摘要
【研究背景】目前恶性胶质瘤的治疗仍然面临巨大挑战,寻找比传统治疗更有效的治疗手段一直备受各国神经科学工作者的关注。血管生成不仅在个体发育、损伤修复、组织再生等生理过程中扮演重要角色,同时它也是肿瘤生长和转移过程中的一个重要步骤。由于肿瘤内皮细胞遗传上稳定,不易产生耐药,抗肿瘤血管生成治疗已成为肿瘤治疗领域很有前途的治疗方法之一。目前已有多种抑制新生血管生成的药物用于抗肿瘤研究。肿瘤抑素(Tumstatin)是继血管生成抑素(Angiostatin)和内皮抑素(Endostatin)之后,新发现的源于血管基膜Ⅳ型胶原α3链的内源性血管生成抑制因子,具有明显的抑制肿瘤血管生成和诱导其内皮细胞调亡的活性。由于采用血管生成抑制因子直接投药的方式治疗肿瘤存在所需剂量过大,同时生产有完整活性的血管生成抑制因子存在技术困难,因此通过基因转染的方法提供有完整生物活性的血管生成抑制因子在体内持续、较高浓度的表达,是肿瘤抗血管生成治疗的理想策略。利用全长Tumstatin片段抑制胶质瘤血管生成的基因治疗实验目前国内外尚未见报导;故本课题试图克隆人Tumstatin基因,构建其真核细胞表达载体(pcDNA3.1-Tumstatin);观察Tumstatin体外抗血管生成的生物活性及了解此基因转染对U251胶质瘤细胞株的自身增殖有无影响,并观察Tumstatin在肿瘤局部持续、较高浓度表达对U251裸鼠移植瘤生长有无抑制作用,探讨Tumstatin基因治疗胶质瘤的可行性。
目的构建抗血管生成的人肿瘤抑素(Tumstatin)基因真核表达载体,为后续体外Tumstatin转染U251细胞及其在荷瘤裸鼠(BALB/N)的体内表达创造条件。
方法1、从HEK293细胞提取总RNA,通过RT-PCR扩增出Tumstatin基因。2、将该基因导入克隆载体pUC57-T中,转化大肠杆菌DH5α进行扩增,并提取纯化,对所获重组质粒进行酶切鉴定和DNA序列分析鉴定。3、用内切酶NheI和NotI对重组质粒pUC57-Tumstatin双酶切,并纯化目的基因片段,同时用内切酶NheI和NotI对质粒pcDNA3.1(-)进行双酶切。取纯化的目的基因片段与表达载体pcDNA3.1(-)在T_4连接酶的作用下进行连接反应。经菌落PCR、酶切鉴定和DNA测序证实后,制备和纯化重组质粒。
结果1、提取的RNA经电泳有明显的28S、18S、5S三条带,分光光度法测得A_260/A_280=1.8933,提示RNA无明显降解;RT-PCR产物电泳显示在750bp处有一特异性条带。2、重组质粒pUC57-TumstatinDNA测序结果显示插入的目的基因与GenBank收录的人Tumstatin基因cDNA序列完全一致,无任何突变。3、重组质粒pcDNA3.1-Tumstatin经酶切鉴定后,DNA序列分析结果与GenBank收录的人Tumstatin基因cDNA序列亦完全一致,无任何突变。分光光度法测得此重组质粒溶液A_260/A_280=1.823。
结论成功克隆了人Tumstatin基因并构建了Tumstatin基因的真核表达载体pcDNA3.1-Tumstatin。
目的观察血管生成抑制因子Tumstatin体外抗血管生成的生物功能及了解此基因转染对于U251胶质瘤细胞株的自身增殖有无影响。
方法以脂质体法将分泌型真核表达载体pcDNA3.1-Tumstatin转染至人胶质瘤细胞株U251,并用G418筛选出稳定转染的细胞株,观察转染后的U251细胞的生物学特性(MTT法观察U251细胞株的增殖活性、流式细胞仪检测其细胞周期),并以Western blot检测培养上清中Tumstatin的分泌表达,进一步提取U251细胞的培养上清液,加至脐静脉内皮细胞(HUVECs)的培养液中,采用MTT法观察HUVECs的增殖活性、细胞划痕法检测其迁移能力、体外管腔形成抑制实验检测其成管能力及流式早期细胞凋亡检测法检测HUVECs的早期细胞凋亡率。
结果1、成功获得可表达Tumstatin的U251细胞株,转染组U251细胞的增殖活性及细胞周期与未转染组的无显著差别(P＞0.05);2、Tumstatin在体外不能抑制HUVECs的迁移,但它能显著抑制HUVECs的增殖,诱导HUVECs细胞凋亡及抑制其形成毛细血管的能力。
结论Tumstatin基因转染对U251细胞自身增殖无显著影响,Tumstatin在体外有较强的抗血管生成的活性。
目的探讨Tumstatin抑制U251裸鼠移植瘤生长的作用。
方法BALB/N裸鼠随机分成三组,每组10只,然后将未转染的U251细胞株及pcDNA3.1-Tumstatin稳定转染和pcDNA3.1稳定转染的U251细胞株各分别以1×10~7/只接种裸鼠。成瘤后每隔一周测量移植瘤体积一次,于接种后第28天处死裸鼠,将获取的移植瘤标本称重后,做肿瘤切片HE染色、TUNEL法检测肿瘤细胞凋亡、CD31免疫组化检测MVD。
结果1、pcDNA3.1-Tumstatin组移植瘤的平均体积及瘤重都比pcDNA3.1组及未转染组低(p＜0.05)。2、肿瘤切片HE染色三组无明显区别,但pcDNA3.1-Tumstatin组肿瘤细胞平均凋亡率比其它两组高(26.60±2.02 vs.11.21±1.57,9.69±0.86,P＜0.05),同时微血管密度(MVD)比其它两组低(5.62±1.32 vs.23.84±1.71,29.33±4.45,P＜0.05)。
结论人Tumstatin有抑制U251移植瘤生长的作用,通过抑制肿瘤血管生成而间接诱发肿瘤细胞凋亡可能是其主要机制。
[Background]
Treatment of malignant gliomas remains a formidable challenge and it deserves special attention to search for more effective therapeutic methods.Angiogenesis is required for a variety of physiological processes such as development,wound repair,and tissue regeneration.Moreover,it is also one major pathogenic step in the process of tumor growth and metastasis.Because nonmalignant endothelial cells are genetically stable and unlikely to mutate into drug-resistant variants,anti-tumor angiogenesis therapy has become a promising method in cancer treatment. Several anti-angiogenic proteins have been identified that supress the growth of cancer.Tumstatin,the NC1 domain ofα3 chain of typeⅣcollagen,was identified as prossessing anti-angiogenic activity.Therapy with endogenous angiogenesis inhibitor is not clinically practical because of the high doses required and the difficulty in producing them in a fully functional state.Alternatively,It is an ideal strategy that if endogenous angiogenesis inhibitors(such as angiostatin or endostatin) can be expressed in vivo by gene transfection method and provided therapeutic levels of the functionally actively proteins.We cloned the cDNA of human tumstatin gene and constructed a recombinant plasmid which expressed tumstatin in eukaryotic cells.U251 glioma cells were transfected with this recombinant plasmid and transplanted into the nude mice's armpits.Then the inhibitory effect of Tumstatin on the growth of U251 glioma xenograft nude mice model was investigated.
