fMLP诱导中性粒细胞极性及其机制研究
发布时间：2018-08-09 11:02
【摘要】： 研究背景 细胞极性是多种不同类型细胞的基本特征，对多数细胞的功能发挥是必需的。细胞极性是指细胞为了行使特殊的生理功能或产生定向的分化，在胞内的信号分子不对称分布致使细胞在表型上的不对称。具体表现为细胞的形状、细胞内的蛋白质、脂质以及产生极性所必需的细胞骨架等的不对称分布，细胞结构出现重构的变化。细胞极性形成的过程通常是由肌动蛋白细胞骨架和细胞皮质所介导，在这一进程中，细胞膜首先形成绉褶，同时在绉褶处肌动蛋白聚集增加，从而形成一个前极具有片足后极出现尾足的极化形态。细胞极性在细胞迁移、轴突生长、胚胎发育、血管发生、伤口修复等细胞基本生命活动过程中发挥着重要的调控作用。 中性粒细胞极性对于其生理功能的发挥有着重要的意义。临床上多种疾病如败血症、哮喘、缺血／再灌注损伤和器官移植的排斥反应、类风湿性关节炎及一些肿瘤的发生和转移都与中性粒细胞极性功能的增强和减弱有关。当机体遭受外来细菌和其他病原体入侵时，中性粒细胞充当了机体的第一道防线，这种作用是建立在细胞的极性和趋化性基础上的。在各种趋化物的作用下，中性粒细胞凭借胞内分子对极化信号的优化和放大，能够准确向炎症部位形成极性化，，并最终向此处迁移，而中性粒细胞极性是这种定向迁移功能的前提条件。 目前有关中性粒细胞极性形成机制的研究主要包括参与胞内极性形成的信号传导通路的研究，参与胞内钙离子(Ca~(2+))信号调节的钙库操纵性钙离子流入(SOCE)机制的探讨以及胞膜上的特殊脂质结构脂筏在其中的作用。但中性粒细胞极性各种机制之间的关系，尤其是参与SOCE机制的瞬时受体电位离子通道(TRP)在极化过程中的明确作用，以及其与脂筏的相互联系还有待于进一步探讨。 目的 1．采用4℃温度对急性分离的人外周血嗜中性粒细胞进行预处理，探讨温度处理对细胞的极性和膜通道有何影响。 2．探讨均一浓度的趋化剂fMLP对中性粒细胞极性形成的影响。 3．探讨fMLP诱导的中性粒细胞胞内游离钙离子浓度([Ca~(2+)]_i)变化与细胞极性化过程启动的关系。 4．探讨TRPC1和脂筏在fMLP诱导的中性粒细胞极性中的作用及二者之间的关联性。 方法 1．采用重复梯度密度离心的方法分离中性粒细胞，进行台盼蓝和瑞氏染色，分析细胞的活性和纯度。分离后的细胞进行4℃温度预处理。通过倒置显微镜观察fMLP诱导的中性粒细胞的变化情况，采用单通道和全细胞模式膜片钳记录中性粒细胞的钾通道的电流活动。 2．采用激光共聚焦显微镜测量中性粒细胞[Ca~(2+)]_i。 3．应用免疫荧光方法检测对照组、fMLP刺激组和脂筏破坏后fMLP刺激组的TRPC1的表达情况。 4．使用超高速冷冻离心机分离脂筏，并应用Western-blotting方法检测对照组、fMLP刺激组和脂筏破坏后fMLP刺激组的TRPC1蛋白在脂筏中的含量变化。 结果 1．中性粒细胞分离后，对细胞进行台盼蓝染色，细胞的活性大于94％，瑞氏—姬姆萨染色，细胞纯度大于95％。对分离后的细胞进行4℃温度预处理，结果表明4℃温度预处理的细胞出现片足的时间(45.93±7.87msec，n=35)和常温组(33.75±6.02msec，n=28)相比有显著性差异(P＜0.01)。4℃温度预处理的细胞极性周期所用时间(538.26±27.74sec，n=23)和常温组(260.25±25.87sec，n=23)相比有显著性差异(P＜0.01)。全细胞模式记录温度预处理和常温对照组中性粒细胞的电压依赖性钾电流，两组的Ⅰ-Ⅴ曲线，电流幅度均随钳制电位升高而增高，电流幅度没有显著性差异(n=5，P＞0.05)。采用单通道记录BKCa电流，两组细胞的电导值大约为210pS(n=3，P＞0.05)。在钳制电位为40mV、60mV、80mV下，温度处理组和常温组的NPo分别进行t检验，两组之间均没有显著性差异(n=30，P＞0.05)。 2．在三种浓度(10nM，50nM，100nM)下的fMLP激活中性粒细胞，极化中性粒细胞的50％极化率所用的时间(51.16±6.49sec，45.83±4.95sec，37.33±5.16sec；n=6，P＜0.01)，伪足的平均极化长度(1.62±0.050μm，1.73±0.036μm，1.81±0.027μm；n=30，P＜0.01)以及极性振荡周期(209.37±17.70sec，n=52；234.00±21.42sec，n=50；286.15±19.44sec，n=52；P＜0.01)均具有显著性差异。