葫芦巴碱诱导烟草Bright Yellow-2细胞凋亡及其分子机制研究
摘要
葫芦巴碱(Trigonelline,TRG)是从豆科植物葫芦巴(Trigonellafoenum-graecum L.)的干燥种子中分离的一种生物碱,它不仅存在于葫芦巴属植物,而且广泛存在于其它植物中。葫芦巴碱是尼克酸和尼克酰胺的代谢产物,与NADP+/NADPH和NAD+/NADH的代谢循环密切相关。在外界特殊环境下NADP+/NADP和NAD+/NADH积累过多后,其中一条代谢途径就会引起葫芦巴碱增加,葫芦巴碱进而作为信号分子作用于呼吸链引起细胞凋亡。为了证明这种假说,本文以外源葫芦巴碱为供试材料,以烟草Bright Yellow-2(BY-2)悬浮细胞为实验材料,探讨了葫芦巴碱诱导烟草细胞凋亡的形态变化与生化特征,初步探索了细胞凋亡发生的机制,得到以下几方面的结果：
1.烟草BY-2悬浮细胞的生长受转速、光照、温度、培养液、传代的体积比、传代时间等因素的影响。研究发现,振荡培养的速度为130rpm、暗培养和25±2℃为最适合烟草BY-2悬浮细胞生长的条件。悬浮细胞培养液采用MS改良后的培养液,另加2mg/L的2,4-D激素能为细胞生长提供良好的营养。细胞的传代体积比为1：4,每隔7天传代一次,可以保证细胞汲取新的营养物质以及避免大的细胞团释放有毒物质对新生细胞毒害。通过对细胞鲜重、干重和FDA染色活力检测,发现细胞生长在3-4天时处于对数期,此时的细胞最稳定、活性强可以用于进一步的实验。
2.TRG诱导的烟草细胞死亡表现为细胞凋亡的典型特征。20mmol/LTRG可诱导烟草BY-2细胞凋亡,处理72小时后Trypan blue染色,可观察到细胞膜皱缩与细胞壁间有空隙,类似于质壁分离的现象,细胞核凝集甚至细胞器不完整。Hoechst33258/PI双染色经荧光显微镜检测观察到细胞核染色质固缩,边缘化,空泡化。处理108小时提取其基因组DNA进行琼脂糖凝胶电泳,观察到DNA Ladder现象,表明DNA在核小体间发生断裂；TUNEL检测可观察到较强的阳性信号,进一步表明其细胞核内发生了DNA3'-OH末端断裂；TUNEL/Hoechst33258双染观察到与Hoechst33258/PI双染相同的形态学特征。以上结果表明TRG可以诱导BY-2烟草悬浮细胞发生细胞程序性死亡。
3.探讨了TRG诱导烟草细胞凋亡的机制。经TRG处理后烟草细胞内发生了活性氧迸发,TRG处理8小时内,胞内的H202出现两个峰值,第一个峰值出现在处理后0.25h,第二个峰值出现在处理后6小时,且第二个峰值大于第一个峰值；TRG处理后检测到胞外的H202也分别在0.25小时和1小时达到积累的高峰,而后H202达到了相对稳定的状态；另外,在TRG处理诱导活性氧(ROS)的积累动态变化过程中发现,TRG处理12小时后检测ROS的迸发量最高,此后直到120小时ROS的含量相对稳定,说明在烟草细胞凋亡过程中H202和ROS都参加了细胞凋亡的信号转导途径；与此同时在TRG诱导了烟草细胞凋亡过程中30分钟内就检测到NO的产生,到2.5小时时发生了NO迸发,在6小时时检测到得NO含量达到最大值,此变化趋势基本与H202相同,表明NO与H202协同作用促进了细胞凋亡；为明确氧化反应在烟草细胞死亡中的作用,进一步用抗氧化剂抗坏血酸盐((+)-Sodium L-ascorbate简称AsA)对葫芦巴碱诱导的烟草细胞凋亡进行了分析,结果发现用AsA预处理后加入TRG,从AsA抑制TRG诱导细胞凋亡率的变化图可以看出AsA对抑制TRG诱导细胞凋亡有显著效果。随着时间的延长AsA抑制TRG诱导细胞凋亡的比率也相应增加,由开始的抑制率6.84%增加到96h的抑制率16.14%,从推迟凋亡的速度上讲,AsA预处理后可使细胞发生凋亡的时间延缓20小时左右。AsA预处理后加入TRG诱导凋亡,同时检测ROS,发现AsA+TRG组处理释放的ROS比TRG单独处理释放的ROS要少,由此可见AsA可以抑制TRG诱导细胞凋亡过程中释放ROS,从而抑制凋亡的产生。另外葫芦巴碱诱导凋亡的过程中细胞培养液的pH值先变碱而后变酸。
Trigonelline (Trigonelline, TRG) is an alkaloid separed from dry seeds of a leguminous plant, fenugreek (Trigonellafoenum-graecum L.). Trigonelline is not only accumulates in the leguminous plants but also widely in other plants. Trigonelline is the metabolic products of niacin and nicotinamide, and is closely related to the metabolic cycle of NADP+/NADPH and NAD+/NADH. In special external environmental condition, NADP+/NADPH and NAD+/NADH often accumulate in cytoplasm of living cell, one of which metabolic pathway will cause trigonelline increases and then as the signal molecules to induce apoptosis in the respiratory chain.To prove this hypothesis, taking tobacco BY-2 suspension cells as the test materials, morphological and biochemical characteristics of PCD induced by trigonelline were investigated. Also the mechanisms of PCD in tobacco cells induced by trigonelline were analyzed.The main results were as follows:
1. Growth of tobacco BY-2 suspension cells was affected by the rotate speed, light, temperature, culture medium, the volume ratio of passage, cycle of passage and other factors. It was found that the optima culture conditions of tobacco BY-2 suspension cells were in speed of 130rpm with dark culture and temperature 25±2℃. Suspension cells was cultured in MS liquid medium, and 2 mg/L 2,4-D plant hormone added to MS liquid medium could provide good nutrition to cells. Cell passage with the volume ratio of 1:4 and generation time with every 7days could provide new cells nutrition to avoid cell aggregate releases of toxic substances to the newborn cells. Cell growth in 3-4 days in the log phase was found according to testing cell fresh weight, dry weight and vigor test of FDA staining, and during this phase cells grew very well, and were in the condition of stable and strong activity. It suggested test cells can be used for further experiments.
