重组腺病毒载体介导血管抑素基因治疗大鼠脑胶质瘤的实验研究
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摘要
目的：构建重组缺陷型腺病毒血管抑素基因（angiostatin）载
体，进行血管抑制基因治疗脑胶质瘤的体内及体外的实验研究。方
法：RT-PCR法克隆血管抑素基因，同源重组共转染293细胞构建携
带血管抑素基因的腺病毒载体，体外检测血管抑素的重组缺陷型腺病
毒载体对内皮细胞增殖的抑制作用。建立大鼠皮下及脑内C6胶质瘤
模型，给予体内基因治疗，观察及评价治疗脑胶质瘤的效果。结果：克
隆得到约1.2Kb的血管抑素cDNA。构建携带血管抑素基因重组腺病
毒载体AdhCMV-AGS，体外试验表明，重组载体可以强烈抑制内皮细
胞的增殖。体内试验表明重组载体可以在体内表达血管抑素，并且有
效抑制皮下胶质瘤的生长，使脑内荷C6胶质瘤大鼠长期存活。结论：
以重组腺病毒为载体的血管抑素基因治疗脑胶质瘤效果显著，将是一
种基因治疗肿瘤的新策略。
Objective To study the therapy for C6 glioma with the
recombinant adenovirus containing angiostatin eDNA (AdhCMV-
AGS). Methods Angiostatin gene was cloned into vector
pAdCMV-AGHpA to make pAdhCMV-AGS. Recombinant adenovirus was
constructed in 293 cells contransfected with pAdCMV(AGS) and
pJM17. Detected the effect of AdhCMV-AGS on proliferation of
endothelial cells(ECV3O4) in vitro. Made a model of rat C6 brain
glioma, then observed tumors size and survival of tumor-bearing
rats treated with AdhCMV-AGS. Results AdhCMV-AGS could
inhibited the proliferation of ECV3O4 cells significantly, but
not C6 cells. The size of tumors injected with AdhCMV-AGS
decreased. Some of it even disappeared, while the size of control
(injected with saline solution) was larger significantly. the
survival time of groups were beyond 90 days (with AdhCMV-AGS),
16.8 ±3.3 days(with Ad-null), 16.7±2.5 days(with
saline) (p<0.001). Conclusion AdhCMV-AGS could inhibit the
proliferation of endothelial cells and the angiogenesis by
which it suppressed C6 glioma. Ours results suggest that
antiangiogenesis gene therapy will be a promising way of
treating tumors that have abundant capillary.
体，进行血管抑制基因治疗脑胶质瘤的体内及体外的实验研究。方
法：RT-PCR法克隆血管抑素基因，同源重组共转染293细胞构建携
带血管抑素基因的腺病毒载体，体外检测血管抑素的重组缺陷型腺病
毒载体对内皮细胞增殖的抑制作用。建立大鼠皮下及脑内C6胶质瘤
模型，给予体内基因治疗，观察及评价治疗脑胶质瘤的效果。结果：克
隆得到约1.2Kb的血管抑素cDNA。构建携带血管抑素基因重组腺病
毒载体AdhCMV-AGS，体外试验表明，重组载体可以强烈抑制内皮细
胞的增殖。体内试验表明重组载体可以在体内表达血管抑素，并且有
效抑制皮下胶质瘤的生长，使脑内荷C6胶质瘤大鼠长期存活。结论：
以重组腺病毒为载体的血管抑素基因治疗脑胶质瘤效果显著，将是一
种基因治疗肿瘤的新策略。
Objective To study the therapy for C6 glioma with the
recombinant adenovirus containing angiostatin eDNA (AdhCMV-
AGS). Methods Angiostatin gene was cloned into vector
pAdCMV-AGHpA to make pAdhCMV-AGS. Recombinant adenovirus was
constructed in 293 cells contransfected with pAdCMV(AGS) and
pJM17. Detected the effect of AdhCMV-AGS on proliferation of
endothelial cells(ECV3O4) in vitro. Made a model of rat C6 brain
glioma, then observed tumors size and survival of tumor-bearing
rats treated with AdhCMV-AGS. Results AdhCMV-AGS could
inhibited the proliferation of ECV3O4 cells significantly, but
not C6 cells. The size of tumors injected with AdhCMV-AGS
decreased. Some of it even disappeared, while the size of control
(injected with saline solution) was larger significantly. the
survival time of groups were beyond 90 days (with AdhCMV-AGS),
16.8 ±3.3 days(with Ad-null), 16.7±2.5 days(with
saline) (p<0.001). Conclusion AdhCMV-AGS could inhibit the
proliferation of endothelial cells and the angiogenesis by
which it suppressed C6 glioma. Ours results suggest that
antiangiogenesis gene therapy will be a promising way of
treating tumors that have abundant capillary.
