TIE2受体介导的靶向性非病毒载体的构建
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摘要
恶性肿瘤是正常染色体受多重损伤的一种疾病，它的发生和发展是外界环境和遗传因素共同作用所致。对于这样一种复杂的疾病，单纯依靠少数几种手段进行治疗是不够的，所以在手术、放疗、化疗等方法之外，用基因疗法治疗肿瘤是现在研究比较集中的领域。到目前为止，全世界范围内已有六百多种基因治疗方案获准进入临床试验，其中恶性肿瘤治疗方案占63.1%[1]。但遗憾的是，尚未有一种方案真正成为临床治疗有明显效果并成为常规治疗的方案。所以针对基因治疗中所使用的载体和治疗基因的研究具有重要的意义和紧迫性。
目前，肿瘤基因治疗的靶细胞主要是肿瘤细胞，通过导入细胞因子，抑癌基因，癌基因的反义基因或"自杀基因"等进行治疗。但是由于肿瘤的异质性，一种生物疗法往往只能针对某一种肿瘤。
为解决这个问题，Folkman博士提出了抗血管生成疗法，希望通过"饿死"肿瘤的办法来进行治疗。他认为，各种肿瘤有一个共性，就是当实体肿瘤生长到体积大于1-2mm3后，必须有新生血管形成，为其生长提供足够的养料，并为肿瘤细胞进入血液循环和转移到远处提供可能，抑制肿瘤新生血管生成可能会抑制多种肿瘤的生长[2,3,4]。虽然目前认为，并不是所有的肿瘤的生长都依赖于血管新生，但这种办法仍有相当重要的价值。
Angiopoietin-1(Ang1)是Tie-2受体的配体，在体外不能直接促进培养的内皮细胞生长，据推测它可能在血管生成过程的后期起作用，使血管的官腔形成及网络稳定。Angiopoietin-2(Ang2)，也可与Tie2受体结合，它的功能可能是与Ang1拮抗，使血管网处于一种不稳定的状态，有利于内皮细胞的分裂和血管网的重新建立。这两种配体的受体-Tie2，属于蛋白质酪氨酸激酶家族。它的表达具有相对特异性，在正常人体内主要表达于血管内皮细胞和一些造血细胞表面。在多种肿瘤，如乳腺癌、肺癌、膀胱癌等，及这些肿瘤内的血管中，
Ang1和Tie2受体的表达均增高。基于以上这些因素，我们决定尝试构建一种新的非病毒基因转移系统GA，该系统可将外源基因导入Tie2受体高表达的新生血管内皮细胞和肿瘤细胞。
研究主要包括以下三个部分：
1. 通过GA1基因转移系统转导报告基因(-gal，观察该系统是否可以在体内外将外源基因导入靶细胞，同时观察报告基因在体内主要脏器的分布和不同时间的分布情况。
2. GA1基因转移系统体内转导pCEP-p53基因，观察导入基因能否抑制肿瘤的生长。
3. 配体寡肽GA1与Tie2受体的结合试验。
第一部分
TIE2受体介导的GA1基因转移系统转导(-gal的体内外导入实验
由于促血管生成素与受体的结合区域尚属未知。所以首先，根据文献的报道，确定了一个促血管生成素中可能与受体结合的一个大致的范围，然后通过同源序列比较和疏水性分析，确定了一段可能的序列。当然这段序列是否可行需要通过实验的检验。
用双功能交联剂将配体寡肽GA1、HA20分别与多聚赖氨酸共价结合，然后包裹报告基因CMV-(-gal，进行体外实验。由于我们没有合适的血管内皮细胞株，通过免疫组化筛选，发现SPC-A1肺腺癌细胞有较高的Tie2受体表达。体外培养的细胞共分为三组，一组使用生理盐水，一组使用裸DNA（pCMV-(-gal），另一组使用GA1-PL/HA20-PL/pCMV-(-gal。结果表明，GA1载体系统可以将外源基因导入Tie2受体高表达的细胞。
然后，我们进行体内实验。使用GA1-PL/HA20-PL/pCMV-(-gal，分别对九种肿瘤SPC-A1、SMMC-7721、A375、SGC-7901、SK-OV-3、宫颈癌移植瘤和两种肝癌移植瘤通过瘤周注射进行体内导入实验，结果表明，在多种肿瘤组织的切片中观察到在部分血管内皮细胞中有报告基因的表达；另外经免疫组化检测，在多种肿瘤组织细胞的表面有Tie2受体的表达，在这些细胞内也观察到报告基因的表达。
同时我们对报告基因在不同时间的表达情况和在裸鼠体内不同脏器内的分布情况进行了研究。结果表明，在瘤周注射了GA1基因转移系统后，(-gal在24小时后就有表达，在48小时达到高峰，72小时起开始下降，而到168小时，仍有表达。报告基因在脾脏中有一定数量的表达，而在心、肺、肝、肾中未见明显的表达，胃、肠粘膜虽也可见蓝染，但未进行任何处理的裸鼠对胃、肠染色也可见蓝染，指示上述组织可能存在内源性的半乳糖苷酶活性。
第二部分
GA1基因转移系统介导p53的体内有效实验
使用A375人黑色素瘤裸鼠模型，待肿瘤长到直径约为0.3-0.4cm时，挑选出瘤体均匀的动物，随机分为5组，每组6只备用。分组如下：
①生理盐水组，②非病毒载体组(1μg GA1-PL)，③单纯质粒pCEP-p53组(1μg pCEP-p53质粒)，④低剂量组（每只注射相当于0.2μg质粒的四元复合体基因转移系统），⑤高剂量组（每只注射相当于1μg质粒的四元复合体基因转移系统）。每周注射一次，持续1个月。每周用游标卡尺测量裸鼠皮下瘤体的长(a)、短(b)径，根据V=(ab2/6计算瘤体积。
结果表明，五个组的平均值分别为：① 1.498 ± 0.600cm3 ② 1.734 ± 0.811cm3 ③ 1.727 ± 0.526 cm3 ④ 1.169 ± 0.381cm3 ⑤ 0.731 ± 0.328cm3，高剂量组与①、②、③组相比，抑制率分别为51.2%、57.8%、57.7%，并且与三?
