扇贝裙边GAG对碱性成纤维细胞生长因子诱导的血管平滑肌细胞增殖的影响
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摘要
目的：研究栉孔扇贝裙边糖胺聚糖(SS-GAG)对体外培养的血管平滑肌细胞(VSMC)增殖的抑制作用，并探讨SS-GAG抗动脉粥样硬化(AS)的作用机理。
方法：1．体外培养血管平滑肌细胞，建立由碱性成纤维细胞生长因子(bFGF)诱导的VSMC增殖模型，运用MTT比色分析法研究SS-GAG对VSMC增殖活性的影响。
2．采用bFGF诱导的VSMC增殖模型，通过黄嘌呤氧化酶法、硫代巴比妥酸显色法等多种生化的方法观察SS-GAG对VSMC增殖过程中的超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-PX)和总抗氧化能力(T-AOC)的影响。
3．应用bFGF诱导的VSMC增殖模型，用硝酸还原酶法等化学的方法观察SS-GAG对VSMC增殖过程中细胞内总一氧化氮合酶(NOS)活力和一氧化氮(NO)含量的影响。
4．采用透射电镜观察SS-GAG对bFGF诱导增殖的VSMC超微结构的影响。
5．运用流式细胞仪检测SS-GAG对增殖的VSMC的细胞增殖动力学的影响。
6．采用原位杂交的方法，观察SS-GAG对bFGF诱导增殖的VSMC内的血小板源性生长因子(PDGF)A链mRNA表达的影响。
结果：1．与模型组比较，SS-GAG(50μg／ml、100μg／ml和200μg／ml)可降低bFGF所诱导的VSMC的增殖活性(P＜0.05，P＜0.01)，以100μg／ml抑制作用最强。
2．与模型组相比，SS－GAG (50μg／ml、100μg／ml和200μg／ml)可使bFGF诱导增殖的VSMC的脂质过氧化物—丙二醛(MDA)产生减少(P＜0.05，P＜0.01)：SS-GAG(100μg／ml和200μg／ml)可使bFGF诱导增殖的VSMC的谷胱甘肽过氧化物酶(GSH-PX)活力和总抗氧化能力(T-AOC)升高(P＜0.05)。
中文摘要
3.与模型组相比，55一GAG(1 00林g/ml和200林g/ml)可使bFGF所诱导增殖的VSMC
内的，Q， Nos活力升高(P<0.05，P<0.01);而100林g/ml的55一GAG还可使bFGF所
诱导增殖的VSMC的NO释放量明显增加(P<0.01)。
4.透射电镜显示，55一GAG(100林岁ml)作用后血管平滑肌细胞内肌丝较模型组增多，
而粗面内质网和高尔基复合体等细胞器明显少于模型组，表明55一GAG可明显抑制
VSMC由收缩表型向合成表型转化。
5一与模型组比较，55一GAG(50林g/ml、100卜g/ml和200卜g/ml)可使bFGF所诱导增
殖的VSMC中处于S期的细胞明显减少(P<0.01)。
6.原位杂交结果显示，bFGF所诱导增殖的VSMC经55一GAG(100林g/ml和200林g/ml)
作用后，细胞内PDGF一A链mRNA表达较模型组减少(P<0.05，P<0.01)，阳性细
胞数量也较模型组减少。
结论:55一GAG可降低bFGF所诱导的VSMC的增殖活性，减少MDA的生成，使
GSH一PX活力增加，而且细胞的总抗氧化能力也升高;同时细胞内的总NOS活力和
NO的分泌量均增加。表明55一GAG的抗动脉粥样硬化作用可能与其减少脂质过氧
化物产生、增强抗氧化系统的活性、提高细胞内总NOS活力和增加NO释放有关。
55一GAG还可通过抑制VSMC由收缩表型向合成表型转化、抑制细胞内DNA合成、
减少S期细胞和减少细胞内PDGF一A链基因的转录，从而抑制VSMC增殖，达到抗
动脉粥样硬化的效果。
Objective To investigate the inhibitory effects of scallop skirt glycosaminoglycan (SS-GAG) on the proliferation of vascular smooth muscle cells (VSMC) cultured in vitro,and to discuss its mechanisms of anti-atherosclerosis .
