魟鱼软骨多糖对血管形成的影响及机制研究
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摘要
血管形成是一个重要的生理学过程。血管形成刺激因子如VEGF、bFGF、MMP等触发一系列级联反应导致局部基底膜降解、内皮细胞迁移、增生、微血管形成。组织中血管网内环境稳定取决于血管生成因子与抗血管生成因子的平衡。血管形成调节机制失衡与一些疾病的发生有关,包括慢性炎症、鳞癣、类风湿性关节炎、老年性黄斑病变和癌症。
目前,许多合成的、生物的、天然来源的化合物已被证明对血管形成具有抑制作用。第一个血管形成调节因子是在软骨中发现的。本文就是以魟鱼软骨作为研究对象。魟鱼为脊椎动物门Verterata,软骨鱼纲Chondrichthyes,板鳃亚纲Elasmobranchii,鳐目(或魟目)Rajiformes,魟科Dasyatidae,属暖温类底层鱼。以往我们的研究发现魟鱼软骨提取物对鸡胚绒毛尿囊膜血管生成具有抑制作用。本实验所研究的魟鱼软骨多糖采用盐酸胍抽提,膜超滤、丙酮分级沉淀、Sephadex凝胶柱层析分离纯化,经天青Ⅰ比色法和高效液相色谱法检测分子量为9.7×10.4,纯度为99%以上。本实验探讨魟鱼软骨多糖抗血管形成的生物活性。
目的
本研究目的在于应用培养的人脐静脉内皮细胞、大鼠角膜新生血管模型和小鼠移植瘤模型考察魟鱼软骨多糖对血管形成的影响,并探讨其作用机制。
方法
1.人脐静脉内皮细胞的培养和鉴定
Angiogenesis is a fundamental physiological process. Angiogenic stimulus such as VEGF,bFGF,MMP and so on triggers a cascade of functional responses leading to local basement membrane dissolution, endothelial cell migration, proliferation and micro vessel morphogenesis. Homeostatic equilibrium of the vascular network within tissues depends on the balance between angiogenic and anti-angiogenic factors. Imbalance in angiogenesis regulatory mechanisms has been associated with several pathologies including chronic inflammation, psoriasis, rheumatoid arthrities, aged - related macular degeneration and cancer.Recently, a variety of synthetic, biological and natural compounds have been indentified as inhibitors of angiogenesis, the first biological regulator of neovascularization was found in cartilage. In this study, Ray cartilage has been tested. Ray which is belong to vertebrata, chondrichthyes, elasmobranchii, raji-formes, dasyatidae is ground floor fish of warm temperature. We had observed that the extraction of ray cartilage could inhibit angiogenesis of chicken embryo chorioallantoic membrane effectively. Here, Ray cartilage polysaccharide was extracted, separated and purified by guanidine hydrochloride, membrane ultrafil-trating, acetone classification precipitation and sephadex gels pillar patients, Tianqing I colorimetric method and high performance liquid chromatographic (HPLC) detected Ray cartilage polysaccharide molecular mass was 9. 7 x 10~4 and its purity exceeded ninty - nine percent. In the experiment, we explore Ray cartilage polysaccharide antiangiogenic biological activities.Objectivestudy was designed to investigate the effects of Ray Cartilage Glyco-
saminoglycans ( RCG) on angiogenesis and to explore its mechanism and cultured Human Umbilical Endothelial cell ( HUVEC) , Wistar rat corneal neovas-cularization ( CNV) and transplanting tumor mouse models were used in our work.Methods1. Culture and Identification of the Endothelial Cells of Human Umbilical VeinEndothelial cells were isolated from human umbilical cords with 0. 1% I type collagenase and were digested by 0. 05% tripsin and 0. 02% EDTA when the endothelial cells confluent. The cells are cultivated in Ml99 medium containing Endothelial Cells Growth Supplement(ECGS) and fetal bovine serum. Endothelial cells were identified by morphologic and immunohistochemical assays .2. The effect of RCG on angiogenesis in vitroThe experiments were classified into normal group, RCG groups at varied concentrations (100,50,25,10, 2mg/mL ) and SCG group ( 50mg/mL ). MTT, endothelial cell migration and capillary formation experiments were adopted to e-valuate the effects of RCG on HUVEC. Meantime,we also detected the effect of RCG on HUVEC cycle by means of Flow cytometry.3. The effects of RCG on corneal neovascularization(CNV)in Wistar rats The RCG eye drop was prepared. Corneal neovascularization was inducedby sutures on Wistar rats-. The experimental rats were randomly divided into saline group,RCG groups at varied concentrations( 100,50,25mg/mL)and Hepa-rin + Corlin group. CNV area, histopathological changes and VEGF immunohisto-chemistry were used to evaluate the RCG therapeutic effects on CNV in Wistar rats.4. The effects of RCG on implanted Lewis lung carcinoma in C57BL/6J mice.The model of mouse with Lewis lung carcinoma was made;the experimental mice were randomly divided into saline group,RCG groups at varied concentrations (500,250,125mg/kg) and CTX group. Tumor growth states were observed;