PartⅠConstruction of eukaryotic expression vector of human tumstatin gene
Objective To construct the eukaryotic expression vector of human antiangiogenic tumstatin gene,which will be used in the next section experiments,including transfection U251 cell line with tumstatin in vitro and its expression in BALB/N mice.
Methods 1.Total RNA was extracted from HEK293 cell lines, then the target gene were amplified by RT-PCR technique.2.The target gene was linked to pUC57-T vector,and transformed into E.coli DH5α.The purified plasmid,named as pUC57-Tumstatin,was identified by restriction endonuclease and the sequence of the target gene was identified by dideoxy chain termination method.3.pUC57-Tumstatin and pcDNA3.1(-) were digested by enzyme NheI and NotI respectively,then coupled reaction between the target gene and pcDNA3.1(-) segment was carried out by T4 DNA joinase.The recombinant plasmid,named as pcDNA3.1-Tumstatin,was identified by restriction endonuclease and the sequence of the target gene was identified by dideoxy chain termination method,then the recombinant plasmid was amplified and purified.
Results 1.Total RNA of HEK293 cells was extracted successfully. Electrophoresis and OD value ratio(A_(260)/A_(280)=l.8933) demonstrated the integrity of RNA.A fragment of DNA about 750bp was expanded in reverse-transcript polymerase chain reaction.2.The target gene in the recombinant plasmid pUC57-Tumstatin was comfirmed to be the cDNA of Tumstatin by sequencing.plasmid pUC57-Tumstatin was expanded and purified.3.The target gene in the recombinant plasmid pcDNA3.1-Tumstatin was also comfirmed to be the cDNA of Tumstatin by sequencing.This plasmid was expanded and purified.
Conclusion The target gene was cloned successfully.Its sequence was completely consistent with that Human tumstatin gene listed in GenBank database.pcDNA3.1-Tumstatin,a recombinant eukaryonic expression plasmid,was constructed with the corrected sequence.
Objective To study the antiangiogenic functions of Tumstatin and to explore the biological influence of the genetic-modification with Tumstatin cDNA on U251 cell line in vitro.
Methods The pcDNA3.1-Tumstatin was transfected into the U251 cell with liposome,followed by G418 method to select definitely the positive-transformed clones.The proliferation ability of transfected cell line was measured by MTT assay and its cell cycle was analyzed by flow cytometry(FCM).Then Western blot method was adopted to detect the protein expressed in cell culture medium.Finally,HUVECs was exposed to culture medium which was extracted from culture supernatant of positive-transformed U251 cells and contains tumstatin protein.The proliferation ability of HUVECs was measured by MTT assay,the migration ability of HUVECs was observed by the scratch assay,the endothelial tube formation ability of HUVECs was observed,and early cell apoptosis ratio of HUVECs was observed by FCM.
Results 1.The U251 cell line with the character of tumstatin expression was obtained,meanwhile,the proliferation ability and cell cycle of the transfected U251 cell line was not significant different from the untransfected cell line(P>0.05).2.Although tumstatin was found having no effects on migration of HUVECs,it was found having inhibitory effects on the proliferation of HUVECs,and it could inhibit endothelial tube formation and introduce endothelial cell apoptosis.