2. Programmed cell death (PCD) in tobacco cells induced by TRG showed the typical features of apoptosis. Concentration of TRG in 20mmol/L subjecte to tobacco Bright Yellow-2 (BY-2) cells after 72hour treatment added Trypan blue staine cells. In the early phase, dead cells showed that the cell membrane retracts and moves away from the cell wall leaving a visible gap.In the later phase the cytoplasm shrinkage, cell nucleus agglutination or organelles incomplete.
Hoechst33258/PI staining was used to observed nuclear chromatin condensation, marginali-zation, and vacuolization by fluorescence microscopy. Treated with TRG 108 hour, extracted genomic DNA on agarose gel electrophoresis, DNA Ladder phenomenon was observed, indicating that DNA fracture between the nucleosome; TUNEL detection observed a strong positive signal, and indicated that cell nucleus takes place fracture in the end of DNA 3 '-OH; the results of TUNEL/Hoechst33258 stained is the same morphology aspects as Hoechst33258/PI stained. These results suggest that the TRG can induce tobacco BY-2 suspension cells occurred programmed cell death.
3. In this paper we discussed the mechanism of apoptosis induced by TRG in tobacco cells. After TRG treatment in tobacco cells occurred the oxidative burst, the kinetics of intracellular H2O2 burst within 8 hour treatment was biphasic:first phase peaked at 0.25 hour and second peaked at 6hour post-treatment,and second peak higer than first peak; TRG treatment to detect ed that extracellular H2O2 were also reached peak at 0.25 hour and 1h, then H2O2 accumulation to achieve a relatively stable state.In addition, we found that TRG treatment induced accumulation of ROS dynamics changes in the BY-2 cells, ROS burst within 12 hour treatment, then relatively stable until the 120hour. It indicated that H2O2 and ROS participated in the apoptosis signaling pathway in tobacco cell apoptosis; and we detected NO production within 30 min with TRG treatment in tobacco cells during apoptosis process, the NO burst at 2.5 hour, then detected NO concentration have reached the maximum at 6 hour. kinetics of NO is same as H2O2, It indicated that NO and H2O2 synergy promotes apoptosis.Also we use anti-oxidant ascorbic acid salt to test the correlation of oxidation with PCD.we found that the rate of death of suspension cell was greatly reduced after the cells were treated by ascorbic acid salt before the TRG induced the cells, With the time longer AsA inhibited the rate of apoptosis become increase from the 6.84% increase to 16.14% at 96hour, and delay the time of PCD to 20 hours. AsA pretreatment 30 min added TRG induced apoptosis, we detected ROS of AsA + TRG group found less than TRG group, we can see that AsA inhibited ROS release during apoptosis induced by TRG, thus inhibition the produce of apoptosis. Also during apoptosis induced by trigonelline pH values of cell culture fluid change alkaline to sour.