Methods 1. The vascular smooth muscle cells had been cultured in vitro , and induced by basic Fibroblast Growth Factor (bFGF) , then the proliferatory model had been established . MTT chromatometry was used to study the effects of SS-GAG on the VSMC proliferatory activity . 2. In vitro it took a model of VSMC proliferation induced by bFGF , many methods including xanthine oxidase method and TBARs were used to test the activities of superoxide dismutase (SOD) and the levels of malanyldiadeyhde (MDA). 3. In vitro it took a model of VSMC proliferation induced by bFGF , some chemical methods were used to analyze the activities of glutathione peroxidase (GSH-PX) and the levels of the total anti-oxidation capacity (T-AOC) . 4. In vitro it took a model of VSMC proliferation induced by bFGF , some chemical methods including nitrate reductase method were used to observe the total activities of nitric oxide synthase (NOS) and the secretion of nitric oxide (NO) . 5.With the help of electron microscope , the changes of the ultrastructure of VSMC proliferation induced by bFGF had been observed after the incubation of SS-GAG . 6.By means of flow cytometry (FCM) , we observed the effects of SS-GAG on the VSMC proliferation dynamics .7.In situ hybridization assay was used to observe the effects of SS-GAG on the mRNA expressions of the A chain of PDGF within proliferatory vascular smooth muscle cells.
Results 1. Compared with model group, the VSMC proliferatory activities induced by bFGF can be lessened by SS-GAG (terminal concentration were 50ug/ml 100ug/ml and 200u/ml) (P<0.05, p<0.01). 2. Compared with model group, the MDA levels of VSMC
proliferation induced by bFGF can be decreased by SS-GAG (terminal concentration were 50ug/mk 100ug/ml and 200ug/ml) (P<0.05, P<0.01); and there was no changes of the activities of SOD. The GSH-PX activities and the T-AOC of VSMC proliferation induced by bFGF can be increased by SS-GAG (terminal concentration were 100ug/ml and 200ug/ml) (P<0.05) . 3. Compared with model group, the activities of cellular NOS and the secretion of NO were higher (P<0.05, P<0.01) after the incubation of SS-GAG (terminal concentration were 100ug/ml and 200ug/ml) . 4. Under the electron microscope, SS-GAG (terminal concentration was 100ug/ml) can greatly inhibit the conversion of contractile type to synthetic type of VSMC . 5. Compared with model group , SS-GAG (terminal concentration were 50ug/mh 100ug/ml and 200ug/ml) can make the number of VSMC in S period of cell cycle increasing (P<0.01) . 6.Compared with model group, the expressions of the gene for the PDGF-A chain within the proliferatory VSMC induced by bFGF can be reduced
by SS-GAG (terminal concentration were 100ug/ml and 200ug/ml) (P<0.05, P<0.01).
Conclusion SS-GAG can lessen the proliferatory activities of VSMC induced by bFGF ,decrease the levels of MDA , promote the activities of GSH-PX and T-AOC ; and increase the excretion of NO , enhance the activities of NOS. These studies indicate that the anti-atherosclerosis effects may be related to the decreasing of the production of MDA ,. the enhancing of the activities of anti-oxidative system , the increasing of the activities of cellular NOS and the secretion of NO . SS-GAG can inhibit the proliferation of VSMC by inhibiting the conversion of contractile type to synthetic type of VSMC , decreasing the DNA syntheses in S period of cell cycle and reducing the transcription of the gene for PDGF-A chain so as to achieve the effects of anti-atherosclerosis .
