【作者】 杨煌建；

【导师】 瞿伟菁；

【作者基本信息】 华东师范大学 ， 植物学， 2006， 硕士

【摘要】 蒺藜（Tribulus terrestris L．）为入载我国药典的传统中药，主要药用部位的药理药效研究甚多，而对蒺藜全草入药的认识尚属起步。课题组前期发现蒺藜全草皂苷（saponins from Tribulus terrestris L．简称STT）可有效降低高血糖病理状态下糖基化水平的工作基础，提示本文就STT干预糖基化终产物（Advanced Glycation End P roducts，简称AGEs）导致糖尿病肾病恶性病变的潜在效应进行深入追踪，为此拟以人肾癌细胞786-0为靶细胞，从细胞水平和分子水平层面上展开STT对肾癌的体外抑制效应及其作用机制的研究。首先采用MTT法、SRB法观察了STT体外对于786-0肾癌细胞生长的抑制作用，并比较了STT对人胚胎肾细胞体外生长的影响，结果显示：25～100μg／ml的STT对于786-0细胞具有显著的抑制作用和明显的剂量依赖关系；25～200μg／ml的STT对于人胚胎肾细胞的毒作用明显小于对786-0的细胞毒作用。为了进一步探明STT的抑癌机制，研究采用wright染色法、吖啶橙染色法和透射电子显微镜观察了STT作用肿瘤细胞后细胞形态和超微结构的变化；利用琼脂糖凝胶电泳和二苯胺法测定DNA的片断化；应用碘化丙啶（PI）染色的流式细胞术法检测了STT对786-0细胞周期的影响；结合免疫荧光标记，以流式细胞术检测STT对786-0细胞周期蛋白表达的影响和STT作用后细胞抗细胞凋亡蛋白Bcl-2含量及细胞内钙离子浓度的变化、线粒体膜电位的差异。结果显示：①786-0细胞经STT作用后呈早期凋亡特征：细胞变小皱缩，核固缩、浓聚，核仁裂解，染色质凝集边集；但琼脂糖凝胶电泳及流式细胞术结果表明，STT致786-0细胞凋亡率不高。②STT通过下调786-0细胞的cyclin D1蛋白水平表达，从而影响786-0细胞周期调控，即下调G₀／G₁期细胞比例，使细胞DNA的合成受到阻滞；G₂／M期细胞比例减少，可减少细胞有丝分裂。③抗凋亡蛋白Bcl-2的表达下调是STT抑癌作用的分子机理之一。④STT能够引起786-0细胞线粒体跨膜电位丧失，激活线粒体通路，继而诱导786-0走向凋亡。⑤STT可显著升高细胞内Ca²⁺水平，同时细胞内外Ca²⁺水平的改变均可增强STT对786-0细胞的杀伤作用，在培养基中加入2m mol／L CaCl₂可显著抑制STT所致786-0细胞的DNA片段化，而EGTA或ZnSO₄不影响STT诱导786-0细胞DNA片段化程度，提示STT可激活钙离子依赖性核酸内切酶（如DNaseⅠ或DNaseⅩ等），引起一系列的生化反应。药效物质基础的含量稳定可靠并有严格的质量标准，是中药现代化的一个重要环节。为此，针对STT在相关适应症存在有效剂量不稳，利用本实验室建立的反相高效液相色谱法[Kromasil C18（250mm×4.6mm，5um）和示差折光检测结合的技术支撑，采用100％甲醇洗脱，流速为1mL／min]，分析不同产地、不同保存时间对STT主要甾体皂苷元成分的影响，结果发现生药和提取物不同保存时间对STT的HPLC图谱和抑癌生物学活性均存在差异，STT单体药效学追踪发现，海柯皂苷元是STT体外抗肿瘤作用的主要物质基础之一。已经共识，中药所含微量元素亦是中药药效物质基础的重要组成部分。研究根据中药配位化学学说的理论指导，针对蒺藜全草资源在该领域的信息盲点，结合本实验室的六种材料提取物对AGEs形成的干预作用，运用模糊聚类分析探讨这几种材料中微量元素与其药效的相关性。采用电感耦合等离子体发射光谱仪（Inductively Coupled Plasma—Atomic Emission Spectrometry，简称ICP—AES）测定铜、镁、锌、锰、铬、钼、钒的含量，结合锌／铜及锌／锰比值，以数据极差法进行统计指标的标准化处理；以数量积法算出被分类对象之间的相关系数，确定论域上的模糊矩阵进行聚类分析。研究结果显示：几种材料对AGEs的形成有抑制作用，刺梨黄酮和沙棘果渣黄酮的作用效果接近；相关系数λ=0.75时，上述两种物质聚类相似。STT对AGEs的形成抑制作用远不如其它材料，只在GXL体系中有效果，但在体外抗肿瘤作用上蒺藜提取物明显强于其它材料。STT分离的两种皂苷元含有羟基等配位基团，与微量元素可能存在配位效应，STT中所含有机成分、微量元素含量和种类与抑制786-0细胞增殖效应、干预AGEs形成的作用等密切相关。更多还原

