【作者】 孙丽华；

【导师】 吉爱国；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2013， 硕士

【摘要】 岩藻聚糖硫酸酯是一种普遍存在于褐藻细胞壁基质和棘皮动物体中的水溶性硫酸杂多糖。它具有多种生物活性,例如抗肿瘤、抗凝血等等。最近研究表明岩藻聚糖硫酸酯可以诱导人淋巴癌细胞、人结肠癌细胞、人乳腺癌细胞等的凋亡,从而发挥抗肿瘤作用。有文献报道岩藻聚糖硫酸酯可以诱导人结肠癌细胞HT-29发生凋亡,本实验在观察岩藻聚糖硫酸酯诱导HT-29细胞凋亡时发现存在自噬现象。因此本实验研究了岩藻聚糖硫酸酯诱导HT-29凋亡和自噬的关系。论文的主要内容就结果如下：1.岩藻聚糖硫酸酯诱导HT-29凋亡MTT结果显示岩藻聚糖硫酸酯可以显著地抑制HT-29的存活。倒置显微镜下观察发现,岩藻聚糖硫酸酯作用后,HT-29增殖缓慢,细胞皱缩、体积缩小、细胞间隙增大,细胞内空泡增多。通过测定培养基中LDH的含量检测细胞膜的完整性,结果表明的释放量并没有明显增加,只有高浓度岩藻聚糖硫酸酯作用48h后,LDH的释放量明显增加。Hoechst33342染色荧光显微镜下观察结果显不,400μg/mL岩藻聚糖硫酸酯作用48h后,HT-29细胞部分呈致密浓染或呈碎块状致密浓染。流式细胞仪检测线粒体膜电位,结果表明岩藻聚糖硫酸酯作用24h时,线粒体膜电位明显降低。本实验利用分光光度法检测细胞内caspase-3、 caspase-8及caspase-9的活性,结果显示岩藻聚糖硫酸酯作用于HT-29细胞后,caspase-3、caspase-8和caspase-9的活性明显升高。Annexin V-FITC/PI双染的流式检测,岩藻聚糖硫酸酯作用于HT-29后凋亡率明显增高。Western blot结果表明岩藻聚糖硫酸酯作用于HT-29细胞后,Bcl-2的表达量减少而Bax的表达量增多。一系列的实验结果表明,岩藻聚糖硫酸酯可诱导人结肠癌细胞HT-29发生凋亡,膜受体途径和线粒体途径在此凋亡中都发挥了重要作用；岩藻聚糖硫酸酯可以下调HT-29中Bcl-2的表达量和上调Bax的表达量,从而促进细胞凋亡2.岩藻聚糖硫酸酯诱导HT-29自噬MDC染色结果表明,岩藻聚糖硫酸酯作用于HT-29后,细胞内出现许多点状荧光颗粒,用自噬抑制剂3-MA预处理后,点状荧光颗粒明显减少。Western blot结果显示,岩藻聚糖硫酸酯作用于HT-29细胞后,LC3-Ⅰ向LC3-Ⅱ的转化明显升高,LC3-Ⅱ的表达量明显升高：用自噬抑制剂3-MA预处理后,LC3-Ⅰ向LC-Ⅱ转化明显降低,LC3-Ⅱ的表达量明显降低。由此可以推断岩藻聚糖硫酸酯可诱导人结肠癌细胞HT-29发生自噬。3.岩藻聚糖硫酸酯诱导HT-29自噬和凋亡的关系3-MA抑制自噬后,MTT结果显示岩藻聚糖硫酸酯对HT-29细胞的抑制活性明显增强,线粒体膜电位的下降程度明显增强,caspase-3、caspase-8、caspase-9活性明显升高,凋亡率明显升高。一系列的实验结果表明,自噬在岩藻聚糖硫酸酯诱导的HT-29细胞凋亡的过程中起保护细胞的作用。凋亡抑制剂Z-VAD-FMK抑制caspase依赖的凋亡后,MTT结果表明,抑制caspase诱导的凋亡后,自噬程度增强,引起了自噬性程序式细胞死亡。本实验研究结果表明自噬在岩藻聚糖硫酸酯诱导的HT-29细胞凋亡的过程中起保护细胞的作用,因此可以将自噬抑制剂与凋亡诱导剂结合来抑制肿瘤细胞。更多还原