Postgraduate Chu Xiang-Hua (Pharmacology) Directed by Prof. Liu Sai
方法：1．体外培养血管平滑肌细胞，建立由碱性成纤维细胞生长因子(bFGF)诱导的VSMC增殖模型，运用MTT比色分析法研究SS-GAG对VSMC增殖活性的影响。
2．采用bFGF诱导的VSMC增殖模型，通过黄嘌呤氧化酶法、硫代巴比妥酸显色法等多种生化的方法观察SS-GAG对VSMC增殖过程中的超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-PX)和总抗氧化能力(T-AOC)的影响。
3．应用bFGF诱导的VSMC增殖模型，用硝酸还原酶法等化学的方法观察SS-GAG对VSMC增殖过程中细胞内总一氧化氮合酶(NOS)活力和一氧化氮(NO)含量的影响。
4．采用透射电镜观察SS-GAG对bFGF诱导增殖的VSMC超微结构的影响。
5．运用流式细胞仪检测SS-GAG对增殖的VSMC的细胞增殖动力学的影响。
6．采用原位杂交的方法，观察SS-GAG对bFGF诱导增殖的VSMC内的血小板源性生长因子(PDGF)A链mRNA表达的影响。
结果：1．与模型组比较，SS-GAG(50μg／ml、100μg／ml和200μg／ml)可降低bFGF所诱导的VSMC的增殖活性(P＜0.05，P＜0.01)，以100μg／ml抑制作用最强。
2．与模型组相比，SS－GAG (50μg／ml、100μg／ml和200μg／ml)可使bFGF诱导增殖的VSMC的脂质过氧化物—丙二醛(MDA)产生减少(P＜0.05，P＜0.01)：SS-GAG(100μg／ml和200μg／ml)可使bFGF诱导增殖的VSMC的谷胱甘肽过氧化物酶(GSH-PX)活力和总抗氧化能力(T-AOC)升高(P＜0.05)。
中文摘要
3.与模型组相比，55一GAG(1 00林g/ml和200林g/ml)可使bFGF所诱导增殖的VSMC
内的，Q， Nos活力升高(P<0.05，P<0.01);而100林g/ml的55一GAG还可使bFGF所
诱导增殖的VSMC的NO释放量明显增加(P<0.01)。
4.透射电镜显示，55一GAG(100林岁ml)作用后血管平滑肌细胞内肌丝较模型组增多，
而粗面内质网和高尔基复合体等细胞器明显少于模型组，表明55一GAG可明显抑制
VSMC由收缩表型向合成表型转化。
5一与模型组比较，55一GAG(50林g/ml、100卜g/ml和200卜g/ml)可使bFGF所诱导增
殖的VSMC中处于S期的细胞明显减少(P<0.01)。
6.原位杂交结果显示，bFGF所诱导增殖的VSMC经55一GAG(100林g/ml和200林g/ml)
作用后，细胞内PDGF一A链mRNA表达较模型组减少(P<0.05，P<0.01)，阳性细
胞数量也较模型组减少。
结论:55一GAG可降低bFGF所诱导的VSMC的增殖活性，减少MDA的生成，使
GSH一PX活力增加，而且细胞的总抗氧化能力也升高;同时细胞内的总NOS活力和
NO的分泌量均增加。表明55一GAG的抗动脉粥样硬化作用可能与其减少脂质过氧
化物产生、增强抗氧化系统的活性、提高细胞内总NOS活力和增加NO释放有关。
55一GAG还可通过抑制VSMC由收缩表型向合成表型转化、抑制细胞内DNA合成、
减少S期细胞和减少细胞内PDGF一A链基因的转录，从而抑制VSMC增殖，达到抗
动脉粥样硬化的效果。
Objective To investigate the inhibitory effects of scallop skirt glycosaminoglycan (SS-GAG) on the proliferation of vascular smooth muscle cells (VSMC) cultured in vitro,and to discuss its mechanisms of anti-atherosclerosis .
Methods 1. The vascular smooth muscle cells had been cultured in vitro , and induced by basic Fibroblast Growth Factor (bFGF) , then the proliferatory model had been established . MTT chromatometry was used to study the effects of SS-GAG on the VSMC proliferatory activity . 2. In vitro it took a model of VSMC proliferation induced by bFGF , many methods including xanthine oxidase method and TBARs were used to test the activities of superoxide dismutase (SOD) and the levels of malanyldiadeyhde (MDA). 3. In vitro it took a model of VSMC proliferation induced by bFGF , some chemical methods were used to analyze the activities of glutathione peroxidase (GSH-PX) and the levels of the total anti-oxidation capacity (T-AOC) . 4. In vitro it took a model of VSMC proliferation induced by bFGF , some chemical methods including nitrate reductase method were used to observe the total activities of nitric oxide synthase (NOS) and the secretion of nitric oxide (NO) . 5.With the help of electron microscope , the changes of the ultrastructure of VSMC proliferation induced by bFGF had been observed after the incubation of SS-GAG . 6.By means of flow cytometry (FCM) , we observed the effects of SS-GAG on the VSMC proliferation dynamics .7.In situ hybridization assay was used to observe the effects of SS-GAG on the mRNA expressions of the A chain of PDGF within proliferatory vascular smooth muscle cells.