Tumor growth curve was described;On the 21th day, tumor was deprived, inhibitory rate of primary tumor and number of lung metastasis focus were measured. Microvessel density (MVD) was quantitated by immunohistochemistry using monoclonal antibodies of CD31.5. The effects of RCG on the expression of VEGF ,bFGF,MMP 9 mRNA and proteinThe expression of VEGF, bFGF, MMP9 were determined with RT - PCR and Western blot.Result1. The effects of RCG on angiogenesis in vitroBy observing morphologic the cells cultivated were endothelial cells typically and by immunohistochemically the cells cultured were stained positively by VI factor monoclonal antibody. 100 - lOmg/mL RCG obviously inhibited the proliferation of HUVEC, the mean MTT OD value of saline group, RCG groups at varied concentrations (100,50,25,10, 2mg/mL ) and SCG group ( 50mg/mL ) were 0.41 ±0.05,0.11 ±0.05,0. 23 ±0.06,0. 31 ±0. 02,0. 35 ±0. 07,0. 36 ±0. 03,0.21 ±0. 04, respectively,ICjo value was 62. 93mg/mL . 100 - lOmg/mL RCG strongly inhibited the migration of HUVEC, the migrated numbers cell of obove groups were 63.80 ± 10. 18,6. 60 ± 3. 58,15.40 ± 4. 61,18. 80 ± 5. 89, 41. 80 ± 3. 70,13. 60 ± 5. 98 ,respectively;100 - lOmg/mL RCG inhibited capillary formation,the mean capillary length of them were 4.63 ±0.31,1.08 ± 0.22,2.31 ±0.11,3.03 ±0. 19,3. 60 ±0. 15,2.11 ±0.12 mm,respectively. RCG could block HUVEC in G2/M phase by Flow cytometry.2. The effects of RCG on corneal neovascularization(CNV)in Wistar rats By means of RCG effect on corneal neovascularization ( CNV) in Wistarrats, We observed that the corneal neovascularization of RCG groups growed slowly and RCG groups showed smaller areas of new blood vessels and fewer inflammatory cells and lower VEGF expression than saline group, there were high expression in corneal epithelium of Saline group, and negative expression in corneal epithelium of lOOmg/mL, 50mg/mL and Heparin + corlin groups, the ex-
pression of VEGF in 25mg/mL RCG group was lower .3. The effects of RCG on implanted Lewis lung carcinoma in C57BL/6J mice.The effects of RCG on the implanted Lewis lung carcinoma showed that tumor growth curves in RCG groups were smooth compared with Saline group, inhibitory rates of primary tumor in 500,250,125mg/kg RCG groups were 43. 36% ,26. 34% , 15. 10% , respectively, there were significant differences in number of lung metastasis focus between RCG groups and Saline group . In MVD assay, the MVD number of RCG groups were markedly reduced compared with Saline group, and had a clear dose - effect relationship.4. The effect of RCG on the expression of VEGF ,bFGF,MMP 9 mRNA and proteinUsing RT - PCR , RCG at various dosage groups Lewis lung cancer tissues VEGF, bFGF, MMP - 9 mRNA fluorescence intensity reduced gradually and relative AUG values were less significantly than Saline group' s (P<0.01) and there were significant differences among these dosage groups;Western blot results showed that protein strap became weaker and weaker with concentrations increased, VEGF, bFGF, MMP -9 protein expression levels in RCG groups were reduced significantly compared with Saline group (P <0.01) 0ConclusionsFrom the experiments results in vitro and in vivo, we conclude that RCG had srong antiangiogenic effect. Its mechanism in vitro might involve in the blocking of HUVEC cell cycle and its mechanism in vivo might be downregulating the expression of VEGF ,bFGF,MMP -9.