Cancer is a disease of genetic and epigenetic disorder. Abnormalities in genes that control cell proliferation lead to the unrestrained growth and other malignant phenotypes. Initiation and development of cancer is caused by both environmental and hereditary factors and the process of transformation from a normal cell to cancer cell remains unknown. Besides surgery, radiotherapy and chemotherapy, a new strategy, gene therapy, is proposed as a new approach to control or cure cancer. However, up to now, there were more than 600 approved gene therapy clinical trials world wide, but unfortunately, most of them are not as satisfied as expected. Therefore, further serious efforts should be made on the cancer-targeting vectors and therapeutic genes.
In 1971, J. Folkman proposed a concept of the role of blood vessels in tumor growth in the form of a hypothesis that tumor growth is angiogenesis dependent. The new blood vasculatures embedded in a tumor bring nutrition to the tumor and provide as well a gateway for tumor cells to metastasize to distant sites. Angiogenesis has been currently concerned as an essential process of the development and progression of cancer.
Angiopoietin-1(Ang1) is an angiogenic factor that signals through the endothelial cell Tie2 receptor tyrosine kinase. Unlike VEGF, Ang1 does not directly promote the proliferation of cultured endothelial cells. However, its expression in close proximity with developing blood vessels implicates the involvement of Angiopoietin-1 in endothelial developmental processes. Angiopoietin-2, a member of Ang family, which can also bind to the same receptor Tie2, was shown to be a naturally occurring antagonist for Ang1. It is reported that Tie2 is specifically expressed in endothelial cells, but by immunohistochemistry, we found that cancer
cells themselves from many kind of malignancies such as breast cancer, lung cancer etc., also have high expression of Tie2 receptor.
Now we attempt to develop a gene delivery system targeting Tie2 of the endothelial cell of the tumor blood vessels, which could be potentially used to inhibit the angiogenesis and cancer growth.
Part I GA1 gene delivery system transducing (-gal gene
mediated by Tie2 receptor in vitro and in vivo
Since the binding domain of angiopoietin to its receptor Tie2 remains unknown, first of all, we have to narrow down the sequence of the binding area based on the available data. By analyzing the sequence homology between Ang1 and Ang2, and the hydrophobility data, we picked up a candidate oligopeptide sequence subjected to further biological studies.