Results 1. Compared with model group, the VSMC proliferatory activities induced by bFGF can be lessened by SS-GAG (terminal concentration were 50ug/ml 100ug/ml and 200u/ml) (P<0.05, p<0.01). 2. Compared with model group, the MDA levels of VSMC
proliferation induced by bFGF can be decreased by SS-GAG (terminal concentration were 50ug/mk 100ug/ml and 200ug/ml) (P<0.05, P<0.01); and there was no changes of the activities of SOD. The GSH-PX activities and the T-AOC of VSMC proliferation induced by bFGF can be increased by SS-GAG (terminal concentration were 100ug/ml and 200ug/ml) (P<0.05) . 3. Compared with model group, the activities of cellular NOS and the secretion of NO were higher (P<0.05, P<0.01) after the incubation of SS-GAG (terminal concentration were 100ug/ml and 200ug/ml) . 4. Under the electron microscope, SS-GAG (terminal concentration was 100ug/ml) can greatly inhibit the conversion of contractile type to synthetic type of VSMC . 5. Compared with model group , SS-GAG (terminal concentration were 50ug/mh 100ug/ml and 200ug/ml) can make the number of VSMC in S period of cell cycle increasing (P<0.01) . 6.Compared with model group, the expressions of the gene for the PDGF-A chain within the proliferatory VSMC induced by bFGF can be reduced
by SS-GAG (terminal concentration were 100ug/ml and 200ug/ml) (P<0.05, P<0.01).
Conclusion SS-GAG can lessen the proliferatory activities of VSMC induced by bFGF ,decrease the levels of MDA , promote the activities of GSH-PX and T-AOC ; and increase the excretion of NO , enhance the activities of NOS. These studies indicate that the anti-atherosclerosis effects may be related to the decreasing of the production of MDA ,. the enhancing of the activities of anti-oxidative system , the increasing of the activities of cellular NOS and the secretion of NO . SS-GAG can inhibit the proliferation of VSMC by inhibiting the conversion of contractile type to synthetic type of VSMC , decreasing the DNA syntheses in S period of cell cycle and reducing the transcription of the gene for PDGF-A chain so as to achieve the effects of anti-atherosclerosis .
Postgraduate Chu Xiang-Hua (Pharmacology) Directed by Prof. Liu Sai
引文
1 Volkhard L, Douglas AL, Andrew B, et al. Role of fibroblast growth factor in vascularlesion formation. Circulation Res, 1991, 68(1):106～113
2 季宁东，陈琪．血管平滑肌细胞的增殖机制．中国基层医药，2002，9(3)：270～271
3 Michael K, Elazer RE. Biological and biochemical properties of fibroblast growth factors. Arteriosclerosis, 1989, 9:269～278
4 Ross R, Glomser JA. The pathogenesis of atherosclerosis. N Engl J Med, 1976,295: 369～377
5 Klagsbrun M, Edelman ER. Biological and biochemical properties of fibroblast growth factors. Arteriosclerosis,1989, 9(3): 269～278
6 Penn MS, Chisolm GM. Oxidized lipoproteins, altered cell function and atherosclerosis. Atherosclerosis, 1994, 108:2～29
7 崔玉芳，高亚兵，等．小鼠胸腺淋巴细胞辐射损伤特点和机理的研究．中国体视学与图像分析，1998，12(3)：208～213
8 Wilcox JN, Subramanian RR, Sundell CL, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol, 1997, 17(11): 2479～2488
9 Berdeaux A. Nitric Oxide: an ubiquitous messenger. Fundam Clin Pharmacol,1993, 7(8): 401～411