Conclusion The transfection of Tumstatin into U251 cell line has no obvious influence on the proliferation of the transfected cells,and Tumstatin has antiangiogenic activity in vitro.
Objective To investigate the inhibitory effect of Tumstatin on the growth of U251 glioma xenograft nude mice model.
Methods BALB/N nude mice were divided into three groups at random,with 10 animals in each group.Then untransfected U251 cells and cells transfected with pcDNA3.1-Tumstatin/pcDNA3.1 were inoculated at the armpit of nude mices,the growth of the subcutaneously transplanted tumor was observed on 7th,14th,21st and 28th day after inoculation and the volumes were calculated.HE staining of xenografts of nude mice was conducted.Apoptosis of tumor cells was detected by terminal dUTP nick end labeling(TUNEL) technique and expression of CD31 was detected by immunohistochemical staining,then apoptosis index and microvessel density(MVD) was calculated among three groups respectively.
Results 1.Compared to pcDNA3.1 group and untransfected group, the average volume and average weight of xenograft in pcDNA3.1-Tumstatin group was significant smaller(p<0.05).2、There are no significant difference in HE staining of xenograft among three groups,but the tumor cell apoptotic index was significantly higher in pcDNA3.1-Tumstatin group than in the rest 2 groups(26.60±2.02 vs. 11.21±1.57 and 9.69±0.86,P<0.05);meanwhile the MVD was significantly less in pcDNA3.1-Tumstatin group than in the rest 2 groups (5.62±1.32 vs.23.84±1.71 and 29.33±4.45,P<0.05).
Conclusion Human Tumstatin has the anti-tumor effect in the U251 xenograft.To inhibit tumor angiogenesis and indirectly induce tumor cell apoptosis may be the major mechanism.
引文
[1]黄强。恶性胶质瘤治疗进展和新的临床治疗指导。中外健康文摘,2007,4(10):175。
[2]Folkman J,Merler Z,Abernathy C,et al.Isolation of a tumor factor responsible for angiogenises.J Exp Med,1971,133(2):275-288.
[3]Maeshima Y,Colorado PC,torre A,et al.Distinct antitumor properties of a type IV collagen domain derived from basement membrane.J Biol Chem,2000,14;275(28):21340-8.
[4]Hamno Y,Kalluri R.Tumstatin,the NC1 domain of alpha3 chain of type Ⅳcollagen,is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth.Biochem Biophys Res Commun,2005,333(2):292-298.
[5]Sudhakar A,Boosani CS.Inhibition of tumor angiogenesis by Tumstatin:insights into signaling mechanisms and implication in cancer regression.Pharmaceutical research,2008,25(12):2731-9.
[6]Kawaguchi T,Yamashita Y,Kanamori M,et al.The PTEN/Akt pathway dictates the direct αVβ3-dependent growth-inhibitory action of an active fragment of tumstatin in glioma cells in vitro and in vivo.Cancer Res,2006,66(23):11331-40.
[7]Hanahan D,Forlkman J.Patters and emerging mechanisms of the angiogenic switch during tumorgenesis.Cell,1996,86(3):353-364.
[8]Satchi-Fainaro R,Puder M,Davies JW,etal.Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470.Nat Med 2004,10:255-261.
[9]Petitelerc E,Boutaud A,Prestayko A,et al.New function for non-collagen type IV.Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo.J Biol Chem,2000,275(11):8501-61.
[10]Maeshima Y,Colorado PC,Kalluri R.Two RGD-independent αVβ3 integrin binding sites on tumstatin regulate distinct anti-tumor properties.J Biol Chem,2000,14;275(31):23745-50.
[11]Pedchenko V,Zent R,Hudson BG.Alpha(V)and alpha(V)beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1)domain of the alpha3 chain of type Ⅳ collgen:implication for the mechanism of endothelia cell adhesion.J Biol Chem,2004,279(4):2772-80.
[12]Boosani CS,Mannam AP,Cosgrove D,et al.Regulation of COX-2 mediated signaling by alpha3 type Ⅳ non-collagenous domain in tumor angiogenesis.Blood,2007,110:1168-77.
[13]Miyoshi T,Hirohata S,Ogawa H,et al.Tumor-specific expression of the RGD-alpha3(Ⅳ)NC 1 domain suppresses endothelial tube formation and tumor growth in mice.J FASEB,20:1904-6.
[14]Ortega N,Werb Z.New functional roles for noncollagenous domain of basement membrane collgens.J Cell Sci,2002,115(22):4201-4214.
[15]Sottile J.Regulation of angiogenesis by extracellular matrix.Biochim Biophus Acta,2004,1654(1):13-22.
[16]Hudson BG,Reeders ST,Tryggvason K.Type IV collagen:structure,gene organization,and the role in human diseases.Moleclar basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol.Chem,1993,268,26033-6.
[17]Kalluri R.Basement membranes:structure,assembly and role in tumor angiogenesis.Nat Rev Cancer,2003,3:422-233.
[18]Kashtan CE.Alport syndrome and thin glomerular basement membrane disease.J Am Soc Nephrol,1998,9(9):1736-1750.
