【作者】 卫东锋；

【导师】 程卫东；

【作者基本信息】 兰州大学 ， 中西医结合临床， 2012， 博士

【摘要】 研究背景中药红芪(Hedysari radix)系豆科植物多序岩黄芪(Hedysarum polybotrys Hand.-Mazz.)的干燥根,属于黄芪的一种,与黄芪同科不同属,具有益气固表、补血、利尿脱毒、排脓及敛疮生肌等功效。红芪多糖是红芪的主要活性成分之一,主要由鼠李糖、木糖、阿拉伯糖、半乳糖及葡萄糖等单糖组成,现代药理学研究证明其具有调节机体免疫、抗肿瘤及抗氧化等作用,是一种具有开发价值的中药新药。蛋白质组学作为后基因时代的一种系统生物学研究方法,为中药多组分、多靶点及多环节的整体动态研究中提供了提供了崭新的思路和强大的技术支撑。前期研究结果表明,红芪多糖具有一定的免疫调节及抗肿瘤作用,但其具体作用机制尚不清楚。为了进一步深入探讨该过程中红芪多糖多靶点作用的分子机制,本研究应用蛋白质组学方法在总体定性及定量水平鉴定不同生物模型系统中具有明显差异表达的蛋白质,为中药红芪及其多糖的临床应用提供理论依据。目的应用蛋白质组学方法分析红芪多糖调节机体免疫及抗肿瘤作用的差异蛋白质并进行鉴定,从而揭示红芪多糖调节机体免疫及抗肿瘤作用的分子机制,从中筛选出评价红芪多糖疗效的特异性蛋白靶标。同时,研究红芪多糖对免疫抑制小鼠免疫功能的影响。方法建立小鼠和肿瘤细胞模型后,设立对照组和实验组,实验组以红芪多糖在体内或体外作用一定时间,收集组织或细胞进行裂解,提取组织或细胞总蛋白进行纯化,Bradford法测定蛋白样品浓度。蛋白定量后,采用17cm双向凝胶电泳分离组织或细胞总蛋白,考马斯亮蓝或银染显色,建立双向凝胶电泳(2-DE)图谱。通过GS-800校准型光密度透射扫描仪获取高精度双向凝胶电泳图谱,使用PDQuest8.0图像分析软件对不同处理组双向凝胶电泳图谱进行对比分析,筛选出差异蛋白点,并对其进行理论分子量、近似等电点及3D结构分析。应用Mascot2.1软件对MALDI-TOF/TOF串联飞行时间质谱仪所得数据进行NCBI蛋白数据库检索,鉴定差异明显的蛋白质,获得差异蛋白质的肽质量指纹图谱和相关鉴定信息,并用蛋白免疫印迹方法对热休克蛋白β-1进行验证。此外,建立免疫抑制小鼠模型,观察红芪多糖对疫抑制小鼠胸腺和脾脏指数、胸腺细胞结构及腹腔巨噬细胞吞噬能力的影响。结果1.红芪多糖可使小鼠胸腺细胞2-DE图谱中69个蛋白点发生明显变化,其中33个上调,26个下调,3个消失,7个仅在红芪多糖组中表达。应用质谱方法成功鉴定了4个蛋白质,分别为蛋白合成起始因子4、碳酸酐酶、过氧化还原蛋白6和蛋白酶体。2.红芪多糖联合环磷酰胺可明显抑制荷瘤小鼠S180瘤组织的生长,具有一定的协同增效作用(P<0.05)。应用蛋白质组学技术筛选出28个差异明显的蛋白点,其中8个上调,19个下调,1个仅在对照组中表达。经质谱分析,成功鉴定了5个蛋白质,分别为热休克蛋白84、未命名蛋白、载脂蛋白A-I、白蛋白和热休克蛋白p-1。热休克蛋白p-1的免疫印迹结果与蛋白质组学结果一致。3.红芪多糖可使Hela细胞2-DE图谱中21个蛋白点发生明显变化,其中14个上调,4个下调,3个仅在对照组中表达。应用质谱方法成功鉴定了4个蛋白质,分别为核仁磷酸蛋白、α-烯醇酶、磷酸丙糖异构酶、乙酰基辅酶A还原酶。4.红芪多糖可使A549细胞2-DE图谱中35个蛋白点发生明显变化,其中24个表达上调,8个表达下调,3个仅在对照组中表达,同时分析得出每个差异蛋白点的理论分子量、近似等电点及3D结构图。5.红芪多糖可明显提高免疫抑制小鼠的胸腺和脾脏指数,减轻免疫抑制小鼠胸腺细胞的损伤,并可增强免疫抑制小鼠腹腔巨噬细胞的吞噬能力。结论1.应用双向电泳方法筛选出的65个差异蛋白点可能是红芪多糖促胸腺细胞增殖的相关蛋白,经质谱鉴定成功的蛋白合成起始因子4、碳酸酐酶2、过氧化还原蛋白6和蛋白酶体与小鼠的机体免疫调节功能相关。2.红芪多糖联合环磷酰胺可增强环磷酰胺对S180瘤细胞的化疗效果。经质谱鉴定成功的热休克蛋白84、未命名蛋白、载脂蛋白A-I、白蛋白和热休克蛋白β-1的与红芪多糖在荷瘤小鼠体内的协同抗肿瘤作用相关。3.应用双向电泳方法筛选出的21个差异蛋白点可能是红芪多糖抑制人宫颈癌Hela细胞增殖的相关蛋白,经质谱鉴定成功的核仁磷酸蛋白、α-烯醇酶、磷酸丙糖异构酶、乙酰基辅酶A还原酶与红芪多糖红芪多糖的体外抗肿瘤作用相关。4.应用双向电泳方法筛选出的35个差异蛋白点可能是红芪多糖抑制人肺腺癌A549细胞增殖的相关蛋白质。5.红芪多糖可明显增强免疫抑制小鼠的机体免疫功能。更多还原