【Abstract】 Tribulus terrestris L. has been recorded in the Pharmacopoeia of China as a traditional Chinese herb. There have been many studies on pharmacology of the fruit so far, but it is still unclear on the pharmacal use of the whole plant as traditional medicine is fruit. Based on our former research about Tribulus terrestris L., the objective of this study is to investigate the effect of sapomns from Tribulus terrestris L （STT） on the renal cancer cell line （786-0） in vitro, which may be caused by diabetic nephropathy, and its mechanisms.At first, the anti-tumor effect of STT was to be evaluated by MTT and SRB assay. By using the two methods we found that the STT showed a significant inhibitory effect on 786-0 which was in dose depentment manner. Then we compared the cytotoxic effect of STT on 786-0 with that on human normal kidney cell line （HEK-293）, it was observed that STT had less inhibitory effect on the proliferation of HEK-293.In order to further investigate STT induced-apoptosis on 786-0, several methods of morphological observation were used, such as Wright staining, acridine orange staining and electron microscope, the results showed that the morphological changes were induced by STT with apoptosis on 786-0, but the DNA Ladder wasn’t tested because the rate of apoptosis was very low. In addition, the effects of STT on the cell cycle, the expression of Bcl-2 protein, the content of Ca²⁺ and the mitochondrial membrane potential（△ψm） in the 786-0 were determined respectively by flow cytometry（FCM）. After the 786-0 were treated by STT, it was found that: 1) The distribution of 786-0 on G₀/G₁ and G₂/M phase was decreased; 2) The expressions of Bcl-2 protein and cycle D1 fell; 3) The△ψm was disrupted; 4) The intracellular Ca²⁺ content was increased. What’s more, adding 2 m mol/L Ca²⁺, or 1m mol/L EGTA, or 250κmol/L Zn²⁺ to the medium, the cytotoxicity of STT against 786-0 was enhanced, whereas the DNA fragment induction by STT was affected only with 2 m mol/L Ca²⁺. It was speculated that the effect of STT on 786-0 was possibly catalyzed by Ca²⁺-independent DNase. To sum up, STT can significantly inhibit the growth of 786-O in vitro, partially, by apoptosis, which was related to the change of cell cycle, the Bcl-2 protein expression, the intracellular Ca²⁺ content and the potential of mitochondrial membrane.Keeping the basal effective content stable and setting down a strict quality standard are important for Chinese herb modernizing. The HPLC-fingerprinting of the main saponins from Tribulus terrestris L. stored in different periods and their cytotoxicity on 786-O were tested respectively by RP-HPLC [Kromasil C18 analytical column（250mm×4.6mm, 5um）, 100％methanol as eluent with flow rate: 1mL/min] and MTT method. The discipline of changes is found during storage: 1) The determination of main saponins from Tribulus terrestris L was decreased; 2) The cytotoxic effect of STT on 786-O was weakened. In short, the period of storage can affect the determination of main saponins from Tribulus terrestris L. and its effect on cancer cell; partially, the determination of Hecogenin can influence the effect.It was well known that the trace elements are effective components of Chinese herbs. In this study, we tried to discuss the interrelation among trace elements, organic compounds and the effect on AGEs in several plant extracts with method of blurred classification. To begin with, we prepared AGEs with glyoxal, fructose or glucose, and added the plant extracts to test the effect. Secondly, we used Inductively Coupled Plasma—Atomic Emission Spectrometry to determine the contents of seven trace elements, Zn、Mg、Mn、Mo、Cu、Cr、V. By range analysis, correlation coefficients and relative fuzzy matrix were calculated to combine with Zn/Cu and Zn/Mn. It was found that most of the components can inhibit the formation of AGEs, especially, the effect of FFH was close to FRR, cluster in the two plant extracts was similar when relative coefficient is 0.75. The finding of this study suggested that trace elements and organic compounds of STT had much to do with the formation of AGEs and its inhibitory effect on 786-O.更多还原