With heterobifunctional crosslinking agent SPDP, we linked GA1 and HA20 to poly-L-lysine. In normal physiological pH, polylysine has positive ion on its surface, while DNA carries negative charges, thereby forming a electrostatic complex. With immunohistochemistry screening, we found lung adenocarcinoma cell line SPC-A1 expresses Tie2 receptor at a high level. With this cell line, we transduce GA1-PL/pCMV-(-gal/HA20-PL in vitro. After 2 days, the activity of (-gal could be detected in cells transduced by GA1 gene delivery system but not in cells treated with normal saline and naked DNA of plasmid CMV-(-gal.
After animals received injection of GA1-PL/CMV-(-gal/HA20-PL around the subcutaneously transplanted tumor, the expression of reporter gene in tumor and other tissues was examined. No (-gal activity was observed in heart, liver, lung, kidney, stomach and small intestine except spleen. The expression of (-gal at various times was also examined, at the first day after injection, blue staining could be found and it reached its peak at the second day. 1 week after injection, the activity still could be found.
We also injected the GA1 gene delivery complexes subcutaneously to various tumor-bearing nude mice, including SPC-A1, SMMC-7721, A375, SGC-7901, SK-OV-3, cervical xenograft and two heptocarcinoma xenograft. Results showed that
GA1 gene delivery system could transduce reporter gene to some of the endothe
目前，肿瘤基因治疗的靶细胞主要是肿瘤细胞，通过导入细胞因子，抑癌基因，癌基因的反义基因或"自杀基因"等进行治疗。但是由于肿瘤的异质性，一种生物疗法往往只能针对某一种肿瘤。
为解决这个问题，Folkman博士提出了抗血管生成疗法，希望通过"饿死"肿瘤的办法来进行治疗。他认为，各种肿瘤有一个共性，就是当实体肿瘤生长到体积大于1-2mm3后，必须有新生血管形成，为其生长提供足够的养料，并为肿瘤细胞进入血液循环和转移到远处提供可能，抑制肿瘤新生血管生成可能会抑制多种肿瘤的生长[2,3,4]。虽然目前认为，并不是所有的肿瘤的生长都依赖于血管新生，但这种办法仍有相当重要的价值。
Angiopoietin-1(Ang1)是Tie-2受体的配体，在体外不能直接促进培养的内皮细胞生长，据推测它可能在血管生成过程的后期起作用，使血管的官腔形成及网络稳定。Angiopoietin-2(Ang2)，也可与Tie2受体结合，它的功能可能是与Ang1拮抗，使血管网处于一种不稳定的状态，有利于内皮细胞的分裂和血管网的重新建立。这两种配体的受体-Tie2，属于蛋白质酪氨酸激酶家族。它的表达具有相对特异性，在正常人体内主要表达于血管内皮细胞和一些造血细胞表面。在多种肿瘤，如乳腺癌、肺癌、膀胱癌等，及这些肿瘤内的血管中，
Ang1和Tie2受体的表达均增高。基于以上这些因素，我们决定尝试构建一种新的非病毒基因转移系统GA，该系统可将外源基因导入Tie2受体高表达的新生血管内皮细胞和肿瘤细胞。
研究主要包括以下三个部分：
1. 通过GA1基因转移系统转导报告基因(-gal，观察该系统是否可以在体内外将外源基因导入靶细胞，同时观察报告基因在体内主要脏器的分布和不同时间的分布情况。
2. GA1基因转移系统体内转导pCEP-p53基因，观察导入基因能否抑制肿瘤的生长。
3. 配体寡肽GA1与Tie2受体的结合试验。
第一部分
TIE2受体介导的GA1基因转移系统转导(-gal的体内外导入实验
由于促血管生成素与受体的结合区域尚属未知。所以首先，根据文献的报道，确定了一个促血管生成素中可能与受体结合的一个大致的范围，然后通过同源序列比较和疏水性分析，确定了一段可能的序列。当然这段序列是否可行需要通过实验的检验。
用双功能交联剂将配体寡肽GA1、HA20分别与多聚赖氨酸共价结合，然后包裹报告基因CMV-(-gal，进行体外实验。由于我们没有合适的血管内皮细胞株，通过免疫组化筛选，发现SPC-A1肺腺癌细胞有较高的Tie2受体表达。体外培养的细胞共分为三组，一组使用生理盐水，一组使用裸DNA（pCMV-(-gal），另一组使用GA1-PL/HA20-PL/pCMV-(-gal。结果表明，GA1载体系统可以将外源基因导入Tie2受体高表达的细胞。
然后，我们进行体内实验。使用GA1-PL/HA20-PL/pCMV-(-gal，分别对九种肿瘤SPC-A1、SMMC-7721、A375、SGC-7901、SK-OV-3、宫颈癌移植瘤和两种肝癌移植瘤通过瘤周注射进行体内导入实验，结果表明，在多种肿瘤组织的切片中观察到在部分血管内皮细胞中有报告基因的表达；另外经免疫组化检测，在多种肿瘤组织细胞的表面有Tie2受体的表达，在这些细胞内也观察到报告基因的表达。
同时我们对报告基因在不同时间的表达情况和在裸鼠体内不同脏器内的分布情况进行了研究。结果表明，在瘤周注射了GA1基因转移系统后，(-gal在24小时后就有表达，在48小时达到高峰，72小时起开始下降，而到168小时，仍有表达。报告基因在脾脏中有一定数量的表达，而在心、肺、肝、肾中未见明显的表达，胃、肠粘膜虽也可见蓝染，但未进行任何处理的裸鼠对胃、肠染色也可见蓝染，指示上述组织可能存在内源性的半乳糖苷酶活性。
第二部分
