【作者】 李小雨；

【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， 2013， 博士

【摘要】 电离辐射通过诱导人体产生过量的ROS，攻击机体中的生命物质，如蛋白质、脂质和糖类，引发自由基链状反应，破坏机体内环境稳态。科学研究发现长期暴露在电离辐射环境下能够诱导人体产生各种疾病，如衰老、炎症、心脑血管疾病，甚至引发癌症，筛选出高效低毒的辐射防护剂成为科研工作者研究的热点。据研究报道多糖能够通过提升机体的抗氧化系统和免疫系统的活性，从而发挥辐射防护的作用。因此，本论文以食用菌为研究对象，通过在体外建立多重抗氧化模型，从8种食用菌中筛选出抗氧化活性最强的中性元蘑多糖（NTHSP）作为后续研究目标；采用响应面设计优化NTHSP提取工艺，并对其进行纯化和结构表征；系统的研究了NTHSP在体外和体内对辐射诱导氧化应激的防护功效并揭示其辐射防护作用机制。采用酸碱度不同的3种溶剂从产自中国不同地区具有代表性的8种食用菌中分离23种食用菌多糖，成分分析结果表明食用菌多糖样品中仅含有少量蛋白质，无多酚类物质；通过在体外建立抗氧化模型，研究了食用菌多糖对生理性OH·自由基、超氧自由基和非生理性ABTS自由基的清除活性、对脂质过氧化反应的抑制作用以及铁离子还原能力，结果表明NTHSP的抗氧化活性最强，选择其作为后续研究对象。采用响应面设计对NTHSP提取工艺进行优化，得到最佳提取条件为：提取温度94℃、提取时间3.0h、液料比110:1和超声功率480W，所得到的多糖提取得率为17.45±0.18%，而通过传统方法提取NTHSP的得率仅为10.21±0.36%；通过离子交换纤维素DEAE-52及葡聚糖凝胶层析Sephadex G-100对NTHSP进行纯化，采用凝胶渗透色谱（GPC）、气质联用（GC-MS）、红外光谱（FT-IR）、核磁共振光谱（NMR）和原子力显微镜（AFM）对其结构进行表征，结果表明NTHSP为一种具有高分支结构的葡聚糖，分子量为8.09×103，由阿拉伯糖：甘露糖：葡萄糖：半乳糖（3.8:15.8:28.4:10.5）组成，主链由→3,6)-α-D-Glcp-(1→组成，其侧链→2)-α-L-Arap-(1→、α-D-Manp-(1→和→6)-β-D-Galp-(1→分别连接在→3,6)-α-D-Glcp-(1→的3位、3位和6位上，其中，→2)-α-L-Arap-(1→和→6)-β-D-Galp-(1→的2位和6位分别连接甲基。通过在体外建立辐射诱导细胞氧化损伤模型，研究NTHSP对大鼠外周血白细胞、大鼠脾细胞和人脐静脉内皮细胞ECV304的辐射防护作用，通过对辐射后大鼠脾细胞中SOD和CAT的活性以及MDA的水平进行研究，发现NTHSP能够显著提升辐射后大鼠脾细胞中抗氧化酶SOD和CAT的活性，并降低脾细胞脂质过氧化反应的发生；NTHSP还能够有效的降低外周血白细胞中异形淋巴细胞、带空泡分叶细胞和退行性变细胞的数量，提升正常白细胞的数量，对外周血白细胞起到有效的防护；通过DNA ladder研究NTHSP对辐射后脾细胞DNA的防护作用，结果表明NTHSP对电离辐射诱导脾细胞DNA凋亡起到显著的防护功效，并不同程度的促进了脾细胞和内皮细胞增殖，显著的提升辐射后脾细胞和内皮细胞的存活率。通过在体内建立辐射诱导小鼠氧化损伤模型，研究NTHSP的辐射防护途径，结果显示NTHSP能够有效的提升辐射后小鼠体内的抗氧化酶系（SOD、CAT和GSH-Px）的活性和增强抗氧化物质（GSH、VE和铜蓝蛋白）的合成能力，并降低小鼠体内脂质过氧化产物MDA的水平，表明NTHSP能够增强辐射后小鼠抗氧化系统的活性；对辐射后小鼠特异性免疫和非特异性免疫进行研究，发现NTHSP能够有效的提升小鼠血液系统免疫能力、增加小鼠免疫器官指数、促进脾淋巴细胞增殖并增强单核细胞的吞噬作用；通过对小鼠骨髓微核、骨髓染色体畸变和DNA含量进行分析，结果显示NTHSP能够对辐射诱导小鼠产生的遗传毒性起到有效的防护。通过免疫组织化学方法和流式细胞仪检测脾细胞周期研究NTHSP的辐射防护机制进行研究，结果发现NTHSP能够有效的抑制电离辐射诱导小鼠脾细胞停滞在G0/G1期，并对电离辐射诱导的脾细胞凋亡起到防护功效，从而恢复细胞的正常生理代谢过程；通过对细胞凋亡线粒体途径相关蛋白表达进行分析，研究发现NTHSP能够通过抑制促凋亡蛋白Bax、细胞色素c和Caspase-3活化亚基的表达，促进抗凋亡蛋白Bcl-2的表达而抑制细胞凋亡，从而对辐射诱导氧化损伤具有防护功效。本课题研究发现NTHSP可以降低电离辐射诱导活性氧ROS的产生，调节细胞内氧化还原状态，提升机体免疫活性，通过影响线粒体凋亡信号途径相关蛋白表达而抑制细胞凋亡，从而对辐射诱导的氧化损伤具有显著的防护功效。另外，关于NTHSP对辐射诱导小鼠脾细胞凋亡的其他凋亡途径的影响还需进一步研究和探索。本研究对于揭示电离辐射防护途径具有一定的理论价值，对进一步开发天然高效辐射防护剂具有现实意义。更多还原

【Abstract】 Ionizing radiation could induce the body to produce excessive of ROS, whichattack the biological substances such as proteins, lipids and carbohydrates that cancause a chain reaction and damage the body homeostasis. Studies have shown thatlong-term exposure to ionizing radiation environment could induce the body toproduce a variety of diseases such as aging, inflammation, cardiovascular andcerebrovascular diseases, and even cancer. Existing chemical radioprotectors werenot suitable for long-term use due to their side effects on normal tissues and organs.Therefore, screening natural radioprotectors with efficiency and low toxicitybecome a focus. This subject sreened neutral polysaccharide of