【作者】 汲晨锋；

【导师】 季宇彬；

【作者基本信息】 北京中医药大学 ， 中药学， 2010， 博士

【摘要】 肿瘤是目前危害人类健康最严重的疾病之一,据WHO统计,全球平均每年死于恶性肿瘤者达700万人,新发病为800万例,且还在逐年增加,到2020年全世界每年将新发生2000万例肿瘤,成为人类健康头号杀手。传统的化学治疗药物和手术、放疗等结合,成功的提高了多种恶性肿瘤的治愈率,但是由于较强的副作用以及耐药性,使得寻找新药的研究刻不容缓。近年来,植物来源的抗肿瘤药物重新引起重视。芦笋是天门冬科天门冬属多年生草本植物,它是一种质地优良、营养丰富的保健型蔬菜,含有多糖、皂苷、黄酮、组织蛋白、微量元素等多种成分,大量科学研究证实芦笋是一种具有抗肿瘤、增加人体免疫力等功效的天然药物。国内外对芦笋的药用研究,特别是防癌抗癌研究正逐渐增多,但目前的研究多以芦笋原汁或芦笋提取液来进行抗肿瘤作用研究,其抗肿瘤作用的活性成分及具体机制目前还不清楚。课题立足这一研究空白,以芦笋多糖、芦笋皂苷为研究对象,明确芦笋抗肿瘤的有效成分及其抗肿瘤作用机制。肿瘤的发生发展与机体免疫反应密切相关,红细胞作为机体血液循环数量最多的血细胞,调节多种细胞的免疫功能,对机体免疫功能有重要调节作用。红细胞免疫功能的变化在肿瘤发生发展中具有极其重要的作用,红细胞膜表面存在着大量的免疫相关分子,它们是红细胞发挥免疫功能的重要物质基础。抗肿瘤药物通过增强肿瘤机体红细胞免疫功能,对肿瘤病情治疗具有实用价值。多糖的抗肿瘤作用具有整体性的优势,尤其可以增强机体免疫功能。基于这一理论基础,课题首次考察了芦笋多糖对荷瘤小鼠抗肿瘤作用,并从红细胞免疫角度考察了其抗肿瘤的作用机制。肿瘤的发生发展还与细胞凋亡密切相关,细胞凋亡是指为维持内环境稳定,在多种基因调控下的一个严密完整的、主动的、有序的死亡过程。以诱导肿瘤细胞凋亡作为治疗肿瘤的手段,已成为当今抗肿瘤药物作用机制研究的热点。课题首次考察了芦笋皂苷诱导人肝癌HepG2细胞凋亡的作用,并从线粒体介导细胞凋亡的途径着手,通过对凋亡事件中凋亡诱导因素、凋亡生死开关、凋亡启动分子、凋亡效应分子等指标的检测,考察了其抗肿瘤的作用机制。主要包括三部分研究内容：一、芦笋多糖、芦笋皂苷抗肿瘤作用研究；二、芦笋多糖对S180小鼠红细胞免疫及生理功能影响的研究；三、芦笋皂苷通过线粒体途径诱导HepG2细胞凋亡研究。1芦笋多糖、芦笋皂苷抗肿瘤作用研究1.1芦笋多糖、芦笋皂苷对荷瘤小鼠瘤重和生存时间的影响目的：考察芦笋多糖、芦笋皂苷对荷瘤小鼠瘤重和生存时间的影响,确定各组分体内抗肿瘤作用。方法：建立了肿瘤动物模型,分高、中、低剂量给予芦笋多糖、芦笋皂苷7天,计算抑瘤率、生命延长率。结果：①芦笋多糖可以抑制S180小鼠肿瘤的生长,给药组小鼠瘤重降低,与阴性组比较具有明显差异(P<0.05),抑瘤率具有量效性,高剂量组为47.65%；芦笋多糖可以改变H22小鼠的生存时间,给药组小鼠生存时间延长,与阴性组比较具有明显差异(P<0.05),生命延长率具有量效性,高剂量组为66.65%。②芦笋皂苷可以抑制S180小鼠肿瘤的生长,给药组小鼠瘤重降低,与阴性组比较具有明显差异(P<0.05),抑瘤率具有量效性,高剂量组为44.8%；芦笋皂苷可以改变H22小鼠的生存时间,给药组小鼠生存时间延长,与阴性组比较具有明显差异(P<0.05),生命延长率具有量效性,高剂量组为57.05%。结论：芦笋多糖、芦笋皂苷可以降低S180小鼠的瘤重,抑瘤作用明显。芦笋多糖、芦笋皂苷还可以延长H22小鼠的生存时间，生命延长作用明显。1.2芦笋多糖、芦笋皂苷对人肿瘤细胞增殖的影响目的：考察芦笋多糖、芦笋皂苷对HepG2、SGC-7901细胞增殖的影响,确定各组分体外抗肿瘤作用。方法：体外培养HepG2、SGC-7901细胞,分别给予浓度为0.1、1、10、100、1000μg/mL的芦笋多糖和芦笋皂苷,MTT法和SRB测定芦笋各组分的细胞增殖抑制率。结果：①MTT结果表明,芦笋多糖作用于HepG2、SGC-7901细胞72 h,对细胞生长有较弱的抑制作用,细胞毒作用不明显。②MTT结果表明,芦笋皂苷作用于HepG2、SGC-7901细胞72 h,对细胞生长有抑制作用,其中对HepG2细胞的IC50为101.15μg/mL,对SGC-7901细胞的IC50为177.5μg/mL。SRB结果表明,芦笋皂苷作用于HepG2、SGC-7901细胞72 h,对细胞生长有抑制作用,对HepG2细胞的GI50为107.53 gg/mL,对SGC-7901细胞的GI50为157.44 gg/mL。结论：芦笋多糖对人肿瘤细胞增殖无抑制作用,芦笋皂苷对人肿瘤细胞增殖有较强抑制作用,对HepG2细胞作用敏感。2芦笋多糖对S180小鼠红细胞免疫及生理功能影ktm的研究2.1芦笋多糖对S180小鼠红细胞补体受体-1(CR1,CD35)数量及活性的影响目的：研究芦笋多糖对S180小鼠红细胞补体受体-1(CRl)数量、红细胞CR1天然免疫活性及红细胞调控淋巴细胞免疫反应能力的影响。方法：取小鼠抗凝全血制备红细胞悬液,用大鼠抗小鼠CD35一抗和FITC-羊抗大鼠二抗标记小鼠红细胞,应用流式细胞仪测定小鼠红细胞CR1数量；显微镜下观察红细胞C3b受体花环率(RBC-C3bRR)和肿瘤红细胞花环率(DTER);取正常小鼠抗凝全血制备淋巴细胞悬液和血浆,取正常小鼠和芦笋多糖不同剂量组S180小鼠抗凝全血制备红细胞悬液,以癌细胞S180作为激活抗原,正常血浆作为反应基质,正常淋巴细胞作为效应细胞,分别建立抗原激活及无抗原激活红细胞调控淋巴细胞免疫反应体系,流式细胞仪检测淋巴细胞表面CD25表达。结果：①S180小鼠红细胞CR1数量显著低于正常组(P<0.01),芦笋多糖高、中剂量可以增加S180小鼠红细胞CR1数量,与阴性组比较具有明显差异(P<0.05)。②S180小鼠RBC-C3bRR、DTER显著低于正常组(P<0.01),芦笋多糖高、中剂量可以增加S180小鼠CR1天然免疫活性,与阴性组比较具有明显差异(P<0.05)。③在抗原激活情况下,小鼠红细胞能够对淋巴细胞的免疫反应发生正向调控作用,正常组小鼠红细胞作用下的CD25表达量明显高于肿瘤组，芦笋多糖组S180小鼠红细胞作用下的CD25表达量明显高于阴性组;无抗原激活情况下,红细胞同样能够单独对淋巴细胞进行正向调控,使CD25表达量升高,但各组表达量低于抗原激活情况下的表达量。结论芦笋多糖可以增加S180小鼠红细胞CR1的数量,增强S180小鼠红细胞CR1天然免疫活性,增强S180小鼠红细胞CR1调控淋巴细胞免疫反应的能力,改善S180小鼠红细胞免疫功能。2.2芦笋多糖对S180小鼠红细胞膜组分及流动性的影响目的：研究芦笋多糖对S180小鼠红细胞膜组分及流动性的影响。方法：芦笋多糖对S180小鼠腹腔注射给药7天,应用聚丙烯酰胺凝胶电泳测定小鼠红细胞膜带3蛋白及血型糖蛋白A的相对含量,采用试剂盒测定红细胞膜磷脂、胆固醇和唾液酸含量,荧光偏振法结合探针1,6-二苯-1、3、5己三烯测定红细胞膜流动性。结果：①S180小鼠与正常对照组比较红细胞膜磷脂含量降低、胆固醇含量升高,芦笋多糖可以使S180小鼠红细胞膜磷脂含量升高、胆固醇含量降低,与阴性对照组比较具有显著性差异P<0.05,P<0.01)。②S180小鼠红细胞膜带3蛋白、血型糖蛋白A相对含量明显低于正常对照组,芦笋多糖中、高剂量可以显著提高S180小鼠红细胞膜两种蛋白相对含量,与阴性对照组比较具有显著性差异(P<0.05,P<0.01)。③S180小鼠与正常对照组比较红细胞膜唾液酸含量显著降低,芦笋多糖高、中剂量组能够升高S180小鼠红细胞膜唾液酸含量,与阴性对照组比较具有显著性差异(P<0.05,P<0.01)。④S180小鼠与正常对照组比较红细胞膜流动性降低,芦笋多糖中、高剂量组能降低S180小鼠红细胞膜荧光偏振度和微黏度,升高S180小鼠红细胞膜流动性,与阴性对照组比较具有显著性差异(P<0.05,P<0.01)。结论：芦笋多糖可以改善S180小鼠红细胞膜组分的异常变化,从而保持细胞膜的流动性,恢复红细胞的结构和功能。2.3芦笋多糖对S1so小鼠红细胞膜电位的影响目的：研究芦笋多糖对S180小鼠红细胞膜电位的影响。方法：建立了肿瘤动物模型,分高、中、低剂量腹腔给予芦笋多糖7d,采集并制备红细胞悬液,采用DiBAC4(3)作为膜电位荧光探针,应用流式细胞仪检测小鼠红细胞膜电位。结果：阴性对照组S180小鼠红细胞荧光强度明显高于正常对照组,说明Slso小鼠红细胞膜电位高于正常对照组,细胞膜发生去极化,两组之间比较有非常显著性差异(P＜0.01)，芦笋多糖中、高剂量组能够减弱S180小鼠红细胞荧光强度,降低红细胞膜电位,使红细胞膜去极化减弱。结论：芦笋多糖可以改善S180小鼠红细胞膜电位,从而稳定细胞内外离子的跨膜扩散。