1.烟草BY-2悬浮细胞的生长受转速、光照、温度、培养液、传代的体积比、传代时间等因素的影响。研究发现,振荡培养的速度为130rpm、暗培养和25±2℃为最适合烟草BY-2悬浮细胞生长的条件。悬浮细胞培养液采用MS改良后的培养液,另加2mg/L的2,4-D激素能为细胞生长提供良好的营养。细胞的传代体积比为1：4,每隔7天传代一次,可以保证细胞汲取新的营养物质以及避免大的细胞团释放有毒物质对新生细胞毒害。通过对细胞鲜重、干重和FDA染色活力检测,发现细胞生长在3-4天时处于对数期,此时的细胞最稳定、活性强可以用于进一步的实验。
2.TRG诱导的烟草细胞死亡表现为细胞凋亡的典型特征。20mmol/LTRG可诱导烟草BY-2细胞凋亡,处理72小时后Trypan blue染色,可观察到细胞膜皱缩与细胞壁间有空隙,类似于质壁分离的现象,细胞核凝集甚至细胞器不完整。Hoechst33258/PI双染色经荧光显微镜检测观察到细胞核染色质固缩,边缘化,空泡化。处理108小时提取其基因组DNA进行琼脂糖凝胶电泳,观察到DNA Ladder现象,表明DNA在核小体间发生断裂；TUNEL检测可观察到较强的阳性信号,进一步表明其细胞核内发生了DNA3'-OH末端断裂；TUNEL/Hoechst33258双染观察到与Hoechst33258/PI双染相同的形态学特征。以上结果表明TRG可以诱导BY-2烟草悬浮细胞发生细胞程序性死亡。
3.探讨了TRG诱导烟草细胞凋亡的机制。经TRG处理后烟草细胞内发生了活性氧迸发,TRG处理8小时内,胞内的H202出现两个峰值,第一个峰值出现在处理后0.25h,第二个峰值出现在处理后6小时,且第二个峰值大于第一个峰值；TRG处理后检测到胞外的H202也分别在0.25小时和1小时达到积累的高峰,而后H202达到了相对稳定的状态；另外,在TRG处理诱导活性氧(ROS)的积累动态变化过程中发现,TRG处理12小时后检测ROS的迸发量最高,此后直到120小时ROS的含量相对稳定,说明在烟草细胞凋亡过程中H202和ROS都参加了细胞凋亡的信号转导途径；与此同时在TRG诱导了烟草细胞凋亡过程中30分钟内就检测到NO的产生,到2.5小时时发生了NO迸发,在6小时时检测到得NO含量达到最大值,此变化趋势基本与H202相同,表明NO与H202协同作用促进了细胞凋亡；为明确氧化反应在烟草细胞死亡中的作用,进一步用抗氧化剂抗坏血酸盐((+)-Sodium L-ascorbate简称AsA)对葫芦巴碱诱导的烟草细胞凋亡进行了分析,结果发现用AsA预处理后加入TRG,从AsA抑制TRG诱导细胞凋亡率的变化图可以看出AsA对抑制TRG诱导细胞凋亡有显著效果。随着时间的延长AsA抑制TRG诱导细胞凋亡的比率也相应增加,由开始的抑制率6.84%增加到96h的抑制率16.14%,从推迟凋亡的速度上讲,AsA预处理后可使细胞发生凋亡的时间延缓20小时左右。AsA预处理后加入TRG诱导凋亡,同时检测ROS,发现AsA+TRG组处理释放的ROS比TRG单独处理释放的ROS要少,由此可见AsA可以抑制TRG诱导细胞凋亡过程中释放ROS,从而抑制凋亡的产生。另外葫芦巴碱诱导凋亡的过程中细胞培养液的pH值先变碱而后变酸。
Trigonelline (Trigonelline, TRG) is an alkaloid separed from dry seeds of a leguminous plant, fenugreek (Trigonellafoenum-graecum L.). Trigonelline is not only accumulates in the leguminous plants but also widely in other plants. Trigonelline is the metabolic products of niacin and nicotinamide, and is closely related to the metabolic cycle of NADP+/NADPH and NAD+/NADH. In special external environmental condition, NADP+/NADPH and NAD+/NADH often accumulate in cytoplasm of living cell, one of which metabolic pathway will cause trigonelline increases and then as the signal molecules to induce apoptosis in the respiratory chain.To prove this hypothesis, taking tobacco BY-2 suspension cells as the test materials, morphological and biochemical characteristics of PCD induced by trigonelline were investigated. Also the mechanisms of PCD in tobacco cells induced by trigonelline were analyzed.The main results were as follows:
1. Growth of tobacco BY-2 suspension cells was affected by the rotate speed, light, temperature, culture medium, the volume ratio of passage, cycle of passage and other factors. It was found that the optima culture conditions of tobacco BY-2 suspension cells were in speed of 130rpm with dark culture and temperature 25±2℃. Suspension cells was cultured in MS liquid medium, and 2 mg/L 2,4-D plant hormone added to MS liquid medium could provide good nutrition to cells. Cell passage with the volume ratio of 1:4 and generation time with every 7days could provide new cells nutrition to avoid cell aggregate releases of toxic substances to the newborn cells. Cell growth in 3-4 days in the log phase was found according to testing cell fresh weight, dry weight and vigor test of FDA staining, and during this phase cells grew very well, and were in the condition of stable and strong activity. It suggested test cells can be used for further experiments.