Hohenbueheliaserotina from8kinds of mushroom through multiple antioxidant model in vitro asthe object; optimized the extraction process using RSM and characterized itsstructure, systematically studied the in vivo and in vitro protective effect of NTHSPon radiation induced oxidative stress and revealed the mechanism of radiationprotection; the main findings were as follows:23kinds of polysaccharides were isolated from eight kinds of mushrooms withthree kinds of solvents. There was a small amout of protein and no polyphnol inpolysaccharides. By establishing multiple antioxidant model in vitro, we studied thescavenging activity on physiological hydroxyl radicals and non-physiological ABTSradical, as well as lipid peroxidation inhibition activities of23kinds of ediblefungus polysaccharides. The results indicated that HSP had the most significantantioxidant activities in vitro. The ferric reducing ability and superoxide radicalscavenging activity of three kinds of HSP were determined. The results showed thatNTHSP could effectively supply electron and suppress radical chain reaction whichwas the best fraction for antioxidant activities.An ultrasonic-assisted procedure for the extraction of polysaccharides from thefruiting body of Hohenbuehelia serotina was investigated using response surfacemethodology. The effect of four factors on the yield of polysaccharides were studied.The optimized conditions for maximum yield were extraction temperature of94°C,extraction time of3.0h, water to raw material ratio of110:1and ultrasonic power of480W. Under these conditions, the experimental yield of polysaccharides was17.45±0.18%, which was higher than the yield of polysaccharide extracted bytraditional method (10.21±0.36%). Then, the structural features of untreatedmaterials, hot water extraction residue and ultrasonic-assisted extraction residuewere compared by SEM. The results indicated that ultrasonic-assisted extraction technology could be an effective and advisable technique. The NTHSP was purifiedby DEAE-52and Sephadex G-100. The structure of NTHSP-A1was characterizedby HPCPC, GC-MS, FT-IR, NMR and AFM. The results suggested that NTHSP wasa glucan with high branches. The molecular weigh is8.09×103. The polysaccharidewas composed of arabinose, mannose, glucose and galactose with a ratio of3.8:15.8:28.4:10.5. The main chain of NTHSP-A1was→3,6)-α-D-Glcp-(1→, andthe side chains→2)-α-L-Arap-(1→, α-D-Manp-(1→and→6)-β-D-Galp-(1→werelinked at3-position,3-position and6-position of→3,6)-α-D-Glcp-(1→respectively.Furthermore, methyl groups were respectively connected with→2)-α-L-Arap-(1→and→6)-β-D-Galp-(1→at2-position and6-position.By the established model of radiation inducing oxidative stress in cells, westudied the radioprotective effect of HSPs on rat peripheral blood white blood cells,rat spleen cells and human