2.4芦笋多糖对S180小鼠红细胞离子通道的影响目的：研究芦笋多糖对S180小鼠红细胞离子通道的影响。方法：建立了肿瘤动物模型,分高、中、低剂量腹腔给予芦笋多糖7d,采集并制备红细胞悬液,试剂盒结合分光光度计测定S180小鼠红细胞膜Na+,K+-ATPase、Ca2+,Mg2+-ATPase活性,采用Fluo-3/AM、MQAE、BCECF-AM作为荧光探针,应用激光共聚焦显微镜分别观察S180小鼠红细胞内[Ca2+]浓度,[CI-]浓度、细胞内pH。结果：①芦笋多糖能升高S180小鼠红细胞膜Na+,K+-ATPase、Ca2+,Mg2+-ATPase活性,降低S180小鼠红细胞内[Ca2+]浓度,增强红细胞阳离子通道转运活性。②芦笋多糖能够降低S180小鼠红细胞内[CI-]浓度,增强红细胞阴离子通道转运活性。③芦笋多糖还能够降低S180小鼠红细胞内的H+浓度,升高红细胞内pH,维持红细胞酸碱平衡。结论：芦笋多糖正是通过提高S180小鼠红细胞膜离子通道的功能,从而稳定细胞内环境,保护红细胞膜结构,维持红细胞发挥正常功能。2.5芦笋多糖对S180小鼠红细胞合淌度的影响目的：研究芦笋多糖对S180小鼠红细胞合淌度的影响。方法：建立了肿瘤动物模型,分高、中、低剂量腹腔给予芦笋多糖7d,采集并制备红细胞悬液,应用高效毛细管电泳法检测红细胞的合淌度。实验条件：毛细管为75μm×50cm,电泳缓冲液为含2g/L羟丙基甲基纤维素的磷酸盐溶液,压力进样为3.448kPa×10s,分离电压为20kV,柱温为25℃。结果：S180小鼠红细胞的电泳迁移时间比正常小鼠延长,芦笋多糖可以缩短S180小鼠红细胞迁移时间,与阴性组比较具有显著性差异(P<0.05)。S180小鼠红细胞的合淌度比正常组小鼠降低(P<0.01),芦笋多糖可以提高S180小鼠红细胞的合淌度,与阴性组比较具有显著性差异(P<0.05)。结论：芦笋多糖能够改变肿瘤机体红细胞的合淌度,这可能与其改变红细胞表面的电荷密度有关。高效毛细管电泳法可以作为检测红细胞生理状态和功能的一种辅助工具。3芦笋皂苷通过线粒体途径诱导HepG2细胞凋亡研究3.1芦笋皂苷诱导HepG2细胞凋亡的检测目的：研究芦笋皂苷诱导HepG2细胞凋亡的作用。方法:体外培养HepG2细胞,分别给予浓度为50、100、200μg/mL的芦笋皂苷,培养48h、72h。Hoechst 33258染色结合荧光显微镜观察HepG2细胞凋亡形态,透射电子显微镜观察HepG2细胞超微结构,Annexin V-FITC/PI双染结合流式细胞仪测定芦笋皂苷诱导HepG2细胞凋亡作用,PI单染结合流式细胞仪测定芦笋皂苷对HepG2细胞周期及凋亡率的影响。结果：①荧光显微镜观察表明,阴性对照组细胞界限清晰,细胞呈现弥散均匀荧光；芦笋皂苷作用于细胞48 h后,HepG2细胞出现了凋亡形态,随着浓度的增加,凋亡细胞数量增多,出现凋亡小体。②透射电子显微镜下观察表明,阴性对照组细胞有核仁且细胞器丰富,芦笋皂苷作用72h后,HepG2细胞出现了凋亡形态,且随着药物浓度的增加细胞凋亡特征性形态逐渐明显,高剂量组,细胞核裂解为碎块,产生凋亡小体。③芦笋皂苷作用48 h后,凋亡早期细胞数随药物浓度升高而减少,凋亡晚期和坏死细胞数随药物浓度升高而增加,凋亡细胞总数随着药物浓度升高呈现一定的剂量依赖性。④芦笋皂苷作用72 h后HepG2细胞周期发生了明显的变化,各剂量组细胞S期比例增加,G2/M期比例减少,随着芦笋皂苷浓度的增加周期变化趋势明显。各给药组出现亚二倍体凋亡峰,随着芦笋皂苷浓度的增加凋亡率增加。结论：芦笋皂苷可以诱导HepG2细胞凋亡。3.2芦笋皂苷对HepG2细胞内活性氧(ROS)、Ca2+、pH的影响目的：研究芦笋皂苷对HepG2细胞内活性氧(ROS)、Ca2+、pH的影响。方法：体外培养HepG2细胞,分别给予浓度为50、100、200μg/mL的芦笋皂苷,培养24h。采用Fluo-3/AM、BCECF/AM作为荧光探针,应用流式细胞仪、激光共聚焦显微镜测定芦笋皂苷对HepG2细胞内ROSCa2+、pH的影响。结果：①芦笋皂苷作用24 h,各给药组细胞内ROS荧光强度高于空白对照组,具有统计学意义(P<0.01),随着药物浓度的增加细胞内荧光强度增强,ROS水平明显增加。②芦笋皂苷作用24 h,各给药组细胞内Ca2+荧光强度高于空白对照组,具有统计学意义P<0.01),随着药物浓度的增加细胞内的荧光强度增强,Ca2+浓度升高。③芦笋皂苷作用24 h,各给药组细胞内pH荧光强度低于空白对照组,具有统计学意义(P<0.01),随着药物浓度的增加细胞内荧光强度减弱,细胞内pH降低。结论：芦笋皂苷通过对HepG2细胞内ROS、Ca2+、pH的调控,可以进一步诱发下游凋亡相关事件的发生。3.3芦笋皂苷对HepG2细胞内线粒体膜通道孔(MPTP)及线粒体膜电位(MMP)的影响目的：研究芦笋皂苷对HepG2细胞内线粒体膜通道孔(MPTP)及线粒体膜电位(MMP)的影响。方法：体外培养HepG2细胞,分别给予浓度为50、100、200μg/mL的芦笋皂苷,培养24h。采用MPTP荧光检测试剂盒、Rhodamine 123作为荧光探针,应用激光共聚焦显微镜观察芦笋皂苷对HepG2细胞MPTP、MMP的影响。结果：①芦笋皂苷作用HepG2细胞24h后,细胞荧光强度减弱,表明细胞MPTP活性增强,并且随着药物浓度增加MPTP活性逐渐增强,与空白对照组比较差异显著(P<0.05)。②芦笋皂苷作用HepG2细胞24h后,细胞荧光强度减弱,表明细胞MMP水平降低,并且随着药物浓度增加MMP水平逐渐降低,与空白对照组比较差异显著(P<0.05)。结论：芦笋皂苷通过对细胞线粒体通透性转换孔开放,启动细胞的生死开关,降低线粒体膜电位,最终诱导细胞凋亡。3.4芦笋皂苷对HepG2细胞凋亡相关蛋白表达的影响目的：研究芦笋皂苷对HepG2细胞凋亡相关蛋白表达的影响。方法：体外培养HepG2细胞,分别给予浓度为50、100、200μg/mL的芦笋皂苷,培养24h、48h。分别加Bcl-2、Bax、Cyt-c、Caspase-9、Caspase-3一抗孵育,加异硫氰酸荧光素(FITC)标记的二抗孵育,采用流式细胞仪检测细胞内蛋白表达水平。结果：①芦笋皂苷作用HepG2细胞24h后,细胞内Cyt-c蛋白的表达量增加,并且随着药物浓度增加蛋白含量逐渐升高。②芦笋皂苷作用HepG2细胞48h后,细胞内激活型Caspase-9、Caspase-3蛋白的表达量增加,并且随着药物浓度增加蛋白含量逐渐升高。③芦笋皂苷作用HepG2细胞24h后,细胞内Bcl-2蛋白的表达量降低,并且随着药物浓度增加蛋白含量逐渐降低；细胞内Bax蛋白的表达量增加,并且随着药物浓度增加蛋白含量逐渐升高。结论：芦笋皂苷可以调控HepG2细胞内线粒体途径多个凋亡相关蛋白的表达,从而诱导细胞凋亡。3.5芦笋皂苷对HepG-2细胞Caspase-9和Caspase-3活性影响目的：研究芦笋皂苷对HepG2细胞Caspase-9和Caspase-3活性的影响。方法：体外培养HepG2细胞,分别给予浓度为50、100、200μg/mL的芦笋皂苷,培养24h、48h。采用Caspase活性检测试剂盒结合酶标仪测定Caspase-9和Caspase-3活性。结果：①芦笋皂苷作用HepG2细胞后,pNA生成量均增加,Caspase-9的相对活性升高,与空白对照组比较差异显著(P＜0.01)。芦笋皂苷作用48h后Caspase-9的相对活性较作用24h的相对活性高。24h相对活性分别增加为48.21%、63.89%、102.11%,48h相对活性分别增加为55.52%,93.83%,135.89%。②芦笋皂苷作用HepG2细胞后,pNA生成量均增加,Caspase-3的相对活性升高,与空白对照组比较差异显著(P<0.01)。芦笋皂苷作用48h后Caspase-9的相对活性比作用24h的相对活性高。24h相对活性分别增加为46.79%,55.03%,74.24%,48h相对活性分别增加为80.03%,143.32%,218.66%。结论：芦笋皂苷可以增强HepG-2细胞中Caspase-9和Caspase-3的活性,可以通过Caspase依赖的线粒体途径诱导细胞凋亡。结论芦笋具有抗肿瘤的作用。其活性成分及作用机制包括：一、芦笋多糖可以提高S180小鼠红细胞免疫功能：①芦笋多糖可以增加S180小鼠红细胞CR1的数量,增加S180小鼠红细胞C3b受体花环率(RBC-C3bRR)和肿瘤红细胞花环率(DTER),增强S180小鼠红细胞C3b受体调控淋巴细胞免疫反应的能力,从而对S180小鼠红细胞CRl的数量、活性有所增加和提高。②芦笋多糖能增加S180小鼠红细胞膜表面带3蛋白、血型糖蛋白A的含量；升高S180小鼠红细胞膜磷脂含量,降低胆固醇含量；增加S180小鼠红细胞膜表面唾液酸含量；提高S180小鼠红细胞膜流动性。