【作者】 王海桃；

【导师】 刘赛；

【作者基本信息】 青岛大学 ， 药理学， 2007， 硕士

【摘要】 目的：通过观察牡蛎糖胺聚糖（O-GAG）对过氧化氢损伤的血管内皮细胞合成及分泌功能的影响，以进一步探讨牡蛎糖胺聚糖（O-GAG）的抗动脉粥样硬化（AS）作用机理。方法：1、采用体外培养的人脐静脉内皮细胞株（HUVECS，ECV304），建立过氧化氢（H₂O₂）诱导的血管内皮细胞损伤模型，应用MTT法观察分析O-GAG对损伤及正常血管内皮细胞增殖活性的影响。2、用化学比色的方法测定分析O-GAG对H₂O₂损伤的血管内皮细胞乳酸脱氢酶（LDH）释放的影响。3、用黄嘌呤氧化酶法测定血管内皮细胞内的超氧化物歧化酶（SOD）的活性；用硫代巴比妥酸反应物（TBARS）法测定内皮细胞内氧化损伤产物丙二醛（MDA）的含量；用酶促反应的方法测定血管内皮细胞内谷胱甘肽过氧化酶（GSH-PX）的活性；用化学比色法测定内皮细胞内的总抗氧化能力（T-AOC），分析O-GAG对H₂O₂诱导损伤的血管内皮细胞抗氧化功能的影响。4、建立H₂O₂诱导的血管内皮细胞氧化损伤模型，用硝酸还原酶法测定血管内皮细胞的内皮舒张因子—一氧化氮（NO）含量；用化学比色法测定总一氧化氮合酶（T-NOS）及诱导型一氧化氮合酶（iNOS）活性，分析O-GAG对血管内皮细胞氧化损伤后合成分泌NO功能的影响。5、建立H₂O₂所致的血管内皮细胞氧化损伤模型，用放射免疫法测定血管内皮细胞合成分泌的前列环素（PGI₂）的稳定性代谢产物6酮-前列腺素F₁。(6-keto-PGF_1α)和血栓素A₂（TXA₂）的稳定性代谢产物血栓烷素B₂（TXB₂）的含量，分析O-GAG对血管内皮细胞氧化损伤后的前列腺素代谢的影响。6、用放射免疫法测定血管内皮细胞合成分泌的缩血管生物活性多肽-内皮素（ET）的含量，分析O-GAG对H₂O₂诱导的血管内皮细胞氧化损伤后细胞合成分泌ET功能的影响。结果：1．给予H₂O₂后，VECs的增殖活性与正常对照组相比明显降低（P＜0.01）。与损伤对照组比较，除O-GAG最低剂量组（10ug／ml）外，O-GAG各浓度保护组（50、100、200、400、800ug／ml）的血管内皮细胞活性均有所增高（P＜0.05，P＜0.01）。而O-GAG在100、200ug／ml浓度时还可促进正常细胞的生长（与正常对照组相比P＜0.05）。2、H₂O₂损伤模型组血管内皮细胞释放的LDH明显高于正常对照组（P＜0.01）；与损伤模型组比较，O-GAG各保护组（50、100、200ug／ml）血管内皮细胞释放的LDH显著降低（P＜0.01）。3、与正常对照组比较，H₂O₂损伤模型组内皮细胞的SOD活性明显降低，MDA含量明显增加（P＜0.01）；O-GAG各保护组（50、100、200ug／ml）与损伤模型组比较，SOD活性明显增强，MDA含量明显减少（P＜0.01）。H₂O₂损伤模型组细胞内GSH-PX活性及T-AOC活力明显降低于正常对照组细胞（P＜0.01）；与损伤模型组比较，O-GAG各保护组（50、100、200ug／ml）细胞内GSH-PX、T-AOC活力均明显升高（P＜0.01）。4、与正常对照组相比，H₂O₂损伤模型组VECs内T-NOS活性、NO含量明显降低，iNOS活性则明显升高（P＜0.01）。与H₂O₂损伤模型组比较，O-GAG各浓度保护组（50ug／ml、100ug／ml、200ug／ml）VECs内T-NOS活性、NO含量均明显提高，而细胞内iNOS活性显著降低（P＜0.01）。5、加入H₂O₂后的模型组细胞培养液中的6-keto-PGF₁。水平明显低于正常对照组，而TXB₂的含量则显著增加（P＜0.01）；O-GAG在50、100、200ug／ml浓度时可逆转H₂O₂的氧化损伤作用，提高6-keto-PGF_1α水平，降低TXB₂含量（P＜0.01，P＜0.05）。6、H₂O₂损伤组与正常对照组相比，ET含量明显升高（P＜0.01），说明过氧化氢可使血管内皮细胞中的ET表达增强，而预先加入O-GAG（50、100、200ug／ml）可显著降低受损细胞ET的表达（P＜0.01）。结论：牡蛎糖胺聚糖（O-GAG）能够减轻过氧化氢对血管内皮细胞的氧化损伤，并对正常的血管内皮细胞有促增殖作用；O-GAG可以使受损的血管内皮细胞释放的LDH减少；提高受损内皮细胞抗氧化酶SOD和GSH-PX的活性及总抗氧化能力，降低脂质过氧化代谢产物MDA的产生；O-GAG还可有效提高细胞内T-NOS活性，降低炎性产物iNOS活性；明显增加内皮依赖性舒张因子NO和6-keto-PGF_1α等舒张血管物质的表达；显著降低TXB₂和ET等收缩血管物质的生成。结果表明，牡蛎糖胺聚糖对过氧化氢所造成的血管内皮细胞的氧化损伤有明显保护作用。该作用在防治动脉粥样硬化、高血压、脑卒中等心血管疾病方面具有重要意义。更多还原

