【作者】 刘静；

【导师】 王冬梅；

【作者基本信息】 河北农业大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 本试验首先采用小麦(Triticum aestivum L.)品种洛夫林10的悬浮细胞与硝普钠(SNP,NO供体)互作来研究外源一氧化氮对小麦悬浮细胞的影响,并利用该体系建立了以Griess试剂检测法联合DAF-2DA荧光探针标记法检测NO动态变化的试验方法。试验结果表明:一氧化氮供体SNP能够诱导小麦悬浮细胞死亡,且在一定浓度下(2.5 mmol/L)刺激时,细胞表现出核染色质凝聚、边缘化等程序性死亡的形态特征。初步证明外源一氧化氮能够诱导小麦悬浮细胞死亡,并且具有程序性死亡的核形态变化特征。在此基础上以小麦悬浮细胞与感染叶锈菌(Puccinia triticina)生理小种260的小麦叶片细胞间隙液(IWF-260)组成互作体系,课题组已经证明IWF-260具有激发子活性。以Griess试剂检测法联合DAF-2DA荧光探针标记法检测NO的产生及变化。结果显示:未接种的小麦细胞间隙液(IWF-CK)和IWF-260都能诱导小麦悬浮细胞产生NO,并在30min时有一个NO峰,但随着时间的延长NO产生量逐渐下降,由IWF-260诱导产生的NO比IWF-CK诱导的要多,荧光探针标记结果更直观地观察到了这一现象。当使用NO的专一性清除剂cPTIO处理时,可以显著的抑制激发子诱发细胞产生的NO量。然后,我们利用改良苯酚品红染色法,对IWF-CK和IWF-260处理小麦悬浮细胞后不同时间进行细胞形态学观察。结果显示:IWF-CK处理后细胞核形态没有明显的变化,但是,IWF-260处理以后12h就出现染色质凝集现象,处理24h核凝集更加明显,并有颗粒状物体散布在核内,处理后48h大多数细胞核染色质凝集现象明显,到72h核染色质趋于边缘化。以上结果表明,经过激发子(IWF-260)处理后的小麦悬浮细胞表现出核染色质凝聚和边缘化等典型的细胞程序性死亡的形态特征,初步证明激发子刺激诱发产生的NO可能参与了小麦悬浮细胞发生程序性死亡。我们利用药物学试验对该体系中NO的功能和来源进行了初步探讨。选用NO的清除剂cPTIO;NOS的抑制剂L-NAME;NR的抑制剂Na2WO4;胞外Ca2+螯合剂EGTA、Ca2+通道阻断剂LaCl3、Ca2+载体A23187、胞内Ca2+通道阻断剂Heparin以及胞内Ca2+库激活剂Caffeine预处理小麦悬浮细胞,然后再加入IWF-260处理,并以IWF-CK处理作为对照,观察上述药物对IWF-260刺激诱发的NO的产生以及对细胞死亡的影响。主要结果如下:1. cPTIO预处理小麦悬浮细胞后,再用IWF-CK和IWF-260处理,我们发现cPTIO能够显著的抑制小麦悬浮细胞死亡,表明在该体系中NO介导了激发子刺激诱发的小麦细胞死亡过程。2. L-NAME和Na2WO4预处理小麦悬浮细胞后,再用IWF-CK和IWF-260处理,结果发现Na2WO4预处理后能显著的降低NO的产生以及细胞死亡率,而L-NAME的抑制作用要弱于Na2WO4,并且荧光探针标记结果与此相似,说明NR在小麦悬浮细胞受IWF-260刺激诱发的NO的产生过程中较NOS起到更重要作用。3. Ca2+螯合剂EGTA、Ca2+通道阻断剂LaCl3预处理小麦悬浮细胞,再用IWF-CK和IWF-260处理,结果发现EGTA和LaCl3都能够抑制NO的产生以及细胞死亡率。并且发现螯合剂EGTA的效果较阻断剂LaCl3明显。此外,用Ca2+载体A23187处理小麦悬浮细胞后可以诱导细胞死亡,但是明显低于IWF-260诱导的细胞死亡率。单独使用A23187处理细胞也能够诱导小麦悬浮细胞产生NO,但是NO的产生量非常低。此结果表明,胞外Ca2+内流对激发子诱发小麦悬浮细胞产生NO有一定调控作用。4.胞内Ca2+通道阻断剂Heparin、胞内Ca2+库激活剂Caffeine预处理小麦悬浮细胞,再用IWF-CK和IWF-260处理,结果发现用Caffeine预处理后,细胞死亡率随着Caffeine浓度增加而有所增加。而用Heparin预处理后,Heparin对细胞死亡的影响呈浓度依赖型。据此提出,胞内Ca2+可能参与了小麦悬浮细胞抵抗激发子刺激过程中钙信号的形成,这一过程主要通过IP3途径完成。综上所述,外源NO能够诱导小麦悬浮细胞发生死亡,并且具有程序性死亡的特征;而感染叶锈菌的细胞间隙液也能诱发小麦悬浮细胞NO的迸发,并且也导致细胞发生程序性死亡;药物学实验进一步表明,小麦悬浮细胞与感染叶锈菌的小麦细胞间隙液互作过程中NO的产生途径主要通过NR途径,胞外Ca2+内流以及胞内Ca2+库的释放对此过程中NO的产生有一定的调控作用。更多还原