从而改善S180小鼠红细胞膜组分和结构的变化,恢复红细胞的功能。③芦笋多糖能够减弱S180小鼠红细胞荧光强度,降低红细胞膜电位,使红细胞膜去极化减弱。④芦笋多糖能升高S180小鼠红细胞膜Na+,K+-ATPase、Ca2+,Mg2+-ATPase活性,降低S180小鼠红细胞内[Ca2+]浓度,增强红细胞阳离子通道转运活性。芦笋多糖能降低S180小鼠红细胞内[CI-]浓度,增强红细胞阴离子通道转运活性。芦笋多糖还能够降低S180小鼠红细胞内的H+浓度,升高红细胞内pH,维持红细胞酸碱平衡。⑤芦笋多糖可以缩短S180小鼠红细胞迁移时间,提高S180小鼠红细胞的合淌度。芦笋皂苷通过线粒体途径诱导HepG2细胞凋亡：①荧光显微镜和透射电镜的形态学观察结果表明,芦笋皂苷能够诱导HepG2细胞发生凋亡,产生凋亡小体。流式细胞仪检测同样表明,芦笋皂苷能够诱导HepG2细胞发生凋亡,细胞凋亡率随药物浓度升高呈现剂量依赖性,同时芦笋皂苷能够阻滞HepG2细胞周期于S期,使S期细胞比例增加,G2/M期细胞比例减少。②芦笋皂苷能够刺激HepG2细胞内ROS水平升高,增加胞浆内Ca2+浓度,还可以引起细胞内H+增多,导致细胞内酸化,从而诱导HepG2细胞凋亡。③芦笋皂苷能够使HepG2细胞线粒体通透性转换孔开放,启动细胞的生死开关,降低线粒体膜电位,从而进入一个不可逆转的线粒体凋亡途径。④芦笋皂苷能够调控多种线粒体凋亡途径相关蛋白的表达,诱导HepG2细胞凋亡。可以显著增加细胞内Cyt-C蛋白、激活型Caspase-9、Caspase-3蛋白的表达量,同时显著增加细胞内Bax蛋白的表达量,降低细胞内Bcl-2蛋白的表达量。⑤芦笋皂苷可以增强HepG2细胞中Caspase-9和Caspase-3的活性,这种作用具有量效性和时效性,证明芦笋皂苷可以通过Caspase依赖的线粒体途径诱导HepG2细胞凋亡。1.多糖的抗肿瘤作用具有整体性的优势,尤其可以增强机体免疫功能。基于这一理论基础,本课题首次考察了芦笋多糖的抗肿瘤作用,并从红细胞免疫角度考察了其抗肿瘤的作用机制。①首次检测了小鼠红细胞CR1的数量,并考察了芦笋多糖对S180小鼠红细胞CR1数量的影响,国内外尚无相关报道。②血液免疫反应路线图理论是近年来红细胞免疫基础理论的重大突破,是红细胞免疫研究方法的创新。课题结合这一新理论,考察了芦笋多糖对S180小鼠红细胞调控淋巴细胞免疫反应能力的影响,国内外尚无相关报道。③关于肿瘤机体与正常机体红细胞淌度间差别的报道还比较少,课题首次建立高效毛细管电泳检测方法测定了芦笋多糖对S180小鼠红细胞电泳合淌度的影响。2.课题通过实验筛选出对人肿瘤细胞增殖具有直接抑制作用的芦笋皂苷成分,并考察了其诱导细胞凋亡的作用,同时确定线粒体途径为芦笋皂苷诱导细胞凋亡的主要途径,国内外未见相关报道。更多还原

【Abstract】 Tumor is one of the most harmful diseases to human health, as WHO reports, up to seven million people die from cancer per year the entire world, and the new patient are about eight million, which is gradually increasing. In 2020, the new incidence will be twenty million per year all the world, and the cancer will be the first killer for human health. Chemistry drug, operation and radiotherapy had successfully cured many cancers, but the side-effect and drug-resistance were obvious, so founding new drugs is urgent.In recent years, anti-tumor drugs from plant have aroused people’s attention. Asparagus officinalis is a perennial herb plant; it has abundant nutrition and many active components such as polysaccharide, saponin, flavonoids, tissue protein, and trace element. Many scientific studies showed Asparagus officinalis had anti-tumor effect and enhanced people’s immunity. The research is increasing gradually around the would, especially on cancer cure and prevention, but the present work mainly focus on asparagus officinalis juice or extracts, so the exact anti-tumor active components in’Asparagus officinalis and anti-tumor mechanism are unclear. Based on the vain field, we choose asparagus polysaccharide and saponin as study object, and identify anti-tumor active components in Asparagus officinalis and anti-tumor mechanism.There are substantial connections between tumor occurrence and development with organism immunity. Red blood cells is the most cells in blood cycle, it can adjust many immune functions, which play an important role in organism immunity. The change of red blood cells immunity may affect tumor occurrence and development; there are many immune molecules on red blood cells surface, which are the most important material basis for its immune function. Many anti-tumor drugs cured the patients by enhancing their immunity, and polysaccharide from plant had the advantage to enhance organism immunity. So based on this feature, we determined asparagus polysaccharide anti-tumor effect for the first time, and study the mechanism via red blood cells immunity.There are substantial connections between tumor occurrence and development with cell apoptosis. Apoptosis is the process of programmed cell death that may occur in multi-cellular organisms. Use cell apoptosis induction as tumor treatment method has been hotspot for anti-tumor drug discovery. So based on this feature, we determined HepG2 cell apoptosis induced by asparagus saponin for the first time, and study the