umbilical vein endothelial cells. The results indicatedthat HSPs were able to reduce the number of atypical lymphocyte, leaf granulocytecotaining vacuoles and degenerative cell, and effectively protect the peripheralblood leukocytes. Three kinds of HSP also could promote the proliferation of ratsplenocytes and endothelial cells, significantly improved the survival of thesplenocytes and endothelial cells after radiation. NTHSP possessed the mostsignificant effect; SOD and CAT activities and MDA level in spleen cells afterradiation were investigated. The results showed that NTHSP could significantlyincrease the activities of SOD and CAT, and inhibit the occurrence of lipidperoxidation. DNA ladder study further demonstrated that NTHSP could play aprotective effect on ionizing radiation induced DNA damage.The oxidative damage model of mice induced by60Co-γ radiation wasestablished to investigate the radioprotective effect of NTHSP. The results showedthat NTHSP could effectively increase the activities of SOD, CAT and GSH-Px aswell as enhance the synthesis of antioxidants (GSH, Vitamin E and ceruloplasminprotein) and reduce the level of MDA. The results showed that NTHSP can enhancethe activity of anti-oxidative system after radiation. By studying the specificimmunity and non-specific immunity, it was found that NTHSP could significantlyincrease the immune organ index, promote spleen lymphocyte proliferation andenhance monocyte phagocytosis. By studying the mouse micronucleus,chromosome aberrations and DNA content in mice bone marrow, it was obeservedthat NTHSP could protect DNA against damage induced by radiation.The protective mechanism of NTHSP on radiation induced oxidative stress wasanalyzed by the methods of flow cytometry and immunohistochemistry. The resultssuggested that NTHSP could protect splenocytes against the damage caused by radiation through inhibiting more cells arrest into G0/G1phase and restore thephysiology metabolic processes. NTHSP could also play a protective effect for theapoptosis of splenocytes induced by radiation. The related proteins of apoptosis inmitochondrial pathway were determined and the results showed that NTHSP couldsignificantly inhibit the expression of pro-apoptosis protein Bax, Cytochrome c andCaspase-3and as well as promote the expression of anti-apoptosis protein Bcl-2,and then effectively protect the splenocytes away from oxidative damage inducedby radiation.These results indicated that NTHSP could prevent the radiation induceddamage by reducing the production of ROS and regulating the redox state, and theneffectively inhibiting the mitochondrial apoptotic signaling pathway. The presentstudy will play an important theoretical and practical role in revealing protectivemechanism of radiation and exploiting the natural and efficient raidioprotectors.更多还原