【作者】 马红樱；

【导师】 胡春香；

【作者基本信息】 西北师范大学 ， 植物学， 2005， 硕士

【摘要】 目的:荒漠藻在自身的正常代谢过程中,不断地向周围环境中分泌以多糖、蛋白糖为主的胞外聚合物。这些胞外聚合物是它们适应荒漠环境的生命支持系统,具有抗干旱、抗氧化、抗辐射、抗盐碱,及粘结沙粒等生态学功能。本文以皮肤癌细胞A431为供试细胞,研究了M. vaginatus, P. tenue, S. javanicum、Desmoloccus olvaceus和Nostoc sp.五种荒漠藻胞外聚合物对其细胞凋亡过程的影响,目的在于发现这些聚合物的抗肿瘤活性及其发挥作用的机理。方法:使用MTT法检测聚合物对A431细胞存活率的作用,用倒置显微镜直接观察A431细胞形态方面的变化,透射电镜再次揭示了细胞超微结构的改变;利用流式细胞仪分析了对A431细胞的存活率和细胞线粒体膜电位的变化,采用免疫组化法观察了caspase-3、bcl-2和bax蛋白的表达情况,利用琼脂糖凝胶电泳检测了细胞DNA的降解,并测定了A431细胞内超氧阴离子自由基（O₂^-）、SOD、CAT和POD酶活性的变化。结果:EPSs在体外能够杀死A431细胞,并且有剂量-时间-效应依赖关系,凋亡率随时间延长增加;EPSs会造成A431细胞在G₂/M期的停滞。线粒体膜电位的变化为:在作用24小时后,开始降低,到72小时彻底崩溃,96小时线粒体破裂。免疫组化结果显示EPSs能促进caspase-3和bax蛋白的表达,而抑制bcl-2蛋白的表达。96小时后,DNA开始降解成小片段。形态结构的变化如下:A431细胞经EPSs处理后,首先细胞变小成圆形或月牙形,胞间连接消失;超微结构显示细胞表面微绒毛消失,有“胞浆出芽现象”。细胞核凝集,染色质固缩、边缘化,形成不同形状和大小的块状;最后核膜破裂,凝聚的染色质散布到细胞浆中,没有发现凋亡小体的形成。在凋亡过程中,细胞自由基含量升高,抗氧化酶也随着发生了变化。推论:荒漠藻胞外聚合物具有明显的抗肿瘤活性。在A431细胞的凋亡过程中,胞外聚合物引起的细胞周期阻滞和线粒体膜电位降低可能是细胞凋亡的主要原因,其诱发因素很可能是自由基参与活化的caspase-3和bax下调了bcl-2的表达,使得细胞内抗氧化系统失去平衡,最后导致了细胞凋亡。更多还原

【Abstract】 Aim: Algae from desert environment are known to excrete amount of extracellular polymeric substances (EPSs) into the surrounding matrix when they grow. These EPSs are life support system, play important roles in protecting them from drought, oxidation, salt and radiation. And they are also key substance of gluing soil grains together during the formation process of algal crusts. The object of this study is to further investigate their function in antitumor and possible mechanism. Methods: Microcoleus vaginatus, Phormidium tenue, Scytonema javanicum, Desmoloccus olivaceus and Nostoc sp. are dominant species of algal crusts of Shapotou area, Ningxia Hui Autonomous Region of China. They have different cohesion in stabilization fine sand grains. Their EPSs were extracted, separated and purified. The skin cancer cell A431 was bought from Cell Collection Centre of Wuhan University. After culture and exposure to these EPSs, survival rate of A432 cell proliferation was measured by MTT assay. The cell cycle and membrane potential of mitochondrion (△Ψm) was determined by flow cytometry. The changes of cell morpha and ultrastructure were observed under inverted light microscope and transmission electron microscope. DNA laddering of apoptosis was performed by agarose gel electrophoresis. The expression of caspase-3、 bcl-2 and bax were analyzed by immunohistochemistry, and antioxidation system including content of free radicals (O2), enzyme activity of SOD, CAT and POD were measured as well. Results: The EPSs from the five species all could kill A431 cancer cell, and at the dose-time-dependent pattern. Incubation of A431 cell with EPSs caused a G2/M stagnation in cell cycle progression. Membrane potential of mitochondria began fall after exposure to EPSs 24 h, whole mitochondria collapsed after 72h, broken after 96h. And all EPSs facilitated the expression of caspase-3 and bax protein, restrained the expression of bcl-2. So the result of immunohistochemistry further confirmed the apotpsis by EPSs induced. Agarose gel electrophoresis further showed DNA degrade into small fragments as laddering after 96 h. Light microscope showed the apoptosis process was cell diminish in size or becoming round or crescent shape first, then cell-cell juncture disappeared. TEM showed villus of cell surface disappeared first, cell membrane blebbing , and then nucleous aggregated, chromatin condensetion, andedged. Finally karyotheca broke, the aggregated chromatin spread into cytoplasm. During the whole process there were not apoptosis body formation. This apoptosis process was similar to most tumor cells induced by other anti-tumor agents. The content of free radicals increased after exposure to EPSs, and antioxidant enzyme activities also showed obviously changes. Immunohistochemistry assay showed that EPSs treatment up-regulated the expression of caspase-3 and bax protein, down-regulated that of bcl-2 protein. Conclusion: The EPSs had obvious antitumor activity, the stagnation of cell cycle and decrease of membrane potential of mitochondria might be the main reason of tumor cell apoptosis. The apoptosis process was closely related to content of free radicals, because the free radicals participated in the up-regulation of caspase-3 and bax and down-regulation of bcl-2. So the mechanism of apoptosis of cancer cell might be the reason was because of lost balance of antioxidant system.更多还原