苦马豆素诱导大鼠BRL-3A细胞凋亡机理的研究

发布时间：2018-06-22 02:59

本文选题：苦马豆素 + BRL-3A　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：苦马豆素(Swainsonine,SW)为分布于中国西部草地豆科黄芪属和棘豆属等有毒植物的主要毒性成分。家畜采食疯草后,容易出现典型的疯草中毒症状,全身各组织器官细胞出现广泛的空泡变性。前期苦马豆素致BRL-3A细胞毒性损伤的研究表明,苦马豆素能够引起肝细胞的存活率降低,肝细胞形态严重损害及毒性损伤标志酶释放,最终造成肝细胞死亡。但其作用机制还不清楚。为探索苦马豆素抑制细胞存活率及造成细胞死亡的机制,本论文以大鼠BRL-3A细胞系为研究对象,进行苦马豆素诱导细胞凋亡机理的研究,取得以下结果。1.苦马豆素诱导BRL-3A细胞凋亡的细胞核形态学检测:用不同浓度的苦马豆素(700,900和1100μg/mL)攻毒BRL-3A细胞48h及900μg/mL苦马豆素攻毒BRL-3A细胞不同时间(24,48和72h)后,Hoechst 33258对细胞核染色,在荧光显微镜下观察细胞核的形态学变化。结果显示,与对照组相比,苦马豆素可造成细胞核染色质固缩、染色加深、细胞核碎裂等典型的凋亡特征,且细胞出现凋亡呈现时间和剂量依赖性。该试验从形态学的角度说明苦马豆素能够诱导BRL-3A细胞凋亡。2.苦马豆素诱导BRL-3A细胞凋亡的Annexin V-FITC/PI双染检测:用不同浓度的苦马豆素(700,900和1100μg/mL)攻毒BRL-3A细胞48h及900μg/mL苦马豆素攻毒BRL-3A细胞不同时间(24,48和72h)后,Annexin V-FITC/PI双染,采用流式细胞术检测细胞凋亡率的变化。结果显示,与对照组相比,苦马豆素造成BRL-3A细胞凋亡率差异显著或极显著地上升(P㩳0.05或P㩳0.01),并呈时间-剂量依赖性;同时,苦马豆素能够减少正常细胞的比重,差异显著或极显著(P㩳0.05或P㩳0.01);结果表明,苦马豆素能够显著诱导正常细胞凋亡,呈时间-剂量依赖性。该试验从生物化学的角度说明苦马豆素能够诱导BRL-3A细胞凋亡。3.苦马豆素诱导BRL-3A细胞凋亡相关蛋白表达水平的检测:用不同浓度的苦马豆素(700,900和1100μg/mL)攻毒BRL-3A细胞48h及900μg/mL苦马豆素攻毒BRL-3A细胞不同时间(24,48和72h)后,Western blot检测细胞中凋亡相关蛋白的表达情况。结果显示,与对照组相比,细胞中caspase 3,8和9的表达量逐渐上升,procaspase 8的表达量逐渐下降,Fas和FasL的表达量及Bax/Bcl-2的值逐渐上升,表现出差异显著(P㩳0.05),并呈现时间或剂量依赖性;结果表明,苦马豆素能够诱导BRL-3A细胞凋亡,同时,死亡受体通路和线粒体通路共同参与苦马豆素诱导BRL-3A的细胞凋亡。
[Abstract]:Swainsonine SW is the main toxic component of the genus Astragalus and Echinoides in the grassland of western China. After feeding on the wild grass, the animals are prone to the typical toxic symptoms of the wild grass and the extensive vacuolar degeneration of the tissues and organs of the whole body. The previous studies on the cytotoxic injury of BrL-3A cells induced by krisbanin showed that the survival rate of hepatocytes was decreased, the morphological damage of hepatocytes and the release of toxic damage marker enzymes were induced, which resulted in the death of hepatocytes. However, the mechanism of its action is not clear. In order to explore the mechanism of the inhibition of cell survival and cell death induced by krisbanin in rat BRL-3A cell line, the mechanism of cell apoptosis induced by krisbanin was studied in this paper, and the following results were obtained. 1. Nuclear morphology of BRL-3A cells induced by krisbanin: Hoechst 33258 was stained at different concentrations (700900 渭 g / mL and 1100 渭 g / mL) for 48 h and 900 渭 g / mL for BRL-3A cells (24448 and 72 h) after treatment with different concentrations of krisbanin (700900 渭 g / mL and 1100 渭 g / mL), and then the cells were exposed to different concentrations of kaliotocin (700900 渭 g / mL and 1100 渭 g / mL) for 48 h and 900 渭 g / mL respectively. The morphological changes of nucleus were observed under fluorescence microscope. The results showed that, compared with the control group, the canonical apoptotic characteristics of the cells were characterized by chromatin condensation, deep staining and nuclear fragmentation, and the apoptosis appeared in a time-and dose-dependent manner. From the morphological point of view, the experiment showed that kariotin could induce apoptosis of BRL-3A cells. 2. Annexin V-FITC / Pi double staining for apoptosis of BRL-3A cells induced by krisbanin: annexin V-FITC / Pi double staining was applied to BRL-3A cells at different concentrations (700900 渭 g / mL and 1100 渭 g / mL) for 48 h and 900 渭 g / mL (24 h, 48 h and 72 h), respectively. Apoptosis rate was detected by flow cytometry. The results showed that compared with the control group, the apoptosis rate of BRL-3A cells increased significantly or extremely significantly (P0. 05 or P0. 01). The difference was significant (P0. 05 or P0. 01). The results showed that the apoptosis of normal cells was induced in a time-dose-dependent manner. From the biochemistry point of view, the experiment showed that kariotin could induce apoptosis of BRL-3A cells. 3. Detection of apoptosis-related protein expression in BRL-3A cells induced by krisbanin: Western blot was used to detect apoptosis in BRL-3A cells at different concentrations (700900 and 1100 渭 g / mL) for 48 h and 900 渭 g / mL respectively. Expression of related proteins. The results showed that, compared with the control group, the expression of caspase 3O 8 and 9 increased gradually, the expression of procaspase 8 decreased gradually, and the expression of Bax / Bcl-2 increased gradually, showing a significant difference (P0. 05), and presented a time or dose dependent manner. The results showed that the apoptosis of BRL-3A cells could be induced by krisbanin, and the death receptor pathway and mitochondrial pathway were involved in the apoptosis of BRL-3A cells.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.82

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