三种浓度下的伪足延伸率(0.067±0.0073μm／sec，0.107±0.0086μm／sec，0.120±0.0132μm／sec；n=30，P＜0.01)，缩短率(0.051±0.0050μm／sec，0.091±0.0051μtm／sec，0.110±0.0142μm／sec；n=30，P＜0.01)和变化率(0.057±0.0071μm／sec，0.099±0.0059μm／sec，0.114±0.0124μm／sec；n=30，P＜0.01))也均具有显著性差异。 3．加入fMLP(100nM)后对应于[Ca~(2+)]_i变化的不同时相，即静息期(Osec)、快速上升期(10sec)、快速下降期(150sec)、慢速下降期(250sec)和终末期五个阶段，而中性粒细胞表现为极性化和去极性化状态的交替出现。 4．SKF96365(10μM)和mβCD(10mM)能抑制fMLP诱导中性粒细胞[Ca~(2+)]_i增加及中性粒细胞的极化形态的形成。 5．正常中性粒细胞的TRPC1是在膜均匀的表达，而fMLP(100nM)组TRPC1明显聚集到细胞的片足位置，但fMLP+mβCD组TRPC1没有出现聚集现象。同时使用Western-blotting技术检测三组的TRPC1蛋白在脂筏和非脂筏的含量变化，在非脂筏层，对照组(289.00±8.00)和fMLP+mβCD组(283.33±6.02)的TRPC1的表达比fMLP组(76.00±7.00)显著增高，差异具有统计学意义(n=3，P＜0.01)；而在脂筏层，fMLP组(215.00±6.55)的TRPC1的表达比对照组(0.00±0.00)和fMLP+mβCD组(21.33±5.68)显著增高，差异具有统计学意义(n=3，P＜0.01)。 结论 1．采用4℃温度预处理分离后的中性粒细胞，发现细胞极性明显抑制，而离子通道的功能活动不受影响，因此4℃温度预处理可以减少中性粒细胞自发极化的发生，为后续实验提供良好的细胞来源； 2．建立了一组评价细胞极性变化的指标，根据指标所得数据发现中性粒细胞极性形成对fMLP有时间和浓度的依赖性； 3．本实验显示不同浓度趋化剂fMLP诱导的[Ca~(2+)]_i变化没有显著性差异，而中性粒细胞极性化周期与fMLP浓度有关，进一步揭示了[Ca~(2+)]_i的升高与中性粒细胞极性化启动机制有关； 4．证明了SOCE机制和脂筏参与了fMLP诱导中性粒细胞的极性形态的产生及[Ca~(2+)]_i升高过程，进一步阐明了中性粒细胞极性化过程中的关键信号分子(Ca~(2+)]_i、TRPC1)在信号转导过程中的作用； 5．在可掌握的文献范围内本研究首次报道TRPC1在参与中性粒细胞极化时是结合在脂筏上而发挥作用的。
[Abstract]:Research background
Cell polarity is the basic feature of a variety of different types of cells and is necessary for the function of most cells. Cell polarity refers to the asymmetric distribution of signal molecules in the cell in order to exercise special physiological functions or to produce directional differentiation. The asymmetric distribution of proteins, lipids, and cytoskeletons necessary for the generation of polarity, and the changes in the structure of cells. The process of cell polarity is usually mediated by the actin cytoskeleton and cell cortex. In this process, the membrane is first crepe fold, and the actin aggregation at the crepe fold increases. The polarization form of the tail of a front pole is formed. Cell polarity plays an important regulatory role in cell migration, axon growth, embryo development, angiogenesis, wound repair and other cell basic activities.