[19]O'Reilly MS,Boehm T,Shing Y,et al.Endostatin:an endogenous inhibitor of angiogenisis andtumor growth.Cell.1997,88:277-85.
[20]Hanahan D,Weinberg RA.The hallmarks of cancer.Cell,2000,100:57-70.
[21]Colorado,PC.Anti-angiogenic cues from vascular basement membrane collagen.Cancer Res,2000,60(9):2520-6.
[22]刘斌。细胞培养中的研究方法[M]。//司徒镇强,吴军正。细胞培养。西安:世界图书出版公司,2007,197-233。
[23]Montesano R,Vassalli JD,Baird A,et al.Basic fibroblast growth factor induces angiogenesis in vitro[J].Proc Natl Acad Sci USA,1986,83(19):7297-7301.
[24]Sambrook J,Russel D.黄培堂,等译。分子克隆实验指南[M]。第3版,北京:科学出版社,2002。
[25]胡维新。基因工程与基因体外表达。[M]。//冯作化。医学分子生物学。北京:人民卫生出版社,2005,175-198。
[26]Bin Yao,Qiu-Ming He,Ling Tian,et al.Enhanced Antitumor Effect of the Combination of Tumstatin Gene Therapy and Gemcitabine in Murine Models.Human Gene Therapy.September 2005,16(9):1075-1086.
[27]Maeshima Y,Manfredi M,Reimer C,et al.Identification of the Anti-angiogenic Site within Vascular Basement Membrane-derived Tumstatin.J.Biol.Chem,2001,276(18):15240-15248.
[28]Floquet N,Pasco S,Ramont L,et al.The antitumor properties of the alpha 3(IV)-(185-203)peptide from the NC1 domain of IVtype collagen(tumstatin)are comformation-dependent.J BiolChen,2004,279:2091-100.
[29]Kalluri R.Discovery of Type IV collagen non-collagenous domain as movel integrin ligands and endogenous inhibitors of angiogenesis.Cold Spring Symup Quant Biol,2002,67:255-266.
[30]Petitclerc E,Boutud A,Prestayko A,et al.New functions for non-collagenous domain of human collagen type Ⅳ.Novel integrin ligands inhibiting angiogenesis nd tumor growth in vivo.J Biol Chem 2000,275:8051-61.
[31]朱敬,李利,赵昕。抑癌基因PTEN与人类肿瘤关系的研究现状。广东医学,2008,29(10):1756-7。
[32]姚智强,卢亦成。人脑胶质瘤发病分子机制及其临床应用。中国肿瘤生物治疗杂志。2008,15(1):90-94。
[33]Boosani,C.S.and A.Sudhakar,Cloning,purification,and characterization of a non-collagenous anti-angiogenic protein domain from human alpha 1 type Ⅳcollagen expressed in Sf9 cells.Protein Expression and Purification,2006.49(2):211-218.
[34]Cao,J.G.,et al.,Vascular basement membrane-derived multifunctional peptide,a novel inhibitor of angiogenesis and tumor growth.Acta Biochimica Et Biophysica Sinica,2006.38(7):p.514-521.
[35]Caudroy,S.,et al.,Implication of tumstatin in tumor progression of human bronchopulmonary carcinomas.Human Pathology,2004.35(10):1218-1222.
[36]Chang,K.H.,et al.,Improved production of recombinant turnstatin in stably transformed Trichoplusia ni BTI Tn 5B1-4 cells. Protein Expression and Purification, 2004. 35(1): 69-75.
[37] Maeshima Y,Yerramalla UL, DhanabalM,et al. Extracellular matrix-derived peptide binds to alpha(V)beta(3) integrin and inhibits angiogenesis. J Biol Chem,2001,276(34):31959-68.
[38] Cao Y. Endogenous angiogenesis inhibitors and their therapeutic implications.Int J biochem Cell Biol, 2001, 33(4):357-369.
[39] Caudroy, S., et al., Implication of tumstatin in tumor progression of human bronchopulmonary carcinomas. Human Pathology, 2004. 35(10): 1218-1222.
[40] Eikesdal, H.P., et al., Identification of amino acids essential for the antiangiogenic activity of tumstatin and its use in combination antitumor activity. Proceedings of the National Academy of Sciences of the United States of America, 2008. 105(39): 15040-15045.
[41] Maeshima Y, Sudhakar A, Lively JC.Tumstatin, an endothelial cell-specific inhibitor of protein synthesis.Science,2002,5551:140-143.
[42] Hutchings H, Ortega N, Plouet.Extracellular matrix-bound vascular endothelial growth factor promotes endothelial cell adhesion, migration, and survival through integrand ligation. J FASEB, 2003,17:1520-22.
[43] Maeshima Y, Colorado PC, Kalluri R. Two RGD-independent αVβ3 integrin binding sites on tumstatin regulate distinct anti-rumor properties. J Biol Chem,2000,14;275(31):23745-50.
[44] Gingras AC, Raught B, Sonenberg N. Regulation of translation initiation by FRA P/mTOR. Genes Dev, 2001, 15(7):807-26.
[45] Sudhakar A,SugimotoH, Yang C, et al. Human tumanstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha V beta 3 and alpha 5 beta 1 integrins.Proc.Natl.Acad.Sci.USA,2003,100:4766-71.