10 郭国庆，查彩慧，沈伟哉．一氧化氮与动脉粥样硬化的关系．心血管病学进展，2001，22(3)：178～180
11 陈修，李凯，陈维洲．血管内皮细胞药理学．见陈修，陈维洲，曾贵云主编，心血管药理学，人民卫生出版社，1997：124～130
12 Mundt S, Chun S, Fletcher D, et al. Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice. J Immunol,2000, 165 (6): 3430～3435
13 Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med, 1993, 329: 2002～2012
14 Basseneg E. Antiplatelet effects of endothelium-derived relaxing factor and nitric oxide donors. Eur Heart J, 1991, 12:12～21
15 Lefer AM, Ma XL. Decreased basal nitric oxide release in hypercholesterolemia increase neutrophil adherence to rabbit coronary artery endothelium. Arterioscler Throms,1993, 13 (6): 771～779
16 Scott-Burden T, Vanhoutte PM. The endothelium as a regulator of vascular smooth muscle proliferation. Circulation, 1993, 87:51～59
17 Shears LL-2nd, Kibbe MR, Murdock AD, et al. Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J Am Coll Surg, 1998, 187(3): 295～306
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33 Wilcox JN, Subramanian RR, Sundell CL, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol, 1997, 17 (11): 2479～2488
34 Yin ZL, Dusting GJ. A nitric oxide donor (spermine-NONOate) prevents the formation of neointima in rabbit carotid artery. Clin Exp Pharmacol Physiol, 1997,24 (6): 436～438
2 季宁东，陈琪．血管平滑肌细胞的增殖机制．中国基层医药，2002，9(3)：270～271
3 Michael K, Elazer RE. Biological and biochemical properties of fibroblast growth factors. Arteriosclerosis, 1989, 9:269～278
4 Ross R, Glomser JA. The pathogenesis of atherosclerosis. N Engl J Med, 1976,295: 369～377
5 Klagsbrun M, Edelman ER. Biological and biochemical properties of fibroblast growth factors. Arteriosclerosis,1989, 9(3): 269～278
6 Penn MS, Chisolm GM. Oxidized lipoproteins, altered cell function and atherosclerosis. Atherosclerosis, 1994, 108:2～29
7 崔玉芳，高亚兵，等．小鼠胸腺淋巴细胞辐射损伤特点和机理的研究．中国体视学与图像分析，1998，12(3)：208～213
8 Wilcox JN, Subramanian RR, Sundell CL, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol, 1997, 17(11): 2479～2488
9 Berdeaux A. Nitric Oxide: an ubiquitous messenger. Fundam Clin Pharmacol,1993, 7(8): 401～411
10 郭国庆，查彩慧，沈伟哉．一氧化氮与动脉粥样硬化的关系．心血管病学进展，2001，22(3)：178～180
11 陈修，李凯，陈维洲．血管内皮细胞药理学．见陈修，陈维洲，曾贵云主编，心血管药理学，人民卫生出版社，1997：124～130
12 Mundt S, Chun S, Fletcher D, et al. Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice. J Immunol,2000, 165 (6): 3430～3435
13 Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med, 1993, 329: 2002～2012
14 Basseneg E. Antiplatelet effects of endothelium-derived relaxing factor and nitric oxide donors. Eur Heart J, 1991, 12:12～21
15 Lefer AM, Ma XL. Decreased basal nitric oxide release in hypercholesterolemia increase neutrophil adherence to rabbit coronary artery endothelium. Arterioscler Throms,1993, 13 (6): 771～779
16 Scott-Burden T, Vanhoutte PM. The endothelium as a regulator of vascular smooth muscle proliferation. Circulation, 1993, 87:51～59
17 Shears LL-2nd, Kibbe MR, Murdock AD, et al. Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J Am Coll Surg, 1998, 187(3): 295～306
18 吴正泉，法京，俞永富．透射电子显微术．见凌诒萍，俞彰主编，细胞超微结构与电镜技术—分子细胞生物学基础，上海医科大学出版社，2000：78～88
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