Neutrophil polarity plays an important role in its physiological function. Many diseases such as septicaemia, asthma, ischemia / reperfusion injury and rejection of organ transplantation, rheumatoid arthritis and the occurrence and metastasis of some tumors are related to the enhancement and weakening of the neutrophil polarity function. When bacteria and other pathogens invade, neutrophils act as the first line of defense, based on the polarity and chemotaxis of the cells. Under the action of various chemotaxis, the neutrophils can be accurately polarized to the inflammatory sites by the optimization and amplification of the intracellular molecules. Migration is eventually here, and the polarity of neutrophils is a prerequisite for this directional migration function.
Current studies on the mechanism of neutrophil polarity formation mainly include the study of signal transduction pathways involved in the formation of intracellular polarity, the mechanism of calcium ion inflow (SOCE) regulated by intracellular calcium (Ca~ (2+)) signal and the role of lipid rafts with special lipid structure on the membrane. The relationship between various mechanisms, especially the clear role of the transient receptor potential ion channel (TRP) involved in the SOCE mechanism in the polarization process, and the interaction with the lipid rafts, remains to be further explored.
objective
1. The human peripheral blood neutrophils were pretreated at 4 C to investigate the effect of temperature on cell polarity and membrane channel.
2. to explore the effect of homogeneous concentration of chemoattractant fMLP on the polarity formation of polymorphonuclear neutrophils.
3. To investigate the relationship between the changes of intracellular free calcium concentration ([Ca~ (2+)]_i) in neutrophils induced by fMLP and the initiation of cell polarization.
4. to explore the role of TRPC1 and lipid rafts in the polarity of fMLP induced neutrophils and the correlation between the two.
Method
1. the neutrophils were separated by repeated gradient density centrifugation. Trypan blue and Rayleigh staining were used to analyze the activity and purity of the cells. The cells after the separation were pretreated at 4 degrees centigrade. The changes of neutrophils induced by fMLP were observed by inverted microscope, and the neutrophils were recorded by single channel and whole cell mode patch clamp. The current activity of the potassium channel of the cell.
2. laser confocal microscopy was used to measure neutrophil [Ca~ (2+)]_i..
3. immunofluorescence assay was used to detect the expression of TRPC1 in the control group, fMLP stimulation group and fMLP stimulation group after lipid rafting.
4. the lipid rafts were separated by ultra high speed freezer centrifuge, and the control group was detected by Western-blotting method. The content of TRPC1 protein in the lipid raft was changed by the fMLP stimulation group and the fMLP stimulation group after the lipid raft was destroyed.
Result
1. neutrophils were stained with trypan blue, the cell activity was more than 94%, and the cell purity was more than 95%., and the cell purity was more than 95%.. The cells were pretreated at 4 degrees centigrade after the separation. The results showed that the time (45.93 + 7.87msec, n=35) and normal temperature group (33.75 + 6.02msec, n=28) of the pretreated cells at 4 C Compared with the significant difference (P < 0.01), the time used for the cell polar cycle (538.26 + 27.74sec, n=23) and the normal temperature group (260.25 + 25.87sec, n=23) had significant difference (P < 0.01). The temperature preconditioning and the voltage dependent potassium current of the neutrophils in the normal temperature control group and the two group of I - V curved (P < 0.01). Line, the amplitude of current increased with the increase of the clamp potential (n=5, P > 0.05). Using a single channel to record BKCa current, the conductance value of the two groups of cells was about 210pS (n=3, P > 0.05). Under the clamp potential of 40mV, 60mV, and 80mV, the t test was carried out between the temperature treatment group and the normal temperature group, and there was no significant difference between the two groups. Sexual differences (n=30, P > 0.05).