[46] Chang MC, Wang BR, Huang TF. Characterization of endothelial cell differential attachment to fibrin and fibrinogen and its inhibition by Arg-Gly-Asp-containing peptides.Toxicon, 1995, 33(8):1087-98.
[47] Borlado Lr, Redondo C, Alvarea B, et al. Increased phosphoinositide 3-kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo. J FASEB, 2000,14(7):895-903.
[48] Kim JE, Chen J. Cytoplasm-nuclear shuttling of FKBP12-rapamycin-associated protein is involved in repaying-sensitive signaling and translation initiation. Proc Natal Aced Sic USA, 2000, 97(26): 14340-5.
[49] Herbert TP, Fahraeus R, Prescott A, et al. Rapid induction of apoptosis mediated by peptides that bind initiation factor elF4E.Curr Biol, 2000,10(13):793-6.
[50] Dubois RN, Tsujii M, Bishop P, et al. Cloning and characterization of a growth factor-inducible cyclooxygenase gene from rat intestinal epithelial cells.Am.J.Physiol, 1994, 266:G822-7.
[51] Schmedtje JF, Ji YS, Li ZF, et al. Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. J Biol.Chem, 1997,272:601-8.
[52] Wu AW, Gu J, Li ZF, et al.COX-2 expression and tumor angiogenesis in colorectal cancer. World J.Gastroenterol, 2004,10,2323-6.
[53] Subbaramaiah K, Zakim D, Weksler BB, et al. Inhibition of cyclooxygenase: a novel approach to cancer prevention.Proc.Soc.Exp.Biol.Med, 1997,216:201-10.
[54] Harris AL. Hypoxia a key regulatory factor in tumorgrowth.Nat.Rev.Cancer,2002, 2:38-47.
[55] Panda DJ, Mier JW. Canstatin inhibits Aktactivation and induces Fas-dependent apoptosis in endothelial cells.J.Biol.Chem, 2003, 278:37632-6.
[56] Kunz M, Moeller S, Koczan D, et al. Mechanisms of hypoxic gene regulation of angiogenesis factor Cyr61 in melanoma cells.J.Biol.Chem, 2003, 278:45651-60.
[57] Pasco, S., et al., In vivo overexpression of tumstatin domains by tumor cells inhibits their invasive properties in a mouse melanoma model. Experimental Cell Research, 2004. 301(2): 251-265.
[58] Pearce, W.H. and V.P. Shively, Abdominal aortic aneurysm as a complex multifactorial disease - Interactions of polymorphisms of inflammatory genes, features of autoimmunity, and current status of MMP's. Abdominal Aortic Aneurysm: Genetics, Pathophysiology and Molecular Biology, 2006. 1085:117-132.
[59] Suhr, F., K. Brixius, and W. Bloch, Angiogenic and Vascular Modulation by Extracellular Matrix Cleavage Products. Current Pharmaceutical Design, 2009.15(4): 389-410.
[60] Eble A, Ries A, Lichy A, et al. The recognition sites of the integrins alphalbetal and alpha2betal within collagen IV are protected against gelatinase a attack in the native proteinJ.Biol.Chem, 1996, 271:30964-70.
[61] Pasco S, Monboisse JC, Kieffer N. The alpha3 (Ⅳ) 185-203 peptide from noncollagenous domain 1 of integrin alpha Vbeta3 and stimulates focal adhesion kinase and phosphatidylinositol 3-kinase phosphorylation.J Biol Chem,2000, 275(42): 32999-33007.
[62] Pasco S, Han J, Gillery P, Monboisse JC, et al. A specific sequence of the noncollagenous domain of the alpha3 (Ⅳ) chain of type IV collagen inhibits expression and activation of matrix metalloproteinases by tumor cells. Cancer Res, 2000, 60(2):467-473.
[63] Folkman J. . Angiogenesis inhibitors - A new class of drugs. Cancer Biology &Therapy, 2003, 2(4): S127-S133.
[64] Liu, H., B. Chen, and B. Lilly, Fibroblasts potentiate blood vessel formation partially through secreted factor TIMP-1. Angiogenesis, 2008. 11(3): 223-234.
[65] Hamano. Y, Zeisberg M, Sugimoto H, et al. (2003). Physiological levels of tumstatin, a fragment of collagen Ⅳ alpha 3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via alpha V beta 3 integrin. Cancer Cell,2003, 3(6): 589-601.
[66] Colorado PC,Torre A,Kamphaus G, et al.Anti-angiogenetic cues from vascular basement membrane collagen. Cancer Res,2000,60(9):2520-6.
[67] Kamphaus G,Colorado PC,Panka DJ,et al.Canstatin,a novel matrix-derived inhibitor of angiogenesis and tumor growth.J Biol Chem,2000,275:1209-15.
[68] Ramchandran R,Dhanabal M,Volk R,et al.Antiangiogenic activity of restin,NC10 domain of human collagen XV: comparison to endostatin.Biochem Biophys Res Commun,1999,255:735-9.
[69] Mundel, T.M. and R. Kalluri, Type Ⅳ collagen-derived angiogenesis inhibitors.MicrovascularResearch, 2007. 74(2-3):
[70]Twombly R.First clinical trials of endostatin yield lukewarm results.J Natl Cancer Inst,2002,94:1520-1.