目前,许多合成的、生物的、天然来源的化合物已被证明对血管形成具有抑制作用。第一个血管形成调节因子是在软骨中发现的。本文就是以魟鱼软骨作为研究对象。魟鱼为脊椎动物门Verterata,软骨鱼纲Chondrichthyes,板鳃亚纲Elasmobranchii,鳐目(或魟目)Rajiformes,魟科Dasyatidae,属暖温类底层鱼。以往我们的研究发现魟鱼软骨提取物对鸡胚绒毛尿囊膜血管生成具有抑制作用。本实验所研究的魟鱼软骨多糖采用盐酸胍抽提,膜超滤、丙酮分级沉淀、Sephadex凝胶柱层析分离纯化,经天青Ⅰ比色法和高效液相色谱法检测分子量为9.7×10.4,纯度为99%以上。本实验探讨魟鱼软骨多糖抗血管形成的生物活性。
目的
本研究目的在于应用培养的人脐静脉内皮细胞、大鼠角膜新生血管模型和小鼠移植瘤模型考察魟鱼软骨多糖对血管形成的影响,并探讨其作用机制。
方法
1.人脐静脉内皮细胞的培养和鉴定
Angiogenesis is a fundamental physiological process. Angiogenic stimulus such as VEGF,bFGF,MMP and so on triggers a cascade of functional responses leading to local basement membrane dissolution, endothelial cell migration, proliferation and micro vessel morphogenesis. Homeostatic equilibrium of the vascular network within tissues depends on the balance between angiogenic and anti-angiogenic factors. Imbalance in angiogenesis regulatory mechanisms has been associated with several pathologies including chronic inflammation, psoriasis, rheumatoid arthrities, aged - related macular degeneration and cancer.Recently, a variety of synthetic, biological and natural compounds have been indentified as inhibitors of angiogenesis, the first biological regulator of neovascularization was found in cartilage. In this study, Ray cartilage has been tested. Ray which is belong to vertebrata, chondrichthyes, elasmobranchii, raji-formes, dasyatidae is ground floor fish of warm temperature. We had observed that the extraction of ray cartilage could inhibit angiogenesis of chicken embryo chorioallantoic membrane effectively. Here, Ray cartilage polysaccharide was extracted, separated and purified by guanidine hydrochloride, membrane ultrafil-trating, acetone classification precipitation and sephadex gels pillar patients, Tianqing I colorimetric method and high performance liquid chromatographic (HPLC) detected Ray cartilage polysaccharide molecular mass was 9. 7 x 10~4 and its purity exceeded ninty - nine percent. In the experiment, we explore Ray cartilage polysaccharide antiangiogenic biological activities.Objectivestudy was designed to investigate the effects of Ray Cartilage Glyco-
saminoglycans ( RCG) on angiogenesis and to explore its mechanism and cultured Human Umbilical Endothelial cell ( HUVEC) , Wistar rat corneal neovas-cularization ( CNV) and transplanting tumor mouse models were used in our work.Methods1. Culture and Identification of the Endothelial Cells of Human Umbilical VeinEndothelial cells were isolated from human umbilical cords with 0. 1% I type collagenase and were digested by 0. 05% tripsin and 0. 02% EDTA when the endothelial cells confluent. The cells are cultivated in Ml99 medium containing Endothelial Cells Growth Supplement(ECGS) and fetal bovine serum. Endothelial cells were identified by morphologic and immunohistochemical assays .2. The effect of RCG on angiogenesis in vitroThe experiments were classified into normal group, RCG groups at varied concentrations (100,50,25,10, 2mg/mL ) and SCG group ( 50mg/mL ). MTT, endothelial cell migration and capillary formation experiments were adopted to e-valuate the effects of RCG on HUVEC. Meantime,we also detected the effect of RCG on HUVEC cycle by means of Flow cytometry.3. The effects of RCG on corneal neovascularization(CNV)in Wistar rats The RCG eye drop was prepared. Corneal neovascularization was inducedby sutures on Wistar rats-. The experimental rats were randomly divided into saline group,RCG groups at varied concentrations( 100,50,25mg/mL)and Hepa-rin + Corlin group. CNV area, histopathological changes and VEGF immunohisto-chemistry were used to evaluate the RCG therapeutic effects on CNV in Wistar rats.4. The effects of RCG on implanted Lewis lung carcinoma in C57BL/6J mice.The model of mouse with Lewis lung carcinoma was made;the experimental mice were randomly divided into saline group,RCG groups at varied concentrations (500,250,125mg/kg) and CTX group. Tumor growth states were observed;
