【作者】 姚磊；

【导师】 王振宇；

【作者基本信息】 哈尔滨工业大学 ， 化学工程与技术， 2014， 博士

【摘要】 随着核技术的广泛应用，辐射伤害已渗透到各个领域。电离辐射产生过量的活性氧簇（ROS），攻击生命大分子，所引发的氧化应激与人类近百余种疾病密切相关。电离辐射导致的氧化损伤越来越受到人们的重视，筛选具有强抗氧化活性的天然产物成分已成为医学、生物学和食品科学研究的热点。本课题以大豆抗氧化多糖（Polysaccharides from soybean residue，SRP）为研究对象，系统性研究其制备工艺、分离纯化方法、结构表征、抗氧化、抗辐射活性和对辐射诱导氧化应激防护作用机制。采用纤维素酶法降解豆渣制备可溶性SRP，通过响应面法（RSM）优化得到酶法制备SRP的工艺条件：酶用量60U/g、pH值5.10、温度50℃，在此条件下，酶解1h SRP得率为45.71%。通过活性跟踪研究，确定酶解时间8h SRP具有最佳抗氧化活性。利用DEAE-52阴离子纤维素层析、Sephadex G-100葡聚糖凝胶层析对SRP进行纯化，制备得到中性大豆抗氧化多糖SRP-1a。采用HPGPC、GC-MS、FT-IR高碘酸氧化-Smith降解和NMR技术对SRP-1a结构进行表征，研究发现SRP-1a为均一度较高的多糖，其分子量分布宽度Mw/Mn=1.19，重均分子量为3.89×103Da。含有核糖、鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖等7种单糖组分，其摩尔比为0.82:0.97:2.77:0.63:6.38:70.62:17.82。SRP-1a的糖残基构型主要为1,4-β-D-Glcp、1,3,6-α-D-Manp和1,4-α-D-Galp。主链是由Glcp和Manp组成的甘露葡聚糖，(1→)-β-D-Glcp为主链的非还原性末端残基，由1,3,6-α-D-Manp通过O-6位与1,4-α-D-Galp残基构成的侧链相连。通过体外抗氧化筛选模型，研究了SRP-1a清除生理自由基、非生理自由基、总还原能力、金属离子螯合能力和抑制脂质过氧化能力。结果表明，SRP-1a具有较强的传递氢原子能力，可以通过提供氢原子直接清除羟基自由基（·OH）或终止自由基的链反应，从而防止自由基诱导氧化损伤的发生。同时，SRP-1a具有较高的还原能力和金属离子螯合能力，对于脂质过氧化具有一定的抑制作用，并呈良好的剂量效应关系。建立X和60Coγ辐射诱导细胞损伤模型，研究SRP-1a对细胞的保护作用。结果表明，SRP-1a可以拮抗电离辐射损伤作用，提高辐射损伤小鼠脾细胞活力，降低小鼠脾细胞DNA的辐射损伤程度，增加细胞抗氧化酶活性，减少丙二醛（MDA）含量。建立60Coγ辐射诱导氧化损伤动物模型，对SRP-1a的辐射防护作用进行研究。结果表明，SRP-1a能减轻辐射造成小鼠免疫器官的损伤，显著增加小鼠各脏器中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）活性，提高还原型谷胱甘肽（GSH）含量，降低MDA含量，有效激活抗氧化酶系，减少脂质过氧化，减轻细胞膜的氧化损伤，降低小鼠外周血淋巴细胞异形和骨髓细胞微核率。通过免疫组化和蛋白免疫印迹技术（Western blot）对小鼠脾脏、肝脏组织线粒体介导的细胞凋亡信号途径主要相关蛋白表达进行了研究。结果表明，SRP-1a防护辐射诱导机体氧化损伤的作用机制是：SRP-1a通过提高Bcl-2表达水平，抑制Bax表达的上调，降低胞浆Cyt-c表达，继而降低凋亡酶caspase-3活化片段的表达，起到对辐射诱导细胞过度凋亡的抑制作用。SRP-1a能够改善由电离辐射诱导的氧化损伤，降低活性氧自由基的产生，调节细胞氧化还原和凋亡信号转导，抑制细胞线粒体凋亡信号途径，对辐射诱导氧化伤害具有显著的防护作用。本研究结果对于揭示辐射诱导氧化损伤防护机制及开发研制新型抗辐射药物具有重要的理论价值和应用意义。更多还原