[71]Ju JL,Coomber BL,Kerbel RS.A paradigm for therapy-induced microenviromental changes in solid tumors leading to drug resistance.Differentiation,2002,70:599-609.
[72]Shepherd FA,Sridhar SS.Angiogenesis inhibitor under study for the treatment of lung cancer.Lung Cancer,2003,41:S63-S72.
[73]Goto,T.,et al.,Tum-1,a tumstatin fragment,gene delivery into hepatocellular carcinoma suppresses tumor growth through inhibiting angiogenesis.International Journal of Oncology,2008.33(1):p.33-40.
[74]Weidner N,Folkman J,Pozza F,et al.Tumor angiogenesis:a new significant and independent prognostic indicator in early stage breast carcinoma[J].J Natl Cancer,1992,84(24):1875-1887.
[75]钱志远,黄强。胶质瘤血管发生发展机制研究。中国肿瘤,2005,14(2):85-88。
[76]Harrigan MR.Angiogenix factors in the central nervous system.Neurosurgery,2003,53:639-661.
[77]O'Reilly MS,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(2):315-328.
[78]O'Reilly MS,Boehm T,Shing Y,et al.Endostatin:an endogenous inhibitor of angiogenesis and tumor growth.Cell,1977,88(2):277-285.
[79]Kitayama,H.,et al.,Regulation of angiogenic factors in angiotensin Ⅱ infusion model in association with tubulointerstitial injuries.American Journal of Hypertension,2006.19(7):718-727.
[80]Katoh,Y.and M.Katoh,Comparative integromics on Angiopoietin family members.International Journal of Molecular Medicine,2006.17(6):p.1145-1149.
[81]肖芸,王季石。肿瘤抗血管生成疗法的研究进展。国外医学输血及血液学分册,2002,25(1):21-23。
[82]邹岚,陈正棠。新型内源性血管生成抑制剂Tumstatin。国外医学肿瘤分册, 2003,30(3):187-9。
[83]程鑫,李凯。Endostatin抗肿瘤机制进展研究。中国肿瘤临床,2008,35(9):534-536。
[84]王奇璐,刘彦玲。分子靶向治疗。中国现代医学,2008,46(14):1-4。
[85]李勇,孙燕。肿瘤的抗血管生成治疗研究进展。癌症进展杂志,2008,6(1):65-76。
[86]Fox JA.Gene therapy safety issues come to fore.Nat Biotechnol,1999,17:1153-7.
[87]Schatzlein AG.Non-viral vectors in cancer gene therapy:principles and progress.Anticancer Drug,2001,12:275-304.
[88]Zhang ZL,Zou WG,Luo CX,et al.An armed oncolytic adenovirus system,ZD55-gene,demonstrating potent antitumoral efficacy.Cell Res,2003,13:481-489.
[89]梁红雁,卢圣栋。非病毒载体基因治疗[M]。//卢圣栋。现代分子生物学实验技术。北京:中国协和医科大学出版社,1999,650-5。
[90]You,Y.,et al.,Inhibition effect of pcDNA-tum-5 on the growth of S 180 tumor.Cytotechnology,2008.56(2):97-104.
[91]Chung,I.S.,et al.,Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis in an orthotopic oral squamous cell carcinoma model.Oral Oncology,2008.44(12):1118-1126.
[92]胡维新。基因的治疗研究。[M]。//冯作化。医学分子生物学。北京:人民卫生出版社,2005,297-320。
[1] Ortega N, Werb Z.New functional roles for noncollagenous domain of basement membrane collgens.J Cell Sci, 2002,115(22):4201-4214.
[2] Sottile J. Regulation of angiogenesis by extracellular matrix. Biochim Biophus Acta,2004, 1654(1):13-22.
[3] Hamno Y,Kalluri R.Tumstatin,the NCI domain of alpha3 chain of type Ⅳ collagen, is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth. Biochem Biophys Res Commun, 2005, 333(2):292-298.
[4] Satchi-Fainaro R, Puder M, Davies JW, etal. Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470.Nat Med 2004, 10:255-261.
[5] Folkman J, Hanahan D. Switch to the angiogenic phenotype during tumorigenesis. Princess takamatsu Symp, 1991, 22:339-347.
[6] Arap W, Pasqulini R, Ruoslahti E.Cancer treatment by targeteddrug delivery to tumor vasculature in a mouse model.Science,1998,27(9):377-380.
[7] Hanahan and Folkman J.Patterns and emerginging mechanisms of the angiogenic switch during tumorigenesis.Cell, 1996, 86:352-364.
[8] Smith Mc, Cune KK, Weidner N. Demonstration and characterization of the angiogenic properties of cervical dysplsia. Cancer Res, 1994, 54:800-804.
[9] Weidner N. Current pathological methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res treat, 1996, 36:169-180.
[10] Hayes AJ.Antivascular therapy: a new approach to cancer treatment.BMJ, 1999,318:853-856.
[11] Blumberg B, Mackrell AJ, Olson PF, et al.Basement membrane procollagen Ⅳ and its specialized carboxyl domain are conserved in Drosophia, mouse, and human.J. Biol.Chem, 1987, 262:5947-5950.
[12] Netzer OK, Suzuki K, Itoh BG, etal. Comparative analysis of the noncollagenous NCI domain of type Ⅳ collagen: identification of structural features important for assembly, function, and pathogenesis. Protein Sci, 1998,7:1340-1351.
