剪切力对东北红豆杉细胞悬浮培养的影响机理的初步研究
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摘要
为明确剪切力对植物细胞作用的机理以及植物细胞剪切敏感性的潜在机制，
作者通过自制改进型 Couette 式剪切反应器来悬浮培养东北红豆杉细胞，为细胞
提供精确的层流剪切场，进而围绕特定剪切力作用下细胞防御应答反应以及剪切
信号转导展开一系列的研究，以期能初步阐述剪切力对东北红豆杉细胞的作用机
理。并在此研究结果基础上，对东北红豆杉细胞的剪切敏感性以及东北红豆杉细
胞的剪切敏感性的潜在机制进行了分析。
1. 0.1Pa剪切力诱导了东北红豆杉细胞的防御应答反应。活性氧分子（O2 及 -
H2O2）分别在 0.5-1(h)、2.5-3(h)以及 5-5.5(h)出现了三次积累，具有三时相的
特征。其产生方式主要是质膜氧化还原系统中的NADPH氧化酶，细胞壁过
氧化物酶在其中所起的作用较小。伴随着氧迸发的现象，细胞SOD、POD和
CAT酶活也均发生改变，从而保持细胞自身氧化还原态势的平衡。剪切作用
下产生的各种活性氧分子中，超氧阴离子自由基（O2 ）与细胞膜作用导致了
-
细胞膜透性的增大，而剪切力作用下氧迸发所产生的H2O2则可能作为第二信
使诱导了PAL酶活的改变和酚的积累等次级代谢反应。
2. 剪切信号转导过程中，剪切力的响应可能是通过植物细胞的骨架连续体及其
表面存在的机械感应器与受体而完成的，作用时间为 5-15 min。G-蛋白、Ca2+
通道和PLC参与了剪切信号的跨膜或胞内的转导过程，作用时间分别为G蛋
白：10-15 min、Ca2+通道：15-25 min、PLC：20-25 min。G蛋白的激活为Ca2+
通道激活和PLC的活化的上游时间， Ca2+通道激活的和PLC的活化则为剪切
信号转导的不同通路。
3. 建立了一个剪切力作用下东北红豆杉细胞信号转导以及防御应答的生物学
模型，对剪切力对东北红豆杉细胞作用的机理有进行初步阐述，为研究植物
细胞剪切敏感性提供新的思路。
4. 处于指数期的东北红豆杉细胞对剪切力比较敏感，而处于延滞期的东北红豆
杉细胞则相对不太敏感。这种剪切敏感性的差异在细胞的防御应答反应上表
现明显。在信号转导角度上，这种差异的原因在于不同生长期东北红豆杉细
胞剪切信号的感受以及转导速度方面的差异。
To well-understand the mechanism of the effect of shear stress to plant cell and
the mechanism of shear sensitivity of plant cell, some defined laminar shear fields
were created for suspensions of Taxus cuspidata in a Couette-type shear reactor,
which we made ourselves. The defense responses and signal transduction pathway of
shear signal were studied and a potential model for those results was proposed. Based
on those results, the mechanism of shear sensitivities of taxus cell suspensions in
different growth phases was also investigated. From the work above we gained some
valuable results.
1. Taxus cuspidate can answer 0.1Pa shear stress with defense responses. There have
triphasic characteristics in 6 h for both intracellular H2O2 production and
extra-cellular O2 production and the key enzyme responsible for oxidative bursts
-·
under the shear stress is primarily NADPH oxidase and the contribution of
peroxidase for oxidative bursts was less. The O2 burst may account for the change
-·
of membrane permeability, and H2O2 burst plays an important role in inducing
secondary metabolites such as the activation of phenylalanine ammonia lyase
enzyme and phenolic accumulation.
2. Studies focused on shear signal transduction showed that cytoskeleton are
involved in the sense of shear stress with it action time of 5-15min and that
G-protein, Ca2+ channel and PLC were proved to be necessary in shear signal
transduction with their action time of 10-15 min、15-25 min and 20-25 min,
respectively. It was also suggested that the action of G-proteins is the upstream
event to Ca2+ influx and the action of PLC. The action of Ca2+ channel and PLC
may be the two different pathways in transduction of the shear signal for they
have a period of action time in common.
3. Based on results above, a model is proposed to explain the defense responses and
shear signal transduction in cultured T. cuspidata cells challenged with the shear
stress.
4. In testing suspension cultures of various ages, it was found that those cultures in
the stages of exponential growth were more susceptible to shear stress than
cultures in the lag phase. The reasons for their difference lied in the different
recognition time and the speed for action.