Tumor growth curve was described;On the 21th day, tumor was deprived, inhibitory rate of primary tumor and number of lung metastasis focus were measured. Microvessel density (MVD) was quantitated by immunohistochemistry using monoclonal antibodies of CD31.5. The effects of RCG on the expression of VEGF ,bFGF,MMP 9 mRNA and proteinThe expression of VEGF, bFGF, MMP9 were determined with RT - PCR and Western blot.Result1. The effects of RCG on angiogenesis in vitroBy observing morphologic the cells cultivated were endothelial cells typically and by immunohistochemically the cells cultured were stained positively by VI factor monoclonal antibody. 100 - lOmg/mL RCG obviously inhibited the proliferation of HUVEC, the mean MTT OD value of saline group, RCG groups at varied concentrations (100,50,25,10, 2mg/mL ) and SCG group ( 50mg/mL ) were 0.41 ±0.05,0.11 ±0.05,0. 23 ±0.06,0. 31 ±0. 02,0. 35 ±0. 07,0. 36 ±0. 03,0.21 ±0. 04, respectively,ICjo value was 62. 93mg/mL . 100 - lOmg/mL RCG strongly inhibited the migration of HUVEC, the migrated numbers cell of obove groups were 63.80 ± 10. 18,6. 60 ± 3. 58,15.40 ± 4. 61,18. 80 ± 5. 89, 41. 80 ± 3. 70,13. 60 ± 5. 98 ,respectively;100 - lOmg/mL RCG inhibited capillary formation,the mean capillary length of them were 4.63 ±0.31,1.08 ± 0.22,2.31 ±0.11,3.03 ±0. 19,3. 60 ±0. 15,2.11 ±0.12 mm,respectively. RCG could block HUVEC in G2/M phase by Flow cytometry.2. The effects of RCG on corneal neovascularization(CNV)in Wistar rats By means of RCG effect on corneal neovascularization ( CNV) in Wistarrats, We observed that the corneal neovascularization of RCG groups growed slowly and RCG groups showed smaller areas of new blood vessels and fewer inflammatory cells and lower VEGF expression than saline group, there were high expression in corneal epithelium of Saline group, and negative expression in corneal epithelium of lOOmg/mL, 50mg/mL and Heparin + corlin groups, the ex-
pression of VEGF in 25mg/mL RCG group was lower .3. The effects of RCG on implanted Lewis lung carcinoma in C57BL/6J mice.The effects of RCG on the implanted Lewis lung carcinoma showed that tumor growth curves in RCG groups were smooth compared with Saline group, inhibitory rates of primary tumor in 500,250,125mg/kg RCG groups were 43. 36% ,26. 34% , 15. 10% , respectively, there were significant differences in number of lung metastasis focus between RCG groups and Saline group . In MVD assay, the MVD number of RCG groups were markedly reduced compared with Saline group, and had a clear dose - effect relationship.4. The effect of RCG on the expression of VEGF ,bFGF,MMP 9 mRNA and proteinUsing RT - PCR , RCG at various dosage groups Lewis lung cancer tissues VEGF, bFGF, MMP - 9 mRNA fluorescence intensity reduced gradually and relative AUG values were less significantly than Saline group' s (P<0.01) and there were significant differences among these dosage groups;Western blot results showed that protein strap became weaker and weaker with concentrations increased, VEGF, bFGF, MMP -9 protein expression levels in RCG groups were reduced significantly compared with Saline group (P <0.01) 0ConclusionsFrom the experiments results in vitro and in vivo, we conclude that RCG had srong antiangiogenic effect. Its mechanism in vitro might involve in the blocking of HUVEC cell cycle and its mechanism in vivo might be downregulating the expression of VEGF ,bFGF,MMP -9.