引文
1． Takaiya Y.Short MP.MOOLten FL. Et al. An expression model of retrovirus gene therapy for malignant brain tumors. J Neurosurg 1993; 79: 104
2． Kim TS,Halliday AL Hedley-Whyte ET,et al Correlates of survival and the Daumas-Duport grading system for astrocytomas. J Neurosurg 1991;74:27
3． Anderson WF. Gene therapy for AIDS. Hum Gene Ther 1994; 5: 149
4． Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature medicine, 1995,1:27-31
5． O'Reilly MS, Holmgren L, Shing Y, et al: Angiostatin: A novel angiogenesis inhibitor that meditates the supression of metastases by a Lewis Lung carcinoma. Cell , 1994, 79: 315-328．
6． Kerbal RS. A cancer therapy resistant to resistance. Nature (London), 1997,390:335-336．
7． Harris AL. Antiangiogenesis for cancer therapy. Lancet, 1997, 349: 13-15．
8． Hitt M, Bett AJ, Addison CL, et al. Techniques for human adenovirus vetor construction and characterization in: Methods in molecular genetics. Academic Press. 1995;7: 13-45
9． Curiel DT, Agarwal S, Wagner E, et al. Adenovirus enhancement of transferrin-polylysine-mediated gene delivery. Proc. Natl. Acad. Sci USA. 1991, 88 (19) : 8850-8854．
10． Cotton M, Wagner E, Zatlloukal K,et al. High efficiency recceptor mediated delivery of small and large (48kb) gene
constructs using the endosome distruption activity of defective or chemically inactivated adenovirus particles. Proc Natl Acad Sci USA. 1992; 89: 6094-6098．
11． Vile, R. G. , Russell, S. J. Retroviruses as vectors. Br Med. Bull 1995; 51: 12-30．
12． Meiklejohn G. Viral respiratory disease at lowry air force vase in Denver 1952-1982． J Infect.Dis. 1979; 140: 48-53
13． Kremer, E. J. and Perricaudet, M. Adenovirus and adeno-associated virus mediated gene transfer. Br. Med Bull 1995; 51: 31．
14． Efstathiou, S. and Minson, A. C. Herpes virus-based vectors. Br. Med. Bull 1995; 51: 44-45
15． Naldini, L., Blomer, U., Gallay, P., Ory, D. , Mulligan, R., Gage, F. H. , et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272: 263-267．
16． Klauber N, Rohan RM, Flynn E, et al.Critical component of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM-1470． Nature Medicine , 1997, 3: 443-446．
17． Xu M,Kumar D, Srinivas S, et al. Parental gene therapy with P53 inhibits human breast tumors in vivo through a bystand mechanism without evidence of toxicity. Human Gene Therapy, 1997,8: 177-185．
18． Holash J,Maisonpierre PC, Compton D, et al, Vessal cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science (Wash DC) 1999; 284: 1994-1998
19． Asahara T, Masuda H, Takahashi T, et al,Bone marrow orign
of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85: 221-228
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24． Ades EW, Candal FJ, Swerlick RA, et al. HMEC-1:Establishment of an immortalize human microvascular endothelial cell line. J InvestDermatol. 1992; 99:683-690．
25． Edgell CJ, McDonald CC and Graham HB. Permanent cell line expressing human factor Vlll-related antigen eatablished by hybridization. Proc Natl Acad Sci USA. 1983; 80: 3734-3737．
26． Griffioen AW, Damen CA, Martinotti S,et al. Endothelial ICAM-1 expression is suppressed in human malignancies, role of angiogenic factors. Cancer Res. 1996b; 56: 1111-1117
27． Barendsz-Janson AF, Griffioen AW, Muller AD,et al. In vitro tumor angiogenesis assays: Plasminogen lysine binding site I(LBS-1) inhibits in vitro tumor induced angiogenesis. J Vase. 1998; 35: 109-114．
28． Nicosia RF and Ottinetti A. Modulation of microvascular
growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in matrigel, collagen, fibrin, and plasma clot. In Vitro cell Deb Biol 1990; 26: 119-128．
29． Malinda KM, Nominzu M, Chung M, Delgado M, et al. Identification of laminin a1 and B1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting. FASEB J. 1999; 13: 53-62．