mechanism via mitochondrial pathway, such as apoptosis induction factor, apoptosis death switch, apoptosis starting molecule, apoptosis executing molecule and so on.The experiments include three parts:1. Study on anti-tumor effect of Asparagus polysaccharide and Asparagus saponins; 2. Study on the effect of Asparagus polysaccharide on immune and biochemistry function of erythrocyte in S180 mice; 3. Study on apoptosis of HepG2 cell induced by Asparagus saponins via mitochondrial pathway.1 Study on anti-tumor effect of Asparagus polysaccharide and Asparagus saponins1.1 Effect of Asparagus polysaccharide and Asparagus saponins on tumor weight and survival time of tumor model miceOBJECTIVE:To determine the effect of Asparagus polysaccharide and Asparagus saponins on tumor weight and survival time of tumor model mice, confirm the anti-tumor effect in vitro.METHODS:Tumor mice model was made, and they were treated with different dosage of Asparagus polysaccharide and Asparagus saponins for 7 days, the anti-tumor rate and life-lengthening rate was calculated. RESULTS:①Asparagus polysaccharide inhibited tumor growth of S180 mice, which the tumor weight was reduced remarkably compared with control group (P<0.05). Asparagus polysaccharide improved survival time of H22 mice, which the survival time was lengthened remarkably compared with control group (P<0.05).②Asparagus saponins inhibited tumor growth of S180 mice, which the tumor weight was reduced remarkably compared with control group (P<0.05). Asparagus saponins improved survival time of H22 mice, which the survival time was lengthened remarkably compared with control group (P<0.05).CONCLUSION:Asparagus polysaccharide and Asparagus saponins had anti-tumor effect in vitro.1.2 Effect of Asparagus polysaccharide and Asparagus saponins on human tumor cell proliferation OBJECTIVE:To determine the effect of Asparagus polysaccharide and Asparagus saponins on human tumor cell proliferation, confirm the anti-tumor effect in vivo. METHODS: HepG2 and SGC-7901 cells were cultured, and they were treated with different dosage of Asparagus polysaccharide and Asparagus saponins, the cell proliferation inhibitory rate was measured by MTT and SRB methods. RESULTS:①The result of MTT showed Asparagus polysaccharide had little inhibitory effect on cell proliferation, which was unremarkably compared with control group (P>0.05).②Result of MTT showed Asparagus saponins had inhibitory effect on cell proliferation, and the IC50 for HepG2 and SGC-7901 was 101.15μg/mL and 177.5μg/mL. The result of SRB showed Asparagus saponins had inhibitory effect on cell proliferation, and the GI50 for HepG2 and SGC-7901 was 107.53μg/mL and 157.44μg/mL. CONCLUSION:Asparagus polysaccharide had no anti-tumor effect in vitro, while Asparagus saponins had anti-tumor effect in vitro.2 Study on the effect of Asparagus polysaccharide on immune and biochemistry function of erythrocyte in S180 mice2.1 Effect of Asparagus polysaccharide on the number and activity of erythrocyte complement receptor 1(CD35) in S180 miceOBJECTIVE:To study the effect of Asparagus polysaccharide on the number and activity of erythrocyte complement receptor 1 in S180 mice. METHODS:Red blood cells from mice venous blood were labeled by rat anti-mouse CD35 monoclonal antibody and FITC-conjugated goat anti-mouse antibody. Using flow cytometry, we determined the number of ECR1. Using microscope, we studied the the adherence between erythrocyte immunity and C3b receptor or tumor-cell by RBC-C3bRR and DTER. Suspensions of lymphocytes and plasma were separated from anticoaguted whole blood of normal mice. Suspensions of erythrocytes were separated from anticoaguted whole blood of normal mice and S180 mice treated by Asparagus polysaccharide. Usnig S180 cells as activating antigen, plasma as reactive medium, lymphocytes as effective cell, we established two immuno-response systems of lymphocytes regulated by erythrocytes with or without antigen activation. The expression of CD25 on lymphocytes was measured by flow cytometry. RESULTS:①The number of CR1 in S180 mice group was remarkably low compared with that in normal mice group (P<0.01), and Asparagus polysaccharide could increase CR1 number remarkably compared with control group (P<0.05).