[13] Sarras Jr, Zhang X, Huff JK, et al.Extracellular matrix (mesoglea) of Hydr vulgaris Ⅲ, formation and function during morphogenesis of hydra cell aggregates. Dev Biol, 1993,157:383-398.
[14] Hudson BG, Reeders ST, Tryggvason K. Type Ⅳ collagen: structure, gene organization, and the role in human diseases .Moleclar basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol.Chem, 1993,268,26033-6.
[15] Kalluri R. Basement membranes: structure, assembly and role in tumor angiogenesis. Nat Rev Cancer, 2003, 3:422-233.
[16] Filie JD, Burbelo PD, Kozak A. Genetic mapping of the alpha 1 and alpha 2(Ⅳ) collagen genes to mouse chromosome 8.Mamm Genome, 1995,6:487.
[17] Momota R, Sugimoto M, Oohashi T, etal. Two genes,COL4A3 and COL4A4 coding for the human alpha3(Ⅳ) and alpha4(Ⅳ) collagen chains are arranged head -to-head on chromosome2q36.FFBS Lett,1998,424:11-16.
[18] Soininen R, Huotari M, Hostikka SI, et al.The structure genes for alpha 1 and alpha 2 chains of human type Ⅳ collagen are divergently encoded on opposite DNA strands and have overlapping promoter region. J Biol Chem, 1988,263:17217-20.
[19] Maeshima Y, Colorado PC, torre A, et al. Distinct antitumor properties of a type Ⅳ collagen domain derived from basement membrane. J Biol Chem, 2000, 14;275(28):21340-8.
[20] Sudhakar, A. and C. S. Boosani . Inhibition of Tumor Angiogenesis by Tumstatin: Insights into Signaling Mechanisms and Implications in Cancer Regression. Pharmaceu- tical Research 2008, 25(12): 2731-2739.
[21] Monboisse JC, Garnotel R, Bellon G, et al.The alpha 3 chain of type Ⅳ collagen prevents activation of human polymorphonuclear leukocytes. J Biol Chem. 1994,269(410;25475-82.
[22] Han J, Ohno N, Pasco S, et al. A cell binding domain from the alpha 3 chain of type Ⅳ collagen inhibits proliferation of melanoma cells. J Biol Chem, 1997,272(330; 20395-401.
[23] Petitelerc E, Boutaud A, Prestayko A, et al. New function for non-collagen type Ⅳ. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo. J Biol Chem, 2000, 275(11):8501-61.
[24] Mundel, T. M., R. Kalluri (2007). Type Ⅳ collagen-derived angiogenesis inhibitors.Microvascular Reseach, 2007, 74(2-3): 85-89.
[25] Maeshima Y, Sudhakar A, Lively JC.Tumstatin, an endothelial cell-specific inhibitor of protein synthesis.Science,2002,5551:140-143.
[26] Maeshima Y, Colorado PC, Kalluri R. Two RGD-independent αVβ3 integrin binding sites on tumstatin regulate distinct anti-tumor properties. J Biol Chem,2000,14;275(31):23745-50.
[27] Pedchenko V, Zent R, Hudson BG.Alpha(V) and alpha(V) beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1) domain of the alpha3 chain of type IV collgen:implication for the mechanism of endothelia cell adhesion. J Biol Chem, 2004, 279(4):2772-80.
[28] Maeshima Y,Yerramalla UL, DhanabalM,et al. Extracellular matrix-derived peptide binds to alpha(V)beta(3) integrin and inhibits angiogenesis. J Biol Chem, 2001, 276(34):31959-68.
[29] Maeshima Y, Manfredi M, Reimer C, Reimer C, et al. Identification of the anti-angiogenesis site within vascular basement membrane-derived tumstatin. J Biol Chem, 2001, 276(18): 15240-8.
[30] Cao Y. Endogenous angiogenesis inhibitors and their therapeutic implications. Int J biochem Cell Biol, 2001, 33(4):357-369.
[31] Pasco S, Monboisse JC, Kieffer N. The alpha3 (Ⅳ) 185-203 peptide from noncollagenous domain 1 of integrin alpha Vbeta3 and stimulates focal adhesion kinase and phosphatidylinositol 3-kinase phosphorylation.J Biol Chem, 2000,275(42):32999- 33007.
[32] Pasco S, Han J, Gillery P, Monboisse JC, et al. A specific sequence of the noncollagenous domain of the alpha3 (Ⅳ) chain of type IV collagen inhibits expression and activation of matrix metalloproteinases by tumor cells. CancerRes, 2000, 60(2): 467-473.
[33] Kalluri R. Discovery of Type Ⅳ collagen non-collagenous domain as movel integrin ligands and endogenous inhibitors of angiogenesis. Cold Spring Symp Quant Biol, 2002, 67:255-266.
[34] Floquet N, Pasco S, Ramont L, et althea antitumor properties of the alpha3 (Ⅳ)-(185-203) peptide from the NC1 domain of type IV collagen (tumstatin) are conformation-dependent. J Biol Chem, 2004, 279(3):2091-2100.