【Abstract】 With the development of nuclear technology, people may face many potentialthreat of radiation damage in many areas of life. Ionizing radiation could induce thebody to produce excessive of Reactive oxygen species (ROS), which can attack thebiological macromolecules and then result in oxidative stress. Studies have shown thatmore than one hundred kinds of human diseases are associated with oxidative stress.Hence screening and evaluation antioxidant and radiation protective activitycomponents from natural product resources have become a hot spot in the study ofbiology, medicine and food science. In this thesis, preparation, purification andidentification of polysaccharides from soybean residue (SRP) were researched;antioxidant activity and protective effect on radiation-induced oxidative stress of SRPwere also investigated.SRP was produced by enzymatic hydrolysis of soybean residue crude fibre. Theoptimum hydrolysis conditions for SRP were the ratio of enzyme activity unit to massof soybean crude fiber60U/g, temperature50℃and pH5.10. Under these conditions,at degradation time of1h, the experimental yield of SRP was45.71%. At degradationtime of8h SRP fraction showed the best antioxidant activities.SRP was purified byDEAE-52and Sephadex G-100, and one main homogeneous polysaccharide (SRP-1a)was obtained. The structure of SRP-1a was characterized by HPGPC, GC-MS, FT-IR,periodic acid oxidation-smith degradation and NMR spectra. The results indicated thatSRP-1a was a glucan with high branches. The molecular weight was3.89×103Da. Thepolysaccharide was composed of ribose, rhamnose, arabinose, xylose, mannose, glucoseand galactose with a ratio of0.82:0.97:2.77:0.63:6.38:70.62:17.82. The majorconfiguration of glycoside residues of SRP-1a were1,4-β-D-Glcp,1,3,6-α-D-Manpand1,4-α-D-Galp.The main chain is formed through the connection between gluco-and manno-units, the non-reducing terminal residue was (1→)-β-D-Glcp. The branchwas formed through the connection between1,3,6-α-D-Manp and1,4-α-D-Galp, atthe position of O-6.Antioxidant activities of SRP-1a were studied in vitro, such as removal ofphysiological and non-physiological free radicals, total reducing capacity and anti lipidperoxidation activity. The results show that SRP-1a has high scavenging activity onhydroxide radical (·OH). The main mechanism is that SRP-1a is an active hydrogenatom donor, which can break down radical chain reactions through hydrogen atomtransfer. SRP-1a also showed significant total reducing capacity and inhibition activityon lipid peroxidation with a dose-dependent manner.The mouse spleen cell damage model induced by X-ray and60Coγ-ray radiation was established to test and verify SRP-1a’s protective effect on radiation induceddamage. Results showed that SRP-1a can protect radiated mouse spleen cell throughenhancing cell viability and reducing DNA damage. Then, the oxidative damage animalmodel induced by60Coγ-ray radiation was established to investigate the protectiveeffect in vivo.The results showed that SRP-1a can significantly reduce mouse immune organsoxidative damage through enhancing antioxidant enzymes activities such as superoxidedismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). SRP-1a canameliorate glutathione (GSH) and reduce malondialdehyde (MDA) level in organs andserum of mouse to decrease lipid peroxidation and protect cell membrane. SRP-1a alsocan inhibit generation of atypical lymphocytes and bone marrow micronuclei inducedby radiation.Regulating effect of SRP-1a on apoptosis mitochondrial path related proteinsexpress statuses was analyzed through immunohistochemistry and western blottechnique. The protective effect mechanism of SRP-1a on oxidative damage induced byradiation was proposed. SRP-1a can improve expression level of Bcl-2, blockradiation-induced up regulated of Bax, then inhibit the release of Cyt-c and depress theactivities of caspase-3, and thus inhibit cell apoptosis.These results demonstrate that SRP-1a has effective antioxidant and antiradiationactivities through regulating cell oxidoreduction and apoptosis signal transduction. Thepresent study has important theoretical and practical significance to reveal themechanism of preventing radiation induced oxidative damage and developing newradioprotectors.更多还原