②RBC-C3bRR and DTER in S180 mice group was remarkably low compared with that in normal mice group (P<0.01), and Asparagus polysaccharide could increase RBC-C3bRR and DTER remarkably compared with control group (P<0.05).③Under antigen activation, the erythrocytes could positively regulate immunological reaction of lymphocytes, the expression of CD25 on lymphocytes regulated by erythrocytes in normal group was much higher than that in tumor groups, and in tumor groups treated by Asparagus polysaccharide was much higher than that in tumor control group; Under no antigen activation, the erythrocytes could also positively regulate immunological reaction of lymphocytes and increase the expression of CD25, but the effect was weaker than that under antigen activation.CONCLUSION:Asparagus polysaccharide can improve the erythrocyte immune function of S180 mice, which may be one of its most important anti-tumor mechanisms.2.2 Effect of Asparagus polysaccharide on erythrocyte membrane components and fluidity in S180 miceOBJECTIVE:To study the effect of Asparagus polysaccharide on erythrocyte membrane components and fluidity in S180 mice. METHODS:S180 mice were administrated with Asparagus polysaccharide by i.p. for 7d, and we determined band 3 and glycophorin A content by SDS-PAGE, spectrophotometer with test kit was used to measure phospholipids, cholesterol and sialic acid, DPH dye and fluorescence spectrophotometry was used to determine membrane fluidity. RESULTS:①The content of phospholipids in S180 mice group was remarkably low and cholesterol in S180 mice group was remarkably high compared with that in normal mice group, and Asparagus polysaccharide could increase phospholipids content and decrease cholesterol content remarkably compared with control group (P<0.05,P<0.01).②The content of band 3 and glycophorin A in S180 mice group was remarkably low compared with that in normal mice group, and Asparagus polysaccharide could increase both proteins content remarkably compared with control group (P<0.05,P<0.01).③The content of sialic acid in S180 mice group was remarkably low compared with that in normal mice group, and Asparagus polysaccharide could increase the content remarkably compared with control group (P<0.05,P<0.01).④The erythrocyte membrane fluidity in S180 mice group was remarkably low compared with that in normal mice group, and Asparagus polysaccharide could improve membrane fluidity remarkably compared with control group (P<0.05,P<0.01). CONCLUSION:Asparagus polysaccharide can adjust the abnormality of S180 mice, and improve membrane fluidity, restore erythrocyte membrane structure and function.2.3 Effect of Asparagus polysaccharide on erythrocyte membrane potential in S180 miceOBJECTIVE:To study the effect of Asparagus polysaccharide on erythrocyte membrane potential in S180 mice. METHODS:Tumor mice model was made and treated with different dosage of Asparagus polysaccharide for 7 days, suspensions of erythrocytes were separated from anticoaguted whole blood and dyed with DiBAC4(3), flow cytometry was used to determine erythrocyte membrane potential. RESULTS:The erythrocyte fluorescence intensity in S180 mice group was remarkably high compared