2. Programmed cell death (PCD) in tobacco cells induced by TRG showed the typical features of apoptosis. Concentration of TRG in 20mmol/L subjecte to tobacco Bright Yellow-2 (BY-2) cells after 72hour treatment added Trypan blue staine cells. In the early phase, dead cells showed that the cell membrane retracts and moves away from the cell wall leaving a visible gap.In the later phase the cytoplasm shrinkage, cell nucleus agglutination or organelles incomplete.
Hoechst33258/PI staining was used to observed nuclear chromatin condensation, marginali-zation, and vacuolization by fluorescence microscopy. Treated with TRG 108 hour, extracted genomic DNA on agarose gel electrophoresis, DNA Ladder phenomenon was observed, indicating that DNA fracture between the nucleosome; TUNEL detection observed a strong positive signal, and indicated that cell nucleus takes place fracture in the end of DNA 3 '-OH; the results of TUNEL/Hoechst33258 stained is the same morphology aspects as Hoechst33258/PI stained. These results suggest that the TRG can induce tobacco BY-2 suspension cells occurred programmed cell death.
3. In this paper we discussed the mechanism of apoptosis induced by TRG in tobacco cells. After TRG treatment in tobacco cells occurred the oxidative burst, the kinetics of intracellular H2O2 burst within 8 hour treatment was biphasic:first phase peaked at 0.25 hour and second peaked at 6hour post-treatment,and second peak higer than first peak; TRG treatment to detect ed that extracellular H2O2 were also reached peak at 0.25 hour and 1h, then H2O2 accumulation to achieve a relatively stable state.In addition, we found that TRG treatment induced accumulation of ROS dynamics changes in the BY-2 cells, ROS burst within 12 hour treatment, then relatively stable until the 120hour. It indicated that H2O2 and ROS participated in the apoptosis signaling pathway in tobacco cell apoptosis; and we detected NO production within 30 min with TRG treatment in tobacco cells during apoptosis process, the NO burst at 2.5 hour, then detected NO concentration have reached the maximum at 6 hour. kinetics of NO is same as H2O2, It indicated that NO and H2O2 synergy promotes apoptosis.Also we use anti-oxidant ascorbic acid salt to test the correlation of oxidation with PCD.we found that the rate of death of suspension cell was greatly reduced after the cells were treated by ascorbic acid salt before the TRG induced the cells, With the time longer AsA inhibited the rate of apoptosis become increase from the 6.84% increase to 16.14% at 96hour, and delay the time of PCD to 20 hours. AsA pretreatment 30 min added TRG induced apoptosis, we detected ROS of AsA + TRG group found less than TRG group, we can see that AsA inhibited ROS release during apoptosis induced by TRG, thus inhibition the produce of apoptosis. Also during apoptosis induced by trigonelline pH values of cell culture fluid change alkaline to sour.
引文
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