GA1基因转移系统介导p53的体内有效实验
使用A375人黑色素瘤裸鼠模型，待肿瘤长到直径约为0.3-0.4cm时，挑选出瘤体均匀的动物，随机分为5组，每组6只备用。分组如下：
①生理盐水组，②非病毒载体组(1μg GA1-PL)，③单纯质粒pCEP-p53组(1μg pCEP-p53质粒)，④低剂量组（每只注射相当于0.2μg质粒的四元复合体基因转移系统），⑤高剂量组（每只注射相当于1μg质粒的四元复合体基因转移系统）。每周注射一次，持续1个月。每周用游标卡尺测量裸鼠皮下瘤体的长(a)、短(b)径，根据V=(ab2/6计算瘤体积。
结果表明，五个组的平均值分别为：① 1.498 ± 0.600cm3 ② 1.734 ± 0.811cm3 ③ 1.727 ± 0.526 cm3 ④ 1.169 ± 0.381cm3 ⑤ 0.731 ± 0.328cm3，高剂量组与①、②、③组相比，抑制率分别为51.2%、57.8%、57.7%，并且与三?
Cancer is a disease of genetic and epigenetic disorder. Abnormalities in genes that control cell proliferation lead to the unrestrained growth and other malignant phenotypes. Initiation and development of cancer is caused by both environmental and hereditary factors and the process of transformation from a normal cell to cancer cell remains unknown. Besides surgery, radiotherapy and chemotherapy, a new strategy, gene therapy, is proposed as a new approach to control or cure cancer. However, up to now, there were more than 600 approved gene therapy clinical trials world wide, but unfortunately, most of them are not as satisfied as expected. Therefore, further serious efforts should be made on the cancer-targeting vectors and therapeutic genes.
In 1971, J. Folkman proposed a concept of the role of blood vessels in tumor growth in the form of a hypothesis that tumor growth is angiogenesis dependent. The new blood vasculatures embedded in a tumor bring nutrition to the tumor and provide as well a gateway for tumor cells to metastasize to distant sites. Angiogenesis has been currently concerned as an essential process of the development and progression of cancer.
Angiopoietin-1(Ang1) is an angiogenic factor that signals through the endothelial cell Tie2 receptor tyrosine kinase. Unlike VEGF, Ang1 does not directly promote the proliferation of cultured endothelial cells. However, its expression in close proximity with developing blood vessels implicates the involvement of Angiopoietin-1 in endothelial developmental processes. Angiopoietin-2, a member of Ang family, which can also bind to the same receptor Tie2, was shown to be a naturally occurring antagonist for Ang1. It is reported that Tie2 is specifically expressed in endothelial cells, but by immunohistochemistry, we found that cancer
cells themselves from many kind of malignancies such as breast cancer, lung cancer etc., also have high expression of Tie2 receptor.
Now we attempt to develop a gene delivery system targeting Tie2 of the endothelial cell of the tumor blood vessels, which could be potentially used to inhibit the angiogenesis and cancer growth.
Part I GA1 gene delivery system transducing (-gal gene
mediated by Tie2 receptor in vitro and in vivo
Since the binding domain of angiopoietin to its receptor Tie2 remains unknown, first of all, we have to narrow down the sequence of the binding area based on the available data. By analyzing the sequence homology between Ang1 and Ang2, and the hydrophobility data, we picked up a candidate oligopeptide sequence subjected to further biological studies.