【Abstract】 Fucoidan is a water-soluble sulfated polysaccharide found in brown algae and echinoderm; it has been shown to exhibit a number of biological effects, including anti-tumor effects. Recently, fucoidan has been report to exhibit anti-timors effects by inducing apoptosis in several cancer cell lines, including human lymphoma cells, human colon cancer cells and human breast cells. It has been reported that fucoidan could induce apoptosis of HT-29human colon cancer cells. In this study, we found the effects of fucoidan on autophagy in HT-29human colon cancer cells. Then we evaluated the effects of autophagy on apoptosis of fucoidan on HT-29. The main methods and results are as follows:1. Fucoidan induces apoptosis of HT-29human colon cancer cells.To evaluate the effects of fucoidan in HT-29cells viability and cytotoxicity, MTT and LDH assays were performed. Fucoidan induced a reduction in cell viability that was dose-and time-dependent. A significant increase in LDH release was only observed for fucoidan at high concentration after48hours of treatmet. To investigate the morphplogical alterations induced by fucoidan, HT-29were cultured with or without fucoidan and examined by microscopy and Hoechst staining. The presence of non-adherent cells, chromatin condensation and fragmentation were observed. To investigate the involvement of mitochondria in fucoidan treatment, it was analysed the mitochondrial transmembrane potential (ΔΨFm) by flow cytometry. A significant increase of ΔΨm loss was observed, when HT-29were cultured with fucoidan. For the apoptosis analysis, translocation of PS to the ourter surface of plasma membrane was evaluated by Annexin V-FITC binding. Fucoidan induced a significant increase in binding to Annexin V. Fucoidan induced a significant increase in caspase-3, caspase-8and caspase-9on HT-29. The presence of Bcl-2and Bax proteins, in cells treated with ficoidan was evaluated by immunobloting. A significant increase of Bax and reduction of Bcl-2were observed, when HT-29were cultured with fucoidan. All together the results obstained suggest that fucoidan induced apoptosis, when HT-29were cultured with fucoidan.2. Fucoidan induces aqutophagy of HT-29human colon cancer cells.In order to clarify the nature of the cytoplasmatic observed, MDC was used. MDC detects acid vesicular organelles, suggesting the occurrence of autophagy. MDC is protonated and accumulates, forming aggregates that fluorescence bright green. It was observed an increase in AVOs formation. The presence of LC3protein, an autophagic marker, in cells treated with ficoidan was evaluated by immunobloting. The autophagosome formation can be evaluated by the conversion of LC3-1to LC3-Ⅱ. Fucoidan-treated cells presented higher levels of LC3-Ⅱ than LC3-I confirming the presence of autophagosome.3. Effects of autophagic and apoptotic inhibitor in HT-29cells treated with fucoidan.As HT-29cells treated with fucoidan, presented autophagic features, like an increase of AVOs, the effects of the autophagic inhibitor3-methyladenine (3-MA) were investigated. When cells were treated with fucoidan plus3-MA, it was observed a significant reduction in cell viability when comparing to fucoidan without3-MA.3-MA induced a increase in the ΔΨFm loss of cells treated with fucoidan. In addition,-in our conditions,3-MA plus fucoidan induced a significant increase in caspase-3, caspase-8, caspase-9, when campared to fucoidan. By the analysis of Annexin V-FITC assay,3-MA plus fucoidan induced a significant increase, when campared to fucoidan. Thus, it appears that autophagy induced by fucoidan may act as a pro-survival process. Since it has been referred a cross-talk between apoptosis and autophagy, it was also studied the effect of Z-VAD-FMK in HT-29cells viability. When cells were treated with fucoidan plus Z-VAD-FMK, it was observed a significant increase in cell viability when comparing to fucoidan without Z-VAD-FMK, but a significant reduction in cell viability when comparing to without fucoidan.Different interactions between apoptosis and autophagy have been proposed. They may act as partners to induce efficient cell death in a coordinated or cooperative manner, but in this case, if one pathway of cell death is blocked the other assume or may only be activated if the other fails. Autophagy may act as an antagonist to block apoptotic cell death by promoting cell survival and stabilizing genome integrity. This study may also suggest the use of inhibitors of autophagy with fucoidan in a combination therapy to sensitize colon cancer cells to death, being a promising approach for the treatment of human colon cancers.更多还原