with that in normal mice group (P<0.01), which showed erythrocyte membrane potential in S180 mice group was high, Asparagus polysaccharide could decrease erythrocyte fluorescence intensity and lower erythrocyte membrane potential remarkably compared with control group (P<0.05).CONCLUSION:Asparagus polysaccharide can adjust erythrocyte membrane potential and maintain cell inner environment and ion transmembrance diffusion.2.4 Effect of Asparagus polysaccharide on erythrocyte ion channel in S180 miceOBJECTIVE:To study the effect of Asparagus polysaccharide on erythrocyte ion channel in S180 mice. METHODS:Tumor mice model was made and treated with different dosage of Asparagus polysaccharide for 7 days, and suspensions of erythrocytes were separated from anticoaguted whole blood, spectrophotometer with test kit was used to measure Na+,K+-ATPase, Ca2+,Mg2+-ATPase activity, erythrocytes were dyed with Fluo-3/AM, MQAE, BCECF-AM and laser scanning confocal microscope and fluorescence spectrophotometry were used to determine [Ca2+],[CI-],pH. RESULTS:①Asparagus polysaccharide could increase erythrocyte Na+,K+-ATPase, Ca2+,Mg2+-ATPase activity, lower [Ca2+] concentration, improve cation channel transport activity.②Asparagus polysaccharide could lower [CI-] concentration, improve anion channel transport activity.③Asparagus polysaccharide could increase intra-cellular pH, maintain acid-base balance. CONCLUSION:Asparagus polysaccharide can improve erythrocyte ion channel transport activity, maintain cell inner environment and protect cell structure, enhance erythrocyte immune function. 2.5 Effect of Asparagus polysaccharide on complex mobility of erythrocyte in mice OBJECTIVE:To study the effect of Asparagus polysaccharide on complex mobility of erythrocyte in S180 mice. METHODS:Tumor mice model was made and treated with different dosage of Asparagus polysaccharide for 7 days, and suspensions of erythrocytes were separated from anticoaguted whole blood, high performance capillary electrophoresis was used to determine complex mobility of erythrocyte. Experimental conditions included the following:capillaries,75μm×50cm; buffer for electrophoresis, phosphate solution containing hydroxypropylmethyl cellulose (0.1mol/L, pH7.4); injection pressure,3.448kPa; injection time,10s; separation voltage,20kV; column temperature,25℃. RESULTS:The migration time of erythrocyte in S180 mice group was longer than that in normal mice group, and Asparagus polysaccharide could remarkably shorten migration time of erythrocyte compared with control group (P<0.05). The complex mobility of erythrocyte in S180 mice group was longer than that in normal mice group, and Asparagus polysaccharide could remarkably increase complex mobility of erythrocyte compared with control group (P<0.05). CONCLUSION: Asparagus polysaccharide improved complex mobility of erythrocyte in S180 mice, which is possibly related with the change of charges density on erythrocytes surface. It is believed that HPCE can be used as an auxiliary tool for determining the physiological state and functions of erythrocytes.3. Study on apoptosis of HepG2 cell induced by Asparagus saponins via mitochondrial pathway3.1 Determination of apoptosis of HepG2 cell Induced by Asparagus saponins. OBJECTIVE:To study apoptosis of HepG2 cell inducted by Asparagus saponins.METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins for 48h and 72h.Cells were dyed with Hoechst 33258 and observed under fluorescence microscope, and the ultrastructural changes of cells were observed by transmission electron microscopy. Cells were dyed with Annexin V-FITC/PI and PI, flow cytometry was used to determine apoptosis rate and cell cycle. RESULTS:①Under fluorescence microscope, cells in control group showed integrated and clear edge image. After treated with Asparagus saponins for 48h, cells appeared apoptosis image, as concentration increased, the cells morphology changed irregular, and the number of apoptotic bodies increased.