引文
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2. Mousa SA. Mechanisms of angiogenesis in vascular disorders: potential therapeutic targets. Drugs of the Future, 1998;23 (1): 51-60.
3. Thomas KA, Gimenez-Gallego G. Fibroblast growth factors: Broad spectrum mitogens with potent angiogenic activity. Trends Biochem·Sci, 1986;11: 81-84.
4. 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;79:315-328.
5. Ziche M, Donnini S, Morbidelli L. Development of new drugs in angiogenesis. Curr Drug Targets, 2004;5(5): 485-493.
6. Folkman J. Tumor angiogenesis: therapentic implications. N Engl J Med, 1971;285(21): 1182-1186.
7. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 1996;86(3): 353-364.
8. Mancuso A, Sternberg CN. Colorectal cancer and antiangiogenic therapy: what can be expected in clinical practice? Crit Rev Oncol Hematol,2005;55(1):67-81.
9. Brem H, Folkman J. Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med, 1975;141:427-439.
10. Eric Dupont, Pierre Falardeau Shake A, Mousa, et al. Antiangiogenic and antimetastatic properties of Neo vastat (AE-941), an orally active extract derived from cartilage tissue. Clinical & Experimental Metastasis. Dordrecht: 2002,19 (2): 145.
11. Mathews J. Sharks still intrigue cancer researchers. J Natl Cancer Inst, 1992;84(13): 1000
12.郭斌,刘玉玲,贾玉海.缸鱼骨素对鸡胚绒毛尿囊膜血管形成的影响[J].辽宁中医杂志,2002;29(11):688-689
13. Cotran RS. New roles for the endothelium in inflammation and immunity. J Pathol, 1987;129(3): 407
14. Plat KH, Breior G, Weich HA, et al. angiogenesis: Molecule and mode. Nature 1992;359:845-848.
15. Ausprunk DH, Folkman J. Migration and newly formed bolld vessels during tumor angiogenesis. Microvasc Res, 1977;14:53.
16. Danel TO, Abrahamson D. Endothelialsignal integration in bascular assembly[J]. Annu Rev physiol,2000;62:649-671
17. Yang QR, Vanden Berghe D. Effect of temperature on in vitro proliferative activity of human umbilical vein endothelial cells[J]. Experientia, 1995;51:126.
18. Jaffe EA, Nachman RL, Becket CG, et al. Culture of human endothelial cell derived from umbilical veins[J]. J Clin Invest,1973;52:2745-56.
19.程满根,李佩霞,胡向阳,等.人脐静脉内皮细胞体外培养的影响因素[J].苏州医学院学报,1990;10:224-225.
20. Gorman L, Mercer P, Henning B. Growth requirements of endothelial cells in culture: variation in serum and amino acid concentrations[J]. Nutrition,1996;12:266
21. Gospodarowicz D, Cheng J. Heparin protects basic and acidic FGF from inactivation[J]. J Cell Physiol, 1986;128:475.
22. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical application of research on angiogenesis[J]. N Engl J Med, 1995;333(26): 1757-1763.