2. the time (51.16 + 6.49sec, 45.83 + 4.95sec, 37.33 + 5.16sec, n=6, P < 0.01) for the activation of neutrophils at the three concentrations (10nM, 50nM, 100nM), the average polarization length of the pseudo foot (1.62 + 0.050 mu m, 1.73 + 0.036 mu m, 1.81 + 0.027 Mu m), and polarity oscillation cycle 9.37 + 17.70sec, n=52; 234 + 21.42sec, n=50; 286.15 + 19.44sec, n=52; P < 0.01) have significant differences. The elongation rate of pseudo foot under three concentrations (0.067 + 0.0073, M / sec, 0.107 + 0.0086 mu m / sec, 0.120 + 0.0132 mu / M / 0.01) There were also significant differences in the rates of change (0.057.0071/ sec, 0.099.0059/ sec, 0.114.0124/ sec, n = 30, P < 0.01).
3. after adding fMLP (100nM) to the different phases of [Ca~ (2+)]_i, that is, resting period (Osec), rapid rise period (10sec), rapid descent (150sec), slow descent (250sec) and end stage five stages, and neutrophils appear to be polar and depolarization state alternately.
4. SKF96365 (10 mu M) and M beta CD (10 mM) could inhibit the increase of neutrophil [Ca~ (2+)]_iinduced by fMLP and the formation of polarized morphology of neutrophils.
5. the TRPC1 of normal neutrophils was expressed evenly in the membrane, while the TRPC1 in the group of fMLP (100nM) was obviously aggregated to the cell foot position of the cells, but the TRPC1 in the fMLP+m beta CD group did not appear to be aggregated. At the same time, the content of TRPC1 protein in the lipid rafts and non fat rafts in the three groups was detected by Western-blotting technology, in the non fat raft and in the control group (289 + 8). The expression of TRPC1 in group fMLP+m beta CD (283.33 + 6.02) was significantly higher than that in group fMLP (76 + 7), and the difference was statistically significant (n=3, P < 0.01), while in lipid rafts, the expression of TRPC1 in group fMLP (215 + 6.55) was significantly higher than that of the control group (0 + 0) and fMLP+m beta CD group (21.33 + 5.68), and the difference was statistically significant (n=3, P < 0.01).
conclusion
1. when the neutrophils were pretreated at 4 C, it was found that the cell polarity was obviously inhibited and the functional activity of the ion channel was not affected. Therefore, the temperature preconditioning at 4 C could reduce the spontaneous polarization of neutrophils, and provide a good cell source for the follow-up experiment.
2. A set of indices were established to evaluate the changes of cell polarity. According to the data obtained from these indices, it was found that the formation of neutrophil polarity depended on the concentration and time of fMLP.
3. the experiment showed that there was no significant difference in the changes of [Ca~ (2+)]_i induced by different concentrations of chemokine fMLP, but the polarity of neutrophils was related to the concentration of fMLP, which further revealed that the increase of [Ca~ (2+)]_i was related to the mechanism of neutrophil polarity initiation.
4. it was demonstrated that the SOCE mechanism and lipid rafts participated in the formation of the polar morphology of fMLP induced neutrophils and the process of [Ca~ (2+)]_i increase, and further elucidated the role of the key signal molecules (Ca~ (2+)]_i, TRPC1) in the process of signal transduction in the process of neutrophil polarity.
