1995, 146:1029.
[8] Hewett RW, Murry JC. Co-expression of Flt-1,Flt-4 and KDR in freshly isolated and cultured human endothelial cells [J]. Biochem Biophys Res Common, 1996, 221(3): 697～702
[9] 李永东．血管内皮生长因子的研究进展[J]．中国误诊学杂志，2002，2(2)：200～203
[10] 姜恒，郭光金．VEGF及其受体的研究进展[J]．局解手术学杂志，2004，13(2)：126-127
[11] Jiang h, Guo gj. Advances in the studies of VEGF and its receptors [J]. J Regional Anat & Operative Surg, 2004, 13(2): 126～127
[12] Olofsson B, Jeltsch M, Eriksson U, et al. Current biology of VEGF-B and VEGF-C Curr Opin Biotechnol, 1999, 10(6): 528～535
[13] 周泉．血管内皮生长因子的生物学特性及临床研究进展[J]．实用心脑肺血管病杂志，2001，9(1)：51～54．
[14] He Y, Kozaki K, Karpanen T, et al. Suppression of tumor lymph angiogen- esis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling [J]. J Nath Cancer Inst, 2002, 94(11): 819～825
[15] 毛华，赵敏芳，袁爱力，等．血管内皮生长因子对肝癌细胞侵袭能力和同质性粘附作用影响[J]．肿瘤，2002，22(3)：197～199
[16] 陈治，杜钰．血管内皮细胞特异性受体在肿瘤血管靶向治疗中的应用[J]．世界华人消化杂志，2001，9(6)：702～705
[17] Yokoyama Y, Sato S, Futagami M, et al. Prognostic significance of vascular endothelial growth factor and its receptors in endometrial carcinoma [J].GynecolOncol, 2000, 77: 413～418
[18] 胥文春，陈新敏，罗春丽．血管内皮生长因子受体与恶性肿瘤[j]．国外医学临床生物化学与检验学分册，2003，24(3)：158～159
[19] SachieH, Yoshiro M, Akiko O, et al. Involvement of Flt-1 tyrosine kinase(vascular endothelial growth factor receptor-1) in pathological angiogenesis[J].Cancer Res, 2001, 61(3):1207～1213.
[20] Lam PM, Briton-Jones C, Cheuntg CK et al. In vivo regulation of mRNA expression of Vascular endothelial growth factor receptors(KDR and fit-1)in the human oviduct. Fertil Steril, 2004, 81(2): 416～423
[21] Suhandja A, Hoffman H. Role of growth factors and their receptors in proliferation of microvascular endothelial cell. Microsc Res Tech, 2003, 60: 70～75
[22] 尼玛卓玛，李亚里，朱运峰．血管内皮生长因子受体研究进展[J]．武警医学，2005，16(7)：542～544
[23] Makinen T, Jussila L, Veikkola T, et al. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3 [J]. Nat Med, 2001, 7(2): 199～205
[24] Lymboussaki A, Achen MG, Stacker SA, et al. Growth factors regulating lymphatic vessels[J]. Curr Top Microbiol Immunol, 2000, 25(1):75～82
[25] Toster L, Clapp L, Lrickson M, et al. Botulinum toxin A and chronic low back pain: a randomized, double-blind study [J]. Neurology.2001, 56(10): 1290～1293
[26] Baillie R, Harada K, Bradley NJ, et al. Expression of vascular endothelial growth factor in normal and tumor oral tissues with different antibodies[J]. The Histochemical Journal, 2001，33(2)：287～294．
[27] 朱新峰，林坚．血管内皮生长因子与肿瘤关系研究进展[J]．广西医科大学学报，2006，23(2)：333～335
[28] Padro T, Bieker R, Ruiz S, et al. Overexpression of vascular endothelial growth factor(VEGF) and its cellular receptor KDR(VEGFR-2) in the bone marrow of patients with acute myeloid leukemia[J]. Leukemia, 2002, 16(7): 1302～1310.
[29] Alessandra F, Taghi M,Zeev E, et al. High levels of vascular endothelial growth factor receptor-2 correlate with shortened survival in chronic lymphocytic leukemia[J]. ClinC ancer Res, 2001, 7(4):795～799.
[30] Masood R, Cm I, Zheng T, et al. Vascular endothelial growth factor vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma [J]. Pro NatlAcad Sci USA, 1997, 94(3): 979～984
[31] 刘丽军，肖畅，朱迅．VEGF及其受体抗肿瘤血管治疗应用新进展[J]．国外医学肿瘤学分册，2001，28(2)：113～115
[32] Rapisarda A, Uranchimeg B, Scudicro DA, et al. Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway [J]. Cancer Res, 2002, 62:4316～4324
[33] Asano M, Yukita A, Suzuki H. Wide spectrum of anti-tumor activity of neutralizing monoclonal antibody to human vascular endothelial growth factor[J]. Jpn J Cancer Res, 1999, 90(1),93～100
[34] Galdmn CK, Kendall RL, Cabrera G, et al. Panacrine expression of a native soluble vascular endothelial growth factor receptor inhibits turner growth, metastasis, and mortality rate [J]. Proc Nail Acad Sei USA, 1998, 95:879～880
[35] Zhai Y, Yu J, Iruela-Arispel., et al. Inhibition of angiogenesis and breast cancer engraft tumor growth by VEGI, a novel cytokine of the NF superfamily [J]. Int J Cancer, 1999, 82(1): 131～136
[36] Reilly MS, Holmgren L, Shing Y, et al. Angionstatin a novel angiogensis inhibitor that mediates the suooression of metastases by a Lewis lung carcinoma [J]. Cell, 1994, 79(2): 315～328
[37] Xie CG, Wang XP. Effect of selective cyclooxygenase-2 inhibitor celebrex on expression of vascular endothelial growth factor in pancreatic carcinoma [J]. Chinese Journal of Cancer, 2003,22(10): 1042-1046.
[38] Ramakrishnan S, Olson TA, Bautch VI, et al. Vascular endothelial growth factor toxin conjugate specifically inhibits KDR/Flk-1 positive endothelial cell proliferation in vitro and angiogenesis in Vivo [J]. Cancer Res, 1996, 56(6): 1324～1330