作者通过自制改进型 Couette 式剪切反应器来悬浮培养东北红豆杉细胞，为细胞
提供精确的层流剪切场，进而围绕特定剪切力作用下细胞防御应答反应以及剪切
信号转导展开一系列的研究，以期能初步阐述剪切力对东北红豆杉细胞的作用机
理。并在此研究结果基础上，对东北红豆杉细胞的剪切敏感性以及东北红豆杉细
胞的剪切敏感性的潜在机制进行了分析。
1. 0.1Pa剪切力诱导了东北红豆杉细胞的防御应答反应。活性氧分子（O2 及 -
H2O2）分别在 0.5-1(h)、2.5-3(h)以及 5-5.5(h)出现了三次积累，具有三时相的
特征。其产生方式主要是质膜氧化还原系统中的NADPH氧化酶，细胞壁过
氧化物酶在其中所起的作用较小。伴随着氧迸发的现象，细胞SOD、POD和
CAT酶活也均发生改变，从而保持细胞自身氧化还原态势的平衡。剪切作用
下产生的各种活性氧分子中，超氧阴离子自由基（O2 ）与细胞膜作用导致了
-
细胞膜透性的增大，而剪切力作用下氧迸发所产生的H2O2则可能作为第二信
使诱导了PAL酶活的改变和酚的积累等次级代谢反应。
2. 剪切信号转导过程中，剪切力的响应可能是通过植物细胞的骨架连续体及其
表面存在的机械感应器与受体而完成的，作用时间为 5-15 min。G-蛋白、Ca2+
通道和PLC参与了剪切信号的跨膜或胞内的转导过程，作用时间分别为G蛋
白：10-15 min、Ca2+通道：15-25 min、PLC：20-25 min。G蛋白的激活为Ca2+
通道激活和PLC的活化的上游时间， Ca2+通道激活的和PLC的活化则为剪切
信号转导的不同通路。
3. 建立了一个剪切力作用下东北红豆杉细胞信号转导以及防御应答的生物学
模型，对剪切力对东北红豆杉细胞作用的机理有进行初步阐述，为研究植物
细胞剪切敏感性提供新的思路。
4. 处于指数期的东北红豆杉细胞对剪切力比较敏感，而处于延滞期的东北红豆
杉细胞则相对不太敏感。这种剪切敏感性的差异在细胞的防御应答反应上表
现明显。在信号转导角度上，这种差异的原因在于不同生长期东北红豆杉细
胞剪切信号的感受以及转导速度方面的差异。
To well-understand the mechanism of the effect of shear stress to plant cell and
the mechanism of shear sensitivity of plant cell, some defined laminar shear fields
were created for suspensions of Taxus cuspidata in a Couette-type shear reactor,
which we made ourselves. The defense responses and signal transduction pathway of
shear signal were studied and a potential model for those results was proposed. Based
on those results, the mechanism of shear sensitivities of taxus cell suspensions in
different growth phases was also investigated. From the work above we gained some
valuable results.
1. Taxus cuspidate can answer 0.1Pa shear stress with defense responses. There have
triphasic characteristics in 6 h for both intracellular H2O2 production and
extra-cellular O2 production and the key enzyme responsible for oxidative bursts
-·
under the shear stress is primarily NADPH oxidase and the contribution of
peroxidase for oxidative bursts was less. The O2 burst may account for the change
-·
of membrane permeability, and H2O2 burst plays an important role in inducing
secondary metabolites such as the activation of phenylalanine ammonia lyase
enzyme and phenolic accumulation.
2. Studies focused on shear signal transduction showed that cytoskeleton are
involved in the sense of shear stress with it action time of 5-15min and that
G-protein, Ca2+ channel and PLC were proved to be necessary in shear signal
transduction with their action time of 10-15 min、15-25 min and 20-25 min,
respectively. It was also suggested that the action of G-proteins is the upstream
event to Ca2+ influx and the action of PLC. The action of Ca2+ channel and PLC
may be the two different pathways in transduction of the shear signal for they
have a period of action time in common.
3. Based on results above, a model is proposed to explain the defense responses and
shear signal transduction in cultured T. cuspidata cells challenged with the shear
stress.
4. In testing suspension cultures of various ages, it was found that those cultures in
the stages of exponential growth were more susceptible to shear stress than
cultures in the lag phase. The reasons for their difference lied in the different
recognition time and the speed for action.
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攻读硕士期间发表与待发表论文
1：韩荣斌，施中东，元英进。剪切力对植物细胞悬浮培养的影响。化学工
业与工程。2002，19（Sup）: 277-281。
2: 韩荣斌，施中东，元英进。剪切力对南方红豆杉细胞悬浮培养的影响。过
程工程学报。2003,3(2): 135-140。(EI)
3: 韩荣斌，元英进，季艳艳。剪切力作用下的东北红豆杉细胞活性氧迸发及
其清除系统。第二界全国制药工程会议论文。2003: 167-169。
4：Rong-bin Han., Ying-Jin Yuan. Oxidative burst in suspension culture of
Taxus cuspidata induced by a laminar shear stress in short-term.
Biotechnol.Prog (in press bp034242p).
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攻读硕士期间发表与待发表论文
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3: 韩荣斌，元英进，季艳艳。剪切力作用下的东北红豆杉细胞活性氧迸发及
其清除系统。第二界全国制药工程会议论文。2003: 167-169。
4：Rong-bin Han., Ying-Jin Yuan. Oxidative burst in suspension culture of
Taxus cuspidata induced by a laminar shear stress in short-term.
Biotechnol.Prog (in press bp034242p).
5: Rong-bin Han., Ying-Jin Yuan., Zhi-qiang Ge. Differences in biological
and signal responses of Taxus Cuspidata suspension cultures in lag and
exponential phases to a given shear stress. Biotechnol. & Appl. Biochem.
(to be submitted).
6: Rong-bin Han., Ying-Jin Yuan., Zhi-qiang Ge. The involvement of
Cytoskeleton in defense responses of Taxus cuspidate induced by a laminar
shear stress in suspension cultures. (Preparing).