[35] Hutchings H, Ortega N, Plouet.Extracellular matrix-bound vascular endothelial growth factor promotes endothelial cell adhesion, migration, and survival through integrand ligation. J FASEB, 2003,17:1520-22.
[36] Boosani CS, Mannam AP, Cosgrove D, et al. Regulation of COX-2 mediated signaling by alpha3 type IV non-collagenous domain in tumor angiogenesis.Blood,2007,110:1168-77.
[37] Miyoshi T,Hirohata S, Ogawa H, et al. Tumor-specific expression of the RGD-alpha3 (Ⅳ) NC1 domain suppresses endothelial tube formation and tumor growth in mice. J FASEB, 20:1904-6.
[38] Gingras AC, Raught B, Sonenberg N. Regulation of translation initiation by FRA P/mTOR. Genes Dev, 2001,15(7):807-26.
[39] Sudhakar A,SugimotoH, Yang C, et al. Human tumanstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha V beta 3 and alpha 5 beta 1 integrins.Proc.Natl.Acad.Sci.USA,2003,100:4766-71.
[40] Chang MC, Wang BR, Huang TF. Characterization of endothelial cell differential attachment to fibrin and fibrinogen and its inhibition by Arg-Gly-Asp-containing peptides.Toxicon, 1995, 33(8): 1087-98.
[41] Borlado Lr, Redondo C, Alvarea B, et al. Increased phosphoinositide 3-kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo. J FASEB, 2000, 14(7):895-903.
[42] Kim JE, Chen J. Cytoplasm-nuclear shuttling of FKBP12-rapamycin-associated protein is involved in repaying-sensitive signaling and translation initiation. Proc Natal Aced Sic USA, 2000, 97(26):14340-5.
[43] Herbert TP, Fahraeus R, Prescott A, et al. Rapid induction of apoptosis mediated by peptides that bind initiation factor elF4E.Curr Biol, 2000,10(13):793-6.
[44] Dubois RN, Tsujii M, Bishop P, et al. Cloning and characterization of a growth factor-inducible cyclooxygenase gene from rat intestinal epithelial cells.Am.J.Physiol, 1994, 266:G822-7.
[45] Schmedtje JF, Ji YS, Li ZF, et al. Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells.J Biol.Chem,1997,272:601-8.
[46]Wu AW,Gu J,Li ZF,et al.COX-2 expression and tumor angiogenesis in colorectal cancer.World J.Gastroenterol,2004,10,2323-6.
[47]Subbaramaiah K,Zakim D,Weksler BB,et al.Inhibition of cyclooxygenase:a novel approach to cancer prevention.Proc.Soc.Exp.Biol.Med,1997,216:201-10.
[48]Harris AL.Hypoxia a key regulatory factor in tumorgrowth.Nat.Rev.Cancer,2002,2:38-47.
[49]Panda D J,Mier JW.Canstatin inhibits Aktactivation and induces Fas-dependent apoptosis in endothelial cells.J.Biol.Chem,2003,278:37632-6.
[50]Kunz M,Moeller S,Koczan D,et al.Mechanisms of hypoxic gene regulation of angiogenesis factor Cyr61 in melanoma cells.J.Biol.Chem,2003,278:45651-60.
[51]Xu J,Rodriguez,Petitclerc E,et al.Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.J.Cell Biol,2001,154:1069-79.
[52]Eble A,Ries A,Lichy A,et al.The recognition sites of the integrins alphalbetal and alpha2betal within collagen IV are protected against gelatinase a attack in the native protein.J.Biol.Chem,1996,271:30964-70.
[53]Folkman J..Angiogenesis inhibitors-A new class of drugs.Cancer Biology &Therapy,2003,2(4):S127-S133.
[54]Hamano.Y,Zeisberg M,Sugimoto H,et al.(2003).Physiological levels of tumstatin,a fragment of collagen Ⅳ alpha 3 chain,are generated by MMP-9proteolysis and suppress angiogenesis via alpha Ⅴ beta 3 integrin.Cancer Cell,2003,3(6):589-601.
[55]Borza CM,Pozzi A,Borza DB,et al.Integrin alpha3betal:a novel receptor for alpha 3(IV)noncollagenous domain and a transdomaint inhibitor for integrin alphavbeta3.J Biol.Chem,2006,282:20932-9.
[56]邹岚,陈正棠。新型内源性血管生成抑制剂Tumstatin。国外医学肿瘤分册,2003,30(3):187-9。
[57]Jansen M,Wit Hamer P.C de,Witmer AN,et al.Current perspective on antiangiogenesis strategies in the treatment of malignant gliomas.Brain Res Rev, 2004,45:143-163.
[58]Fernando NT,Koch M,Rothrock C,et al.Tumor escape from endogeneous,extracellular matrix-associated angiogenesis inhibitors by up-regulation of multiple proangiogenic factors.Chin.Cancer Res,2008,14:1529-1539.
[59]Hurwita H,Fehernbacher L,Novotny W,et al.Bevacizumab plus irinotecan,fluorouracil,and leucovorin for metastatic colorectal cancer.N.Engl.J.Med,2004,350:2335-2342.
[60]Morabito A,De Maio E,Di Maio M,Normanno,et al.Tyrosine kinas inhibitors of vascular endothelial growth factor receptors in clinical trial;current status and future directions.Oncologist,2006,11:753-764.