With heterobifunctional crosslinking agent SPDP, we linked GA1 and HA20 to poly-L-lysine. In normal physiological pH, polylysine has positive ion on its surface, while DNA carries negative charges, thereby forming a electrostatic complex. With immunohistochemistry screening, we found lung adenocarcinoma cell line SPC-A1 expresses Tie2 receptor at a high level. With this cell line, we transduce GA1-PL/pCMV-(-gal/HA20-PL in vitro. After 2 days, the activity of (-gal could be detected in cells transduced by GA1 gene delivery system but not in cells treated with normal saline and naked DNA of plasmid CMV-(-gal.
After animals received injection of GA1-PL/CMV-(-gal/HA20-PL around the subcutaneously transplanted tumor, the expression of reporter gene in tumor and other tissues was examined. No (-gal activity was observed in heart, liver, lung, kidney, stomach and small intestine except spleen. The expression of (-gal at various times was also examined, at the first day after injection, blue staining could be found and it reached its peak at the second day. 1 week after injection, the activity still could be found.
We also injected the GA1 gene delivery complexes subcutaneously to various tumor-bearing nude mice, including SPC-A1, SMMC-7721, A375, SGC-7901, SK-OV-3, cervical xenograft and two heptocarcinoma xenograft. Results showed that
GA1 gene delivery system could transduce reporter gene to some of the endothe
引文
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25. O'Reilly M.S., Boehm T., Shing Y. et al. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth Cell 1997 88:277-285
26. Dameron K.M., Volpert O.V., Tainsky M.A., et al. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1 Science 1994 265:1582-1584
27. Maione T.E., Gray G.S., Petro J., et al. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides Science 1990 247:77-79
28. Sharpe R.J., Byers H.R., Scott C.F. et al. Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4 J Natl Cancer Inst 1990 82:848-853
29. Kusaka M., Sudo K., Matsutani E., et al. Cytostatic inhibition of endothelial cell gowth by the angiogenesis inhibitor TNP-470(AGM-1470) Br. J. Cancer 1994 69:212-216
30. Takamiya Y., Brem H., Ojeifo J. et al. AGM-1470 inhibits the growth of human glioblastoma cells in vitro and in vivo Neurosurgery 1994 34:869-75
31. Takamiya Y., Friedlander R.M., Brem H., et al. Inhibition of angiogenesis and growth of human nerve-sheath tumors by AGM-1470 J Neurosurg 1993 78:470
32. Qian C.N., Min H.Q., Lin H.L., et al. Primary study in experimental antiangiogenic therapy of nasopharyngeal carcinoma with AGM-1470 (TNP-470) J Laryngol Otol 1998 112:849-53
33. Yoshida T., Kaneko Y., Tsukamoto A., et al. Suppression of hepatoma growth and angiogenesis by a fumagillin derivative TNP470: possible involvement of nitric oxide synthase Cancer Res 1998 58:3751-6
34. Davis S., Aldrich T.H., Jones P.F. et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning Cell 1996 87:1161-9
35. Thurston G, Rudge JS, Ioffe E, et al. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med 2000 6(4):460-3
36. Gamble JR, Drew J, Trezise L Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions. Circ Res 2000 87(7):603-7
37. Maisonpierre P.C., Suri C., Jones P.F., et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis Science 1997 277:55-60
38. Teichert-Kuliszewska K, Maisonpierre PC, Jones N et al. Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis isassociated with activation of Tie2. Cardiovasc Res 2001 49(3):659-70
39. Kim I, Kim JH, Moon SO et al. Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Oncogene 2000 19(39):4549-52
40. Partanen J, Armstrong, E, Makela, TP, et al. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Mol Cell Biol 1992 12:1698-1797
41. Shewchuk LM, Hassell AM, Ellis B, et al. Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail. Structure Fold Des 2000 8(11):1105-13