②Under transmission electron microscopy, cells in control group showed prominent nuclear and abundant organella. After treated with Asparagus saponins for 72h, cells appeared apoptosis image, as concentration increased, the apoptosis morphology was gradually obvious and apoptotic bodies appeared.③After treated with Asparagus saponins for 48h, cells apoptosis rate was gradually high. As concentration increased, the early apoptosis rate decreased and the late apoptosis rate increased.④After treated with Asparagus saponins for 72h, cell cycle changed much, and the ratio of cells increased in S phase and decreased in G2/M phase. As concentration increased, apoptotic peak gradually appeared.CONCLUSION: Asparagus saponins can induce HepG2 apoptosis.3.2 Effect of Asparagus saponins on HepG2 intracellular reactive oxygen species (ROS), Ca2+, pHOBJECTIVE:To study the effect of Asparagus saponins on HepG2 intracellular ROS, Ca2+, pH. METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins for 24h.Cells were dyed with DCFH-DA, Fluo-3/AM, BCECF/AM, flow cytometry and laser scanning confocal microscope were used to determine intracellular ROS, Ca2+, pH.RESULTS:①After treated with Asparagus saponins for 24h, the cells fluorescence intensity intensified, the intracellular ROS level increased remarkably compared with control group (P<0.01).②After treated with Asparagus saponins for 24h, the cells fluorescence intensity intensified, the intracellular Ca2+ level increased remarkably compared with control group (P<0.01).③After treated with Asparagus saponins for 24h, the cells fluorescence intensity weakened, the intracellular pH decreased remarkably compared with control group (P<0.01).CONCLUSIONAsparagus saponins can adjust HepG2 intracellular ROS、Ca2+、pH level and induce downstream apoptosis event happen.3.3 Effect of Asparagus saponins on HepG2 mitochondrial permeability transition pore (MPTP) and Mitochondrial membrane potential (MMP)OBJECTIVE:To study the effect of Asparagus saponins on HepG2. mitochondrial permeability transition pore (MPTP) and mitochondrial membrane potential (MMP). METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins for 24h.Cells were dyed with MPTP fluorescence kit and Rhodamine 123, laser scanning confocal microscope was used to observe MPTP, MMP. RESULTS:①After treated with Asparagus saponins for 24h, the cells fluorescence intensity weakened, the MPTP activity increased remarkably compared with control group (P<0.05).②After treated with Asparagus saponins for 24h, the cells fluorescence intensity weakened, the MMP level decreased remarkably compared with control group (P<0.05). CONCLUSION:Asparagus saponins can open mitochondrial permeability transition pore, turn on the death switch and decrease mitochondrial membrane potential, then induce cell apoptosis.3.4 Effect of Asparagus saponins on apoptosis related protein expression in HepG2OBJECTIVE:To study the effect of Asparagus saponins on apoptosis related protein expression in HepG2. METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins. Cells were incubated with Bcl-2、Bax、Cyt-c、Caspase-9、Caspase-3 antibody respectively, then incubated with FITC-conjugated antibody, flow cytometry was used to determine protein expression. RESULTS:①After treated with Asparagus saponins for 24h, the intracellular Cyt-c expression increased, which was in a dosage dependent manner.②After treated with Asparagus saponins for 48h, the intracellular activated Caspase-9, Caspase-3 expression increased, which was in a dosage dependent manner.