30． Brown KJ, Maynes SF, Bezos A, et al. A movel in vitro assay for human angiogensis. Lab invest 1996;75:539-555．
31． Wartenberg M, Gunther J,Hescheler H, et al. The emvryoid body sd a novel in vitro assay system for antiangiogenic agents. Lab Inbest . 1998; 78: 1301-1314．
32． Risau W. Saropla H, Zerwes HG, et al. Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development. 1988; 102: 471-478．
33． Vittet D, Prandini MH, Berthier R, Schweitzer A, et al. Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood. 1996; 88: 3424-3431．
34． Nguyen M, Shing Y and Folkman J. Quantitatics of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane. Microvasc Res. 1994; 47: 31-40．
35． Conrad TJ, Chandler DB, Corless JM,et al. In vivo measurement of corneal angiogenesis with video data acquisition and computerized image analysis. Lab Invest. 1994; 70: 426-434．
36． Lichtenbekd H, Barendsz-Janson AF, Van Essen H, et al. Angiogenic poteneial of malignant and mon-malignant human breast tissues in an in vivo angiogenesis model. Int J Cancer.
1998; 77: 456-459．
37． Shubik P, Feldman R, Garcia H, et al. Vascularization induced in the cheek poudh of the Syrian hamster by tumor and nontumor substances. J Matl Cancer Inst. 1976; 57:769-774． .
38． Mahadevan V, Hart IR and Lewis GP. Factors influencing blood supply in wound granuloma quantitated by a new in vivo technique. Cancer Res. 1989; 49:415-419．
39． Plunkett ML and Hailey JA. An in vivo quantitative angiogenesis model using tumor cells entrapped in alginate. Lab Invest. 1990; 62: 510-517．
40． Jain RK, Schlenger K, Hockel M,et al. Quantitative angiogenesis assays: Progress and problems. Nat Med. 1997; 3:1203-1208．
41． Ingber D, Fujita T, Kishimoto S, et al. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature (London) 1990; 348: 555-557．
42． Kusaka M, Sudo K, Fujita T, et al. Potent anti-angiogenesis action of AGM-1470: Comparison to the fumagillin parent. Bio chem Biophys Res Commun. 1991; 174: 1070-1076．
43． Castronovo V and Belotti D.TNP-470(AGM-1470) : Mechanisms of action and early clinical development. Eur J fancer. 1996; 32A: 2520-2527．
44． Rastinejad F, Polverini PJ and Bouck N. Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell. 1989; 56: 345-355．
45． Good DJ, Polverini PJ, Rastinejad F, et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci
USA. 1990; 87: 6624-6628．
46． Grossfeld GS, Ginaberg DA, Stein JP.et al. Thrombospendin-1 expression in bladder cancer :Association with P53 alterations, tumor angiogenesis, and tumor progression.H Natl Cancer Inst. 1997; 89: 219-227．
47． Gupta SK, Hassel T and Singh JP. A potent inhibitor of endotheliahl cell proligeration id generated by proteolytic cleavage of the chemokine platelet factor-4． Proc Natl Acad Sci USA. 1995; 92: 7799-7803．
48． Kolber DL, Knisely TL and Maione TE. Inhivition of development of murine metanoma lung metastases by systemic adminstration of recombinant platelet factor-4． J Natl Cance Inst. 1995; 87: 304-309．
49． Luster AD, Greenberg SM and Leder P.The IP-10 chemkine binds to a specific cell surface heparan sulfate site shared with platelet factor-4 and inhibits endothelial cell proliferation. J Exp Med. 1995:182: 219-231．
50． O'Reilly MS, Holmgren L, Shing Y,et al. Angiostatin: a novel angiogenesis inhibitor that mediates suppression of metastases by a Lewis lung carcinoma. Cell. 1994; 79:315-328．
51． O'Reilly MS, Boelim T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997; 88: 2787-285．
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2． Kim TS,Halliday AL Hedley-Whyte ET,et al Correlates of survival and the Daumas-Duport grading system for astrocytomas. J Neurosurg 1991;74:27
3． Anderson WF. Gene therapy for AIDS. Hum Gene Ther 1994; 5: 149
4． Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature medicine, 1995,1:27-31
5． O'Reilly MS, Holmgren L, Shing Y, et al: Angiostatin: A novel angiogenesis inhibitor that meditates the supression of metastases by a Lewis Lung carcinoma. Cell , 1994, 79: 315-328．
6． Kerbal RS. A cancer therapy resistant to resistance. Nature (London), 1997,390:335-336．
7． Harris AL. Antiangiogenesis for cancer therapy. Lancet, 1997, 349: 13-15．
8． Hitt M, Bett AJ, Addison CL, et al. Techniques for human adenovirus vetor construction and characterization in: Methods in molecular genetics. Academic Press. 1995;7: 13-45
9． Curiel DT, Agarwal S, Wagner E, et al. Adenovirus enhancement of transferrin-polylysine-mediated gene delivery. Proc. Natl. Acad. Sci USA. 1991, 88 (19) : 8850-8854．
10． Cotton M, Wagner E, Zatlloukal K,et al. High efficiency recceptor mediated delivery of small and large (48kb) gene
constructs using the endosome distruption activity of defective or chemically inactivated adenovirus particles. Proc Natl Acad Sci USA. 1992; 89: 6094-6098．
11． Vile, R. G. , Russell, S. J. Retroviruses as vectors. Br Med. Bull 1995; 51: 12-30．
12． Meiklejohn G. Viral respiratory disease at lowry air force vase in Denver 1952-1982． J Infect.Dis. 1979; 140: 48-53
13． Kremer, E. J. and Perricaudet, M. Adenovirus and adeno-associated virus mediated gene transfer. Br. Med Bull 1995; 51: 31．
14． Efstathiou, S. and Minson, A. C. Herpes virus-based vectors. Br. Med. Bull 1995; 51: 44-45
15． Naldini, L., Blomer, U., Gallay, P., Ory, D. , Mulligan, R., Gage, F. H. , et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272: 263-267．
16． Klauber N, Rohan RM, Flynn E, et al.Critical component of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM-1470． Nature Medicine , 1997, 3: 443-446．
17． Xu M,Kumar D, Srinivas S, et al. Parental gene therapy with P53 inhibits human breast tumors in vivo through a bystand mechanism without evidence of toxicity. Human Gene Therapy, 1997,8: 177-185．
18． Holash J,Maisonpierre PC, Compton D, et al, Vessal cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science (Wash DC) 1999; 284: 1994-1998
19． Asahara T, Masuda H, Takahashi T, et al,Bone marrow orign
of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85: 221-228
20． Fiedler W,Graeven U, Ergun S, Verago S, et al. Vascular ensothelial growth factor, a possible paracrine growth fator in human acute myeloed leukemia. Blood. 1997; 89: 1870-1875
21． Foss HD, Araujo I,Demel G, et al. Expression of Vascular Endothelial Growth factor in lymphomas and casleman's disease. J Pathol 1997; 183: 44-50．
22． PerezAtyde AR, Salian SE, Tedrow U, et al. Spectrum of tumor angiogensis in the bone marrow of children with acute lymphoblastic leukemia. Am J Pathol. 1997; 150: 815-821．
23． Shing Y, Folkman J, Sullivan R, et al. Heparin affinity: Purification of a tumor-derived capillary endothelial cell growth factor. Science. 1984; 223: 1296-1299
24． Ades EW, Candal FJ, Swerlick RA, et al. HMEC-1:Establishment of an immortalize human microvascular endothelial cell line. J InvestDermatol. 1992; 99:683-690．
25． Edgell CJ, McDonald CC and Graham HB. Permanent cell line expressing human factor Vlll-related antigen eatablished by hybridization. Proc Natl Acad Sci USA. 1983; 80: 3734-3737．
26． Griffioen AW, Damen CA, Martinotti S,et al. Endothelial ICAM-1 expression is suppressed in human malignancies, role of angiogenic factors. Cancer Res. 1996b; 56: 1111-1117
27． Barendsz-Janson AF, Griffioen AW, Muller AD,et al. In vitro tumor angiogenesis assays: Plasminogen lysine binding site I(LBS-1) inhibits in vitro tumor induced angiogenesis. J Vase. 1998; 35: 109-114．
28． Nicosia RF and Ottinetti A. Modulation of microvascular
growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in matrigel, collagen, fibrin, and plasma clot. In Vitro cell Deb Biol 1990; 26: 119-128．
29． Malinda KM, Nominzu M, Chung M, Delgado M, et al. Identification of laminin a1 and B1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting. FASEB J. 1999; 13: 53-62．
30． Brown KJ, Maynes SF, Bezos A, et al. A movel in vitro assay for human angiogensis. Lab invest 1996;75:539-555．
31． Wartenberg M, Gunther J,Hescheler H, et al. The emvryoid body sd a novel in vitro assay system for antiangiogenic agents. Lab Inbest . 1998; 78: 1301-1314．
32． Risau W. Saropla H, Zerwes HG, et al. Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development. 1988; 102: 471-478．
33． Vittet D, Prandini MH, Berthier R, Schweitzer A, et al. Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood. 1996; 88: 3424-3431．
34． Nguyen M, Shing Y and Folkman J. Quantitatics of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane. Microvasc Res. 1994; 47: 31-40．
35． Conrad TJ, Chandler DB, Corless JM,et al. In vivo measurement of corneal angiogenesis with video data acquisition and computerized image analysis. Lab Invest. 1994; 70: 426-434．
36． Lichtenbekd H, Barendsz-Janson AF, Van Essen H, et al. Angiogenic poteneial of malignant and mon-malignant human breast tissues in an in vivo angiogenesis model. Int J Cancer.
1998; 77: 456-459．
37． Shubik P, Feldman R, Garcia H, et al. Vascularization induced in the cheek poudh of the Syrian hamster by tumor and nontumor substances. J Matl Cancer Inst. 1976; 57:769-774． .
38． Mahadevan V, Hart IR and Lewis GP. Factors influencing blood supply in wound granuloma quantitated by a new in vivo technique. Cancer Res. 1989; 49:415-419．
39． Plunkett ML and Hailey JA. An in vivo quantitative angiogenesis model using tumor cells entrapped in alginate. Lab Invest. 1990; 62: 510-517．
40． Jain RK, Schlenger K, Hockel M,et al. Quantitative angiogenesis assays: Progress and problems. Nat Med. 1997; 3:1203-1208．
41． Ingber D, Fujita T, Kishimoto S, et al. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature (London) 1990; 348: 555-557．
42． Kusaka M, Sudo K, Fujita T, et al. Potent anti-angiogenesis action of AGM-1470: Comparison to the fumagillin parent. Bio chem Biophys Res Commun. 1991; 174: 1070-1076．
43． Castronovo V and Belotti D.TNP-470(AGM-1470) : Mechanisms of action and early clinical development. Eur J fancer. 1996; 32A: 2520-2527．
44． Rastinejad F, Polverini PJ and Bouck N. Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell. 1989; 56: 345-355．
45． Good DJ, Polverini PJ, Rastinejad F, et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci
USA. 1990; 87: 6624-6628．
46． Grossfeld GS, Ginaberg DA, Stein JP.et al. Thrombospendin-1 expression in bladder cancer :Association with P53 alterations, tumor angiogenesis, and tumor progression.H Natl Cancer Inst. 1997; 89: 219-227．
47． Gupta SK, Hassel T and Singh JP. A potent inhibitor of endotheliahl cell proligeration id generated by proteolytic cleavage of the chemokine platelet factor-4． Proc Natl Acad Sci USA. 1995; 92: 7799-7803．
48． Kolber DL, Knisely TL and Maione TE. Inhivition of development of murine metanoma lung metastases by systemic adminstration of recombinant platelet factor-4． J Natl Cance Inst. 1995; 87: 304-309．
49． Luster AD, Greenberg SM and Leder P.The IP-10 chemkine binds to a specific cell surface heparan sulfate site shared with platelet factor-4 and inhibits endothelial cell proliferation. J Exp Med. 1995:182: 219-231．
50． O'Reilly MS, Holmgren L, Shing Y,et al. Angiostatin: a novel angiogenesis inhibitor that mediates suppression of metastases by a Lewis lung carcinoma. Cell. 1994; 79:315-328．
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