42. Dumont DJ, Gradwohl G, Fong GH, et al. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, Tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev 1994 8424:1897-1909
43. Patan S. TIE1 and TIE2 receptor tyrosine kinases inversely regulate embryonic angiogenesis by the mechanism of intussusceptive microvascular growth. Microvasc Res 1998 56(1):1-21
44. Sato TN, Tozawa Y, Deutsch U, et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 1995 Jul 6;376(6535):70-4
45. Stratmann A, Risau W, Plate KH et al. Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis Am J Pathol 1998 153(5):1459-66
46. Peters KG, Coogan A, Berry D et al. Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumor angiogenesis. Br J Cancer 1998;77(1):51-6
47. Takahama M, Tsutsumi M, Tsujiuchi T et al. Enhanced expression of Tie2, its ligand angiopoietin-1, vascular endothelial growth factor, and CD31 in human non-small cell lung carcinomas. Clin Cancer Res 1999 (9):2506-10
48. Wurmbach JH, Hammerer P, Sevinc S, et al. The expression of angiopoietins and their receptor Tie-2 in human prostate carcinoma. Anticancer Res 2000 20(6D):5217-20
49. Lin P, Buxton JA, Acheson A, et al. Antiangiogenic gene therapy targeting the endothelium-specific receptor tyrosine kinase Tie2 Proc Natl Acad Sci USA 1998 95:8829-8834
50. 吴善芳等 人体肺腺癌细胞系SPC-A1的建立和生物学特性 中国科学(B辑) 1982 10:913
51. 黄荣春，周荣华，吕发度 SMMC-7721人体肝癌细胞株的建立及其生物学特性的初步观察 第二军医大学学报 1980 1:5-9
52. Goodfellow M. et al. One hundred and twenty-seven cultured human tumor cell lines produing tumors in nude mice J Natl Cancer Inst 1977 59:221-226
53. Lin CH, Fu ZM, Liu YL, et al. Investigation of SGC-7901 cell line established from human gastric carcinoma cells. Chin Med J (Engl). 1984 97(11):831-4.
54. Giard D.J., et al. In vitro cultivation of human tumors: Establishment of cell lines derived from a series of solid tumors J Natl Cancer Inst 1973 51:1417-1423
55. 陈昌炜，张嘉庆，王松霞 人体乳癌体外培养-乳癌细胞株BCaP-37的建立及其生物学特性的初步观察 北京医学院学报 1983 15:161-164
56. Murata M., Kagiwada S., Takahashi S., et al. Membrane fusion induced by mutual interaction of the two charge-reversed amphilic peptides at neutral pH. J Biol Chem 1994 266(22):14353-8
57. Carlsson J, Drevin H, Axen R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobi-functional reagent. Biochem J 1978 173(3):723-37
58. 鄂征 组织培养和分子细胞学技术 北京出版社 1997
59. 萨姆布鲁克 J., 弗里奇 E.F., 曼尼阿蒂斯 T. 分子克隆（第二版） 科学出版社1995
60. 郝柏林，张淑誉 生物信息学手册 上海科学技术出版社 2000
61. 张成岗，贺福初 生物信息学方法与实践 科学出版社 2002
62. http://www.ncbi.nlm.nih.gov/entrez 从该网址检索到蛋白质空间结构的信息，然后使用该网站下载的cn3d软件显示结果。
63. Kyte J, Doolittle RF. A simple method for displaying the hydro-pathic character of a protein. J Mol Biol. 1982 May 5;157(1):105-32.
64. http://www.expasy.org/cgi-bin/protscale.pl
65. http://www.ncbi.nlm.nih.gov/blast/blast2
66. Muller Y.A., Christinger H.W., Keyt B.A., et al. The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 ? resolution: multiple copy flexibility and receptor binding. Structure 1997 5(10):1325-1338
67. Keyt B.A., Nguyen, H.V., Berleau, L.T., et al., Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. J Biol Chem 1996 271(10):5638-5646,
68. Muller Y.A., Li B., Christinger H.W., et al. Vascular endothelial growht factor: Crystal structure and functional mapping of the kinase domain receptor binding site Proc Natl Acad Sci U S A 1997 94:7192-7197
69. Beecken WD, Fernandez A, Joussen AM, et al. Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice. J Natl Cancer Inst 2001 93(5):382-7
70. Folkman J., Shing Y. Angiogenesis J Bio Chem 1992 16:10931-10934
71. Siemeister G., Schirner M., Reusch P., et al. An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells. Proc. Natl. Acad. Sci. USA 1998 95:4625-9
72. Inoue T., Kibata K., Suzuki M., et al. Identification of a vascular endothelial growth factor (VEGF) antagonist, sFlt-1, from a human hematopoietic cell line. FEBS Letters 469 (2000) 14-18
