【作者】 初向华；

【导师】 刘赛；

【作者基本信息】 青岛大学 ， 药理学， 2004， 硕士

【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。更多还原

【Abstract】 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 VSMCproliferation 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更多还原