23. Ushiro S, Ono M, Nakayama J, et al. New nortriterpenoid isolated from antirheumatoid arthrit plant, tripterygium wilfordi, modulates tumor growth and neovascularization[J]. Int J Cancer, 1997;72:657-663.
24. Thaloor D, Singh AK, Sidhu GS, et al. Inhibition of angiogenic differentiation of human umbilical vein endothelial cells by cttrcumin[J]. Cell Growth Diff, 1998;9:305-312.
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2. Mousa SA. Mechanisms of angiogenesis in vascular disorders: potential therapeutic targets. Drugs of the Future, 1998;23 (1): 51-60.
3. Thomas KA, Gimenez-Gallego G. Fibroblast growth factors: Broad spectrum mitogens with potent angiogenic activity. Trends Biochem·Sci, 1986;11: 81-84.
4. 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;79:315-328.
5. Ziche M, Donnini S, Morbidelli L. Development of new drugs in angiogenesis. Curr Drug Targets, 2004;5(5): 485-493.
6. Folkman J. Tumor angiogenesis: therapentic implications. N Engl J Med, 1971;285(21): 1182-1186.
7. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 1996;86(3): 353-364.
8. Mancuso A, Sternberg CN. Colorectal cancer and antiangiogenic therapy: what can be expected in clinical practice? Crit Rev Oncol Hematol,2005;55(1):67-81.
9. Brem H, Folkman J. Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med, 1975;141:427-439.
10. Eric Dupont, Pierre Falardeau Shake A, Mousa, et al. Antiangiogenic and antimetastatic properties of Neo vastat (AE-941), an orally active extract derived from cartilage tissue. Clinical & Experimental Metastasis. Dordrecht: 2002,19 (2): 145.
11. Mathews J. Sharks still intrigue cancer researchers. J Natl Cancer Inst, 1992;84(13): 1000
12.郭斌,刘玉玲,贾玉海.缸鱼骨素对鸡胚绒毛尿囊膜血管形成的影响[J].辽宁中医杂志,2002;29(11):688-689
13. Cotran RS. New roles for the endothelium in inflammation and immunity. J Pathol, 1987;129(3): 407
14. Plat KH, Breior G, Weich HA, et al. angiogenesis: Molecule and mode. Nature 1992;359:845-848.
15. Ausprunk DH, Folkman J. Migration and newly formed bolld vessels during tumor angiogenesis. Microvasc Res, 1977;14:53.
16. Danel TO, Abrahamson D. Endothelialsignal integration in bascular assembly[J]. Annu Rev physiol,2000;62:649-671
17. Yang QR, Vanden Berghe D. Effect of temperature on in vitro proliferative activity of human umbilical vein endothelial cells[J]. Experientia, 1995;51:126.
18. Jaffe EA, Nachman RL, Becket CG, et al. Culture of human endothelial cell derived from umbilical veins[J]. J Clin Invest,1973;52:2745-56.
19.程满根,李佩霞,胡向阳,等.人脐静脉内皮细胞体外培养的影响因素[J].苏州医学院学报,1990;10:224-225.
20. Gorman L, Mercer P, Henning B. Growth requirements of endothelial cells in culture: variation in serum and amino acid concentrations[J]. Nutrition,1996;12:266
21. Gospodarowicz D, Cheng J. Heparin protects basic and acidic FGF from inactivation[J]. J Cell Physiol, 1986;128:475.
22. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical application of research on angiogenesis[J]. N Engl J Med, 1995;333(26): 1757-1763.
23. Ushiro S, Ono M, Nakayama J, et al. New nortriterpenoid isolated from antirheumatoid arthrit plant, tripterygium wilfordi, modulates tumor growth and neovascularization[J]. Int J Cancer, 1997;72:657-663.
24. Thaloor D, Singh AK, Sidhu GS, et al. Inhibition of angiogenic differentiation of human umbilical vein endothelial cells by cttrcumin[J]. Cell Growth Diff, 1998;9:305-312.
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