73. Hammond E.M., Giaccia A.J. Antiangiogenic Therapy and p53. Science 2002 297:471
74. Bargonetti J, Manfredi JJ. Multiple roles of the tumor suppressor p53. Curr Opin Oncol. 2002 14(1):86-91.
75. Hickman ES, Moroni MC, Helin K. The role of p53 and pRB in apoptosis and cancer. Curr Opin Genet Dev 2002 12(1):60-6
76. Horner A., Bord J., Kelsall A.W., et al. Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone. Bone 2001 28(1):65-71
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22. Kim K.J., Li B., Winer J., et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo Nature 1993 362:841-844
23. Millauer B., Longhi M.P., Plate K.H., et al. Dominant-negative inhibition of flk-1 suppresses the growth of many tumor types in vivo Cancer Res 1996 56:1615-1620
24. O'Reilly M.S., Holmgren L., Shing Y., et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma Cell 1994 79:315-328
25. O'Reilly M.S., Boehm T., Shing Y. et al. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth Cell 1997 88:277-285
26. Dameron K.M., Volpert O.V., Tainsky M.A., et al. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1 Science 1994 265:1582-1584
27. Maione T.E., Gray G.S., Petro J., et al. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides Science 1990 247:77-79
28. Sharpe R.J., Byers H.R., Scott C.F. et al. Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4 J Natl Cancer Inst 1990 82:848-853
29. Kusaka M., Sudo K., Matsutani E., et al. Cytostatic inhibition of endothelial cell gowth by the angiogenesis inhibitor TNP-470(AGM-1470) Br. J. Cancer 1994 69:212-216
30. Takamiya Y., Brem H., Ojeifo J. et al. AGM-1470 inhibits the growth of human glioblastoma cells in vitro and in vivo Neurosurgery 1994 34:869-75
31. Takamiya Y., Friedlander R.M., Brem H., et al. Inhibition of angiogenesis and growth of human nerve-sheath tumors by AGM-1470 J Neurosurg 1993 78:470
32. Qian C.N., Min H.Q., Lin H.L., et al. Primary study in experimental antiangiogenic therapy of nasopharyngeal carcinoma with AGM-1470 (TNP-470) J Laryngol Otol 1998 112:849-53
33. Yoshida T., Kaneko Y., Tsukamoto A., et al. Suppression of hepatoma growth and angiogenesis by a fumagillin derivative TNP470: possible involvement of nitric oxide synthase Cancer Res 1998 58:3751-6
34. Davis S., Aldrich T.H., Jones P.F. et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning Cell 1996 87:1161-9
35. Thurston G, Rudge JS, Ioffe E, et al. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med 2000 6(4):460-3
36. Gamble JR, Drew J, Trezise L Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions. Circ Res 2000 87(7):603-7
37. Maisonpierre P.C., Suri C., Jones P.F., et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis Science 1997 277:55-60
38. Teichert-Kuliszewska K, Maisonpierre PC, Jones N et al. Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis isassociated with activation of Tie2. Cardiovasc Res 2001 49(3):659-70
39. Kim I, Kim JH, Moon SO et al. Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Oncogene 2000 19(39):4549-52
40. Partanen J, Armstrong, E, Makela, TP, et al. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Mol Cell Biol 1992 12:1698-1797
41. Shewchuk LM, Hassell AM, Ellis B, et al. Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail. Structure Fold Des 2000 8(11):1105-13
42. Dumont DJ, Gradwohl G, Fong GH, et al. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, Tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev 1994 8424:1897-1909
43. Patan S. TIE1 and TIE2 receptor tyrosine kinases inversely regulate embryonic angiogenesis by the mechanism of intussusceptive microvascular growth. Microvasc Res 1998 56(1):1-21
44. Sato TN, Tozawa Y, Deutsch U, et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 1995 Jul 6;376(6535):70-4
45. Stratmann A, Risau W, Plate KH et al. Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis Am J Pathol 1998 153(5):1459-66
46. Peters KG, Coogan A, Berry D et al. Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumor angiogenesis. Br J Cancer 1998;77(1):51-6
47. Takahama M, Tsutsumi M, Tsujiuchi T et al. Enhanced expression of Tie2, its ligand angiopoietin-1, vascular endothelial growth factor, and CD31 in human non-small cell lung carcinomas. Clin Cancer Res 1999 (9):2506-10
48. Wurmbach JH, Hammerer P, Sevinc S, et al. The expression of angiopoietins and their receptor Tie-2 in human prostate carcinoma. Anticancer Res 2000 20(6D):5217-20
49. Lin P, Buxton JA, Acheson A, et al. Antiangiogenic gene therapy targeting the endothelium-specific receptor tyrosine kinase Tie2 Proc Natl Acad Sci USA 1998 95:8829-8834
50. 吴善芳等 人体肺腺癌细胞系SPC-A1的建立和生物学特性 中国科学(B辑) 1982 10:913
51. 黄荣春，周荣华，吕发度 SMMC-7721人体肝癌细胞株的建立及其生物学特性的初步观察 第二军医大学学报 1980 1:5-9
52. Goodfellow M. et al. One hundred and twenty-seven cultured human tumor cell lines produing tumors in nude mice J Natl Cancer Inst 1977 59:221-226
53. Lin CH, Fu ZM, Liu YL, et al. Investigation of SGC-7901 cell line established from human gastric carcinoma cells. Chin Med J (Engl). 1984 97(11):831-4.