③After treated with Asparagus saponins for 24h, the intracellular Bcl-2 expression decreased and Bax expression decreased. CONCLUSION:Asparagus saponins can adjust apoptosis related protein expression in HepG2, and induce cell apoptosis via mitochondrial pathway.3.5 Effect of Asparagus saponins on Caspase-9 and Caspase-3 activity in HepG2 OBJECTIVE:To study the effect of Asparagus saponins on Caspase-9 and Caspase-3 activity in HepG2. METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins for 24h and 48h. Caspase kit and enzyme-labeled instrument were used to determine Caspase-9 and Caspase-3 activity. RESULTS:①After treated with Asparagus saponins, pNA production increased, Caspase-9 activity increased remarkably compared with control group (P<0.01), and the activity at 48h was higher than that at 24h. The activity at 24h was increased by 48.21%,63.89%,102.11%, and the activity at 48h was increased by 55.52%, 93.83%,135.89%.②After treated with Asparagus saponins, pNA production increased, Caspase-3 activity increased remarkably compared with control group (P<0.01), and the activity at 48h was higher than that at 24h. The activity at 24h was increased by 46.79%, 55.03%,74.24% and the activity at 48h was increased by 80.03%,143.32%,218.66%.CONCLUSION:Asparagus saponins can increase Caspase-9 and Caspase-3 activity in HepG2, and induce cell apoptosis via caspase dependent mitochondrial pathway. Asparagus officinalis had anti-tumor effect, the active component and the mechanism as follows:1. Asparagus polysaccharide can improve immune function of S180 mice:①Asparagus polysaccharide can increase CR1 number, increase RBC-C3bRR and DTER, enhance the regulation of erythrocyte CR1 on immunological reaction of lymphocyte.②Asparagus polysaccharide can increase band 3 protein and glycophorin A content on erythrocyte membrane, increase phospholipids content and decrease cholesterol content, increase sialic acid content, adjust membrane fluidity.③Asparagus polysaccharide can decrease erythrocyte fluorescence intensity and lower erythrocyte membrane potential.④Asparagus polysaccharide can increase erythrocyte Na+,K+-ATPase, Ca2+,Mg2+-ATPase activity, lower [Ca2+] concentration, improve cation channel transport activity; lower [CI-] concentration, improve anion channel transport activity; lower [H+] concentration, increase intra-cellular pH, maintain acid-base balance.⑤Asparagus polysaccharide can shorten erythrocyte migration time, increase erythrocyte complex mobility.2. Asparagus saponins can induce HepG2 cell apoptosis via mitochondrial pathway:①Cell morphology under fluorescence microscope and transmission electron microscope showed Asparagus saponins can induce HepG2 cell apoptosis, and apoptotic bodies appear. Asparagus saponins can change cell cycle and induce S phase arrest, increase the ratio of cells in S phase and decrease in G2/M phase.②Asparagus saponins can increase intracellular ROS level, increase intracellular Ca2+ level, decrease intracellular pH, so induce HepG2 cell apoptosis.③Asparagus saponins can open mitochondrial permeability transition pore, turn on the death switch and decrease mitochondrial membrane potential, then induce cell apoptosis via un-reversible mitochondrial pathway.④Asparagus saponins can adjust apoptosis related protein expression in HepG2 and induce cell apoptosis. It increases intracellular Cyt-C, activated Caspase-9 and Caspase-3, Bax expression, decreases intracellular Bcl-2 expression.⑤Asparagus saponins can increase Caspase-9 and Caspase-3 activity in HepG2 in a dosage and time dependent manner, and induce cell apoptosis via caspase dependent mitochondrial pathway.更多还原