5. In the available literature, we report for the first time that TRPC1 binds to lipid rafts when it participates in neutrophil polarization.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2173871
[Abstract]:Research background
Cell polarity is the basic feature of a variety of different types of cells and is necessary for the function of most cells. Cell polarity refers to the asymmetric distribution of signal molecules in the cell in order to exercise special physiological functions or to produce directional differentiation. The asymmetric distribution of proteins, lipids, and cytoskeletons necessary for the generation of polarity, and the changes in the structure of cells. The process of cell polarity is usually mediated by the actin cytoskeleton and cell cortex. In this process, the membrane is first crepe fold, and the actin aggregation at the crepe fold increases. The polarization form of the tail of a front pole is formed. Cell polarity plays an important regulatory role in cell migration, axon growth, embryo development, angiogenesis, wound repair and other cell basic activities.
Neutrophil polarity plays an important role in its physiological function. Many diseases such as septicaemia, asthma, ischemia / reperfusion injury and rejection of organ transplantation, rheumatoid arthritis and the occurrence and metastasis of some tumors are related to the enhancement and weakening of the neutrophil polarity function. When bacteria and other pathogens invade, neutrophils act as the first line of defense, based on the polarity and chemotaxis of the cells. Under the action of various chemotaxis, the neutrophils can be accurately polarized to the inflammatory sites by the optimization and amplification of the intracellular molecules. Migration is eventually here, and the polarity of neutrophils is a prerequisite for this directional migration function.
Current studies on the mechanism of neutrophil polarity formation mainly include the study of signal transduction pathways involved in the formation of intracellular polarity, the mechanism of calcium ion inflow (SOCE) regulated by intracellular calcium (Ca~ (2+)) signal and the role of lipid rafts with special lipid structure on the membrane. The relationship between various mechanisms, especially the clear role of the transient receptor potential ion channel (TRP) involved in the SOCE mechanism in the polarization process, and the interaction with the lipid rafts, remains to be further explored.
objective
1. The human peripheral blood neutrophils were pretreated at 4 C to investigate the effect of temperature on cell polarity and membrane channel.
2. to explore the effect of homogeneous concentration of chemoattractant fMLP on the polarity formation of polymorphonuclear neutrophils.
3. To investigate the relationship between the changes of intracellular free calcium concentration ([Ca~ (2+)]_i) in neutrophils induced by fMLP and the initiation of cell polarization.
4. to explore the role of TRPC1 and lipid rafts in the polarity of fMLP induced neutrophils and the correlation between the two.
Method
1. the neutrophils were separated by repeated gradient density centrifugation. Trypan blue and Rayleigh staining were used to analyze the activity and purity of the cells. The cells after the separation were pretreated at 4 degrees centigrade. The changes of neutrophils induced by fMLP were observed by inverted microscope, and the neutrophils were recorded by single channel and whole cell mode patch clamp. The current activity of the potassium channel of the cell.
2. laser confocal microscopy was used to measure neutrophil [Ca~ (2+)]_i..
3. immunofluorescence assay was used to detect the expression of TRPC1 in the control group, fMLP stimulation group and fMLP stimulation group after lipid rafting.
4. the lipid rafts were separated by ultra high speed freezer centrifuge, and the control group was detected by Western-blotting method. The content of TRPC1 protein in the lipid raft was changed by the fMLP stimulation group and the fMLP stimulation group after the lipid raft was destroyed.
Result
1. neutrophils were stained with trypan blue, the cell activity was more than 94%, and the cell purity was more than 95%., and the cell purity was more than 95%.. The cells were pretreated at 4 degrees centigrade after the separation. The results showed that the time (45.93 + 7.87msec, n=35) and normal temperature group (33.75 + 6.02msec, n=28) of the pretreated cells at 4 C Compared with the significant difference (P < 0.01), the time used for the cell polar cycle (538.26 + 27.74sec, n=23) and the normal temperature group (260.25 + 25.87sec, n=23) had significant difference (P < 0.01). The temperature preconditioning and the voltage dependent potassium current of the neutrophils in the normal temperature control group and the two group of I - V curved (P < 0.01). Line, the amplitude of current increased with the increase of the clamp potential (n=5, P > 0.05). Using a single channel to record BKCa current, the conductance value of the two groups of cells was about 210pS (n=3, P > 0.05). Under the clamp potential of 40mV, 60mV, and 80mV, the t test was carried out between the temperature treatment group and the normal temperature group, and there was no significant difference between the two groups. Sexual differences (n=30, P > 0.05).