【Abstract】 Objectives: To investigate the influences of oyster glycosaminoglycan（O-GAG） on the synthesize and excretion functions of injured human vassel endothelial cell （VECs）., and to study its mechanism of anti-atherosclerosis （AS）.Methods:1. The endothelial cell strain ofhuman umbilical vein （HUVECs, ECV304） had been cultured in vitro, and we estabilshed a model of endothelial cell oxidative damage induced by hydrogen peroxide （H₂O₂）. We tested the influence of O-GAG on the proliferation activity of injured endothelial cell and normal vascular endothelial cell by means of MTT assay.2. We used chemical methods to analysis the content of Lactate Dehydrogenase （LDH）, and so as to study the effect of O-GAG on endothelial cell oxidative damage induced by H₂O₂.3. To observe the effect of O-GAG on antioxidative function of endothelial cell oxidative damage induced by H₂O₂, xanthne oxidase method was used to analysis the activity of superoxide dismutase （SOD） , chemical methods was used to analysis the level of malanyldiadeyhde （MDA）, the activity of Glutathione Peroxidase （GSH-PX） and the Total Antioxidative Capacity （T-AOC）.4. The secretion of nitric oxide （NO） were measured with nitrate reductase method. Biochemistry methods were used to study the effect of O-GAG on the antioxidant fuction of VECs by testing the activities of the intracellular activities of total nitric oxide synthase （T-NOS） and Inducible nitric oxide synthase （iNOS）.5. The radio-immunity methods was used to observe the 6-keto Prostacyclin F_1α(6-keto-PGF_1α) and Thromboxane-B₂ （TXB₂） contents of the vascular endothelial cells, and we analyse the effect of O-GAG on injured endothelial cells.6. We used radio-immunity methods to analysis the content of Endothelin （ET）, to study the traction effect of O-GAG on endothelial cell oxidative damage induced by H₂O₂.Results:1. Compared with normal control groups, the proliferation activity of model groups injured by H₂O₂ remarkably decreased （P＜0.01） ; While pretreated by O-GAG （except 10μg/ml）, the proliferative activity of VECs obviously increased （P＜0.05, P＜0.01）. O-GAG can proliferate the normal VECs in a certain dosage. 2. LDH testing result showed that the content of LDH in model group was much higher than that in normal control group （P＜0.01 ）; While the groups pretreated by O-GAG （terminal concentration were 50μg/ml, 100μg/ml, 200μg/ml）, compared with model group, the content of LDH obsiously decreased （P＜0.01）3. The activities of SOD in model group remarkably decreased （P＜0.01）and the content of MDA obviously increased, compared with normal control groups （P＜0.01）. While pretreated by O-GAG; the activities of SOD obviously increased.and the levels of MDA decresed. （P＜0.01）. Compared with normal control groups, the activities of GSH-PX and T-AOC in model groups decreased remarkably （P＜0.01） ,but it increased obviously when pretreated by O-GAG （terminal concentration were 50μg/ml, 100μg/ml, 200μg/ml） （P＜0.01）4. Compared with normal control group, H₂O₂ remarkably decreased the levels of NO、the activity of T-NOS and increased the activities of iNOS （P＜0.01） ; Compared with the injured model group, the activities of T-NOS and the secretion of NO were higher （P＜0.01）, and the activities of iNOS was decreased. （P＜0.01）5. The secretion of 6-keto-PGF_1α in model groups was much lower than normal control group （P＜0.01） ; But the levels in O-GAG protective groups （terminal concentration were 50μg/ml, 100μg/ml, 200μg/ml）, was obviously increased, compared with injured model groups. （P＜0.05, P＜0.01） . The content of TXB₂in model groups was much higher than normal control group （P＜0.01） ; While compared with model groups, the levels of TXB₂ in O-GAG protective group （terminal concentration were 50μg/ml, 100μg/ml, 200μg/ml） was obviously decreased （P＜0.05, P＜0.01）6. The secretion of ET in injured model group was much higher than normal control group （P＜0.01）; While the groups pretreated by O-GAG （terminal concentration were 50μg/ml, 100μg/ml, 200μg/ml）, compared with the model group, the content of ET obviously decreased. （P＜0.01）.Conclusion: O-GAG can lessen proliferatory inhibition of endothelial cell induced by H₂O₂, decrease the secretion of MDA and LDH, promote the activities of SOD、GSH-PX and T-NOS, increase the excretion of NO, weaken the activity of iNOS, enhance the level of. 6-keto-PGF_1α, and decrease the contents of ET and TXB₂. According to these studies, they can indicate that O-GAG has the protective action on injuered endothelial cell induced by H₂O₂. This study had important actions on the prevention and treatment of heart and vascular disease such as hypertention, AS and so on.更多还原