【Abstract】 Wheat (Triticum aestivum) suspension cells of Lovrin 10-Nitric Oxide (applied via the NO donors Sodium Nitroprusside, SNP) system was used to study the effects of NO on the wheat suspension cells. The detecting method was established by using Griess reagent and DAF-2DA. The results showed that SNP could induce the cell death of cells. The characteristics of the cell nucleus chromatin condensation and marginalization of PCD was obviously when SNP was the concentration of 2.5 mmol/L. The datas showed that NO might induce the wheat suspension cells to death which presented the characters of PCD.The IWF (Intercellular washing fluids)-260 of wheat which was infected with the incompatible leaf rust fungus races (Puccinia triticina) was used as elicitor to stimulate the suspension cells and the changes of NO was detected by the method of Griess reagent. We also used fluorescent probe DAF-2DA scrutinized the the generation of NO in wheat suspension cells induced by IWF-260 with the help of fluorescence microscope. The results showed that: IWF-260 could induce NO outbreak at 30min and decreased after 30min, in contrast to the cells treated with IWF-CK. The highest fluorescence intensity was detected at the same time, but it can be inhibited by cPTIO.The phenylic alcohol staining was used to observe the cellular morphology of wheat suspension cells after treated with IWF-CK or IWF-260. The morphous of cell nucleus were stay normal after treated with IWF-CK. But it appeared chromatic agglutination at12h after treated with IWF-260. At 24h, it became all the better follow with granulo object spread in the intranuclear. At 48h, most of the cell nucleus had this phenomenon and till 72h it appeared marginalization. The datas showed that the characteristics of the cell nucleus chromatin condensation and marginalization of PCD was obviously after treated with IWF-260. The results demonstrated that NO might induce the programmed cell death of wheat suspension cells.In addition, Pharmacological experiments were carried. NO inhibitor(cPTIO), NOS inhibitor(L-NAME), NR inhibitor(Na2WO4), calcium chelator(EGTA) calcium channel blocker (LaCl3), calcium channel blocker(Heparin) as well as intercellular calcium activator (Caffeine) were separately added before IWF. And add IWF and Ca2 + carrier A23187 at the same time or only add A23187 to study the changes of NO production and cell death rate. The results were given as below. 1. Treatment of cPTIO obviously suppressed the cell death induced by IWF. These results suggested that NO burst might involve in the death of cells caused by IWF.2. Treatment of L-NAME and Na2WO4 could respectively inhibited the NO production and the cell death caused by IWF, but the inhibition of Na2WO4 was stronger than L-NAME′s. The DAF-2DA fluorescence has the same results. The results showed that NR was the important source of NO burst during wheat suspension cells responding to IWF.3. Treatment of EGTA and LaCl3 respectively could also suppressed the NO production and the cell death rate, but the inhibition of EGTA was stronger than LaCl3′s. In addition, calcium ionophore A23187 could induced the cell death, and the higher concentration the more cell death, but the effect was lower than IWF-260. Moreover, the A23187 could increase the NO production, but the content of NO was low. So intracellular Ca2+ appeared to be involve in the signal transduction pathway of wheat suspension cells to IWF by orchestrating the NO burst.4. While caffeine was added to wheat suspension cells, cell death could be detected. The cell death increased when Caffeine concentration was increased. Heparin was added to wheat suspension cells before IWF. The results showed that there was a concentration-depended effect on heparin. So we infered that intercellular calcium might involve in the formation of calcium signal transduction during add of IWF, which used IP3 passway.To sum up, exogenous NO could induced wheat suspension cells′s hypersensitive response (HR) and presented the characters of PCD. IWF could induced the outbreak of NO and the PCD of wheat suspension cells. Pharmacological approaches also suggested that NR might mediate NO generation during wheat suspension cells-IWF, which extracellular calcium influx and intercellular calcium releasing might be involved in.更多还原