【Abstract】 BackgroundHedysari radix. known as "Hongqi’in traditional Chinese medicine, which is the dried root of Hedysarum polybotrys Hand.-Mazz., belongs to the the Fabaceae family but not belongs to the same genus as a species of Astragali radix and was believed to possess the effects of invigorating qi for consolidating superficies, replenishing blood, diuresis for detoxification, drainage, astringing wound for regenerating tissue. Hedysari radix polysaccharide (HPS), which is one of main chemical constituents of Hedysari radix, is composed of rhamnose, xylose, arabinose. galactose and glucose. Pharmacological research indicate that HPS possess the effects of immunomodulatory, antitumor and antioxidant. Furthermore. HPS may be considered as a new drug of traditional Chinese medicine with high value for development.Proteomics, known as an important systems biology tool in the post-genomics era, provided a novel approach for multicomponent, multitargets and multilinks dynamic analysis of traditional Chinese medicine as a powerful biomarker detection tool. Furthermore, Previous research showed that HPS possess the immunomodulatory and antitumor effects and the mechanism remains unclear. In this study, proteomics approach was applied to identify differentially expressed proteins to investigate the complex molecular mechanisms at a global qualitative and quantitative level in various model systems treated by HPS and provided further insights into theoretical progress for clinical application of RH and RHP in traditional Chinese medicine research.ObjectiveTo investigate the differentially expressed proteins in various model systems after treated by HPS through a proteomics approach and provide preliminary results for further study to explore the complex molecular mechanism of immunomodulat ory and antitumor effects of HPS. Furthermore, to explore the immunity in immunosuppressed mice after HPS treatment.MethodsThe target cells or tissues collected from various model systems treated by HPS were lysed by lysis buffer for protein extraction. The protein concentration were measured by Bradford method after acetone precipitation respectively. The proteins extracted from cells or tissues were separated by means of two dimentional gel electrophoresis (2-DE) based on17cm immobilized pH gradient. After Coomassie brilliant blue G250staining or silver staining and image scanning,2-DE image comparison was performed to analyze the differentially expressed proteins including theoretical Mr. theoretical pI value and3D analysis using PDQuest8.0software. Furthermore, mass spectrometry was applied to identify the differentially expressed proteins and the data were searched against NCBInr database by an in-house Mascot search engine. Western blot was employed to assess the expression of heat shock protein beta-1in S180sarcoma. Immunosuppression model was established by administrating cyclophosphamide to normal mice. The thymus gland index and ultrastructure, spleen index, ultrastructure of thymocyte, phagocytosis of the macrophage were determined after HPS treatment.Results1. A total of69protein spots in2-DE images of thymus cells from mice were found to be differentially expressed between control group and RPS-treated group, of which33spots were up-regulated,26spots down-regulated,3spots disappeared,7spots expressed only in HPS-treated group. Finally,4spots were successfully identified to be protein synthesis initiation factor4, carbonic anhydrase, peroxiredoxins6and proteasome by MALDI-TOF/TOF and Mascot software.2. Compared with cyclophsphamide (Cy) group, the HPS combined with Cy group showed obviously inhibitory effect on growth of S180sarcoma (P<0.05). A total of28protein spots in2-DE images of S180sarcoma were found to be differentially expressed between control group and RPS-treated group, of which8spots were up-regulated,19spots down-regulated,1spots expressed only in Cy group. Finally,5spots were successfully identified to be heat shock protein hsp84, unnamed protein product, apolipoprotein A-I, albumin, heat shock protein beta-1by MALDI-TOF/TOF and Mascot software. Results from western blot manifested the same trend as from proteomics analysis.3. A total of21protein spots in2-DE images of HeLa cells were found to be differentially expressed between control group and HRP-treated group, of which14spots were up-regulated,4spots down-regulated,3spots expressed only in control group. Finally,4spots were successfully identified to be nucleophosmin, alpha-enolase, triosephosphate isomerase, acetoacetyl-coenzyme A reductase by MALDI-TOF/TOF and Mascot software.4. A total of35protein spots in2-DE images of A549cells were found to be differentially expressed between control group and RPS-treated group, of which24spots were up-regulated,8spots down-regulated,3spots expressed only in control group and theoretical Mr, theoretical pI value and3D structure of each spot were calculated with PDQuest software.5. HPS treatment significantly raised thymus gland index, spleen index and phagocytosis of the macrophage, and decreased damage of thymocyte.Conclusion1. The protein expression profile of mice thymus was altered by HPS treatment and the successfully identified proteins including synthesis initiation factor4, carbonic anhydrase, peroxiredoxins6and proteasome were associated with immunomodulatory effect.2. HPS treatment strengthened chemotherapy effect of Cy in tumor-bearing mice. The successfully identified proteins including heat shock protein hsp84, unnamed protein product, apolipoprotein A-I, albumin, heat shock protein beta-1were associated with synergistic effect with Cy and immunomodulatory in tumor-bearing mice.3. The protein expression profile of human cervical carcinoma HeLa cells was altered by HPS treatment and the successfully identified proteins including nucleophosmin. alpha-enolase, triosephosphate isomerase, acetoacetyl-coenzyme A reductase were associated with anti-proliferation effec of HPS on HeLa cells.4. The protein expression profile of human lung adenocarcinoma A549cells was altered by HPS treatment and the35differentially expressed spots were associated with anti-proliferation effect of HPS on A549cells.5. HPS treatment significantly increased immunity effect in immunosuppressed mice.更多还原