54. Giard D.J., et al. In vitro cultivation of human tumors: Establishment of cell lines derived from a series of solid tumors J Natl Cancer Inst 1973 51:1417-1423
55. 陈昌炜，张嘉庆，王松霞 人体乳癌体外培养-乳癌细胞株BCaP-37的建立及其生物学特性的初步观察 北京医学院学报 1983 15:161-164
56. Murata M., Kagiwada S., Takahashi S., et al. Membrane fusion induced by mutual interaction of the two charge-reversed amphilic peptides at neutral pH. J Biol Chem 1994 266(22):14353-8
57. Carlsson J, Drevin H, Axen R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobi-functional reagent. Biochem J 1978 173(3):723-37
58. 鄂征 组织培养和分子细胞学技术 北京出版社 1997
59. 萨姆布鲁克 J., 弗里奇 E.F., 曼尼阿蒂斯 T. 分子克隆（第二版） 科学出版社1995
60. 郝柏林，张淑誉 生物信息学手册 上海科学技术出版社 2000
61. 张成岗，贺福初 生物信息学方法与实践 科学出版社 2002
62. http://www.ncbi.nlm.nih.gov/entrez 从该网址检索到蛋白质空间结构的信息，然后使用该网站下载的cn3d软件显示结果。
63. Kyte J, Doolittle RF. A simple method for displaying the hydro-pathic character of a protein. J Mol Biol. 1982 May 5;157(1):105-32.
64. http://www.expasy.org/cgi-bin/protscale.pl
65. http://www.ncbi.nlm.nih.gov/blast/blast2
66. Muller Y.A., Christinger H.W., Keyt B.A., et al. The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 ? resolution: multiple copy flexibility and receptor binding. Structure 1997 5(10):1325-1338
67. Keyt B.A., Nguyen, H.V., Berleau, L.T., et al., Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. J Biol Chem 1996 271(10):5638-5646,
68. Muller Y.A., Li B., Christinger H.W., et al. Vascular endothelial growht factor: Crystal structure and functional mapping of the kinase domain receptor binding site Proc Natl Acad Sci U S A 1997 94:7192-7197
69. Beecken WD, Fernandez A, Joussen AM, et al. Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice. J Natl Cancer Inst 2001 93(5):382-7
70. Folkman J., Shing Y. Angiogenesis J Bio Chem 1992 16:10931-10934
71. Siemeister G., Schirner M., Reusch P., et al. An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells. Proc. Natl. Acad. Sci. USA 1998 95:4625-9
72. Inoue T., Kibata K., Suzuki M., et al. Identification of a vascular endothelial growth factor (VEGF) antagonist, sFlt-1, from a human hematopoietic cell line. FEBS Letters 469 (2000) 14-18
73. Hammond E.M., Giaccia A.J. Antiangiogenic Therapy and p53. Science 2002 297:471
74. Bargonetti J, Manfredi JJ. Multiple roles of the tumor suppressor p53. Curr Opin Oncol. 2002 14(1):86-91.
75. Hickman ES, Moroni MC, Helin K. The role of p53 and pRB in apoptosis and cancer. Curr Opin Genet Dev 2002 12(1):60-6
76. Horner A., Bord J., Kelsall A.W., et al. Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone. Bone 2001 28(1):65-71