2. the time (51.16 + 6.49sec, 45.83 + 4.95sec, 37.33 + 5.16sec, n=6, P < 0.01) for the activation of neutrophils at the three concentrations (10nM, 50nM, 100nM), the average polarization length of the pseudo foot (1.62 + 0.050 mu m, 1.73 + 0.036 mu m, 1.81 + 0.027 Mu m), and polarity oscillation cycle 9.37 + 17.70sec, n=52; 234 + 21.42sec, n=50; 286.15 + 19.44sec, n=52; P < 0.01) have significant differences. The elongation rate of pseudo foot under three concentrations (0.067 + 0.0073, M / sec, 0.107 + 0.0086 mu m / sec, 0.120 + 0.0132 mu / M / 0.01) There were also significant differences in the rates of change (0.057.0071/ sec, 0.099.0059/ sec, 0.114.0124/ sec, n = 30, P < 0.01).
3. after adding fMLP (100nM) to the different phases of [Ca~ (2+)]_i, that is, resting period (Osec), rapid rise period (10sec), rapid descent (150sec), slow descent (250sec) and end stage five stages, and neutrophils appear to be polar and depolarization state alternately.
4. SKF96365 (10 mu M) and M beta CD (10 mM) could inhibit the increase of neutrophil [Ca~ (2+)]_iinduced by fMLP and the formation of polarized morphology of neutrophils.
5. the TRPC1 of normal neutrophils was expressed evenly in the membrane, while the TRPC1 in the group of fMLP (100nM) was obviously aggregated to the cell foot position of the cells, but the TRPC1 in the fMLP+m beta CD group did not appear to be aggregated. At the same time, the content of TRPC1 protein in the lipid rafts and non fat rafts in the three groups was detected by Western-blotting technology, in the non fat raft and in the control group (289 + 8). The expression of TRPC1 in group fMLP+m beta CD (283.33 + 6.02) was significantly higher than that in group fMLP (76 + 7), and the difference was statistically significant (n=3, P < 0.01), while in lipid rafts, the expression of TRPC1 in group fMLP (215 + 6.55) was significantly higher than that of the control group (0 + 0) and fMLP+m beta CD group (21.33 + 5.68), and the difference was statistically significant (n=3, P < 0.01).
conclusion
1. when the neutrophils were pretreated at 4 C, it was found that the cell polarity was obviously inhibited and the functional activity of the ion channel was not affected. Therefore, the temperature preconditioning at 4 C could reduce the spontaneous polarization of neutrophils, and provide a good cell source for the follow-up experiment.
2. A set of indices were established to evaluate the changes of cell polarity. According to the data obtained from these indices, it was found that the formation of neutrophil polarity depended on the concentration and time of fMLP.
3. the experiment showed that there was no significant difference in the changes of [Ca~ (2+)]_i induced by different concentrations of chemokine fMLP, but the polarity of neutrophils was related to the concentration of fMLP, which further revealed that the increase of [Ca~ (2+)]_i was related to the mechanism of neutrophil polarity initiation.
4. it was demonstrated that the SOCE mechanism and lipid rafts participated in the formation of the polar morphology of fMLP induced neutrophils and the process of [Ca~ (2+)]_i increase, and further elucidated the role of the key signal molecules (Ca~ (2+)]_i, TRPC1) in the process of signal transduction in the process of neutrophil polarity.
5. In the available literature, we report for the first time that TRPC1 binds to lipid rafts when it participates in neutrophil polarization.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 邹珍友;黄艳;王勇;张慧;戴威;许露露;程彦伟;汪素美;;趋化剂对细胞伪足极性生长的影响研究[J];现代农业科技;2009年01期
本文编号：2173871
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