钙信号参与中性粒细胞极性化过程的研究
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摘要
研究背景
嗜中性粒细胞(PMNs)是具有定向移动功能的吞噬细胞,在抵抗细菌和真菌感染的宿主反应中起着重要的作用,这种功能是依靠趋化能力来实现的,趋化性是细胞感受信号分子从而具有的定向移动的能力,中性粒细胞能够沿着趋化物浓度梯度定向迁移到急性炎症的位置,这种迁移是中性粒细胞的核心功能。中性粒细胞能够迁移的基础在于其具有接受趋化刺激时具有形成极性化形状的能力。
Ca~(2+)信号在细胞的信号传导和执行功能等各个方面都发挥着重要的作用,影响着从卵子受精到细胞凋亡的整个生命过程。Ca~(2+)的独特性质在于其既能充当第一信使也能充当第二信使;既能调控细胞的生命过程,又能进行自身的调控。胞外Ca~(2+)浓度常超过10~(-3)M,而胞内至少低于胞外四个数量级之多(10~(-7)M),细胞这种Ca~(2+)稳态的维持依靠一些重要的调节区域通过一系列精妙的方式进行严密的控制和调节。在细胞膜或细胞器膜上,一些蛋白质形成的离子通道,其主要功能是通透Ca~(2+),这些离子通道依据门控机制分属于三组:电压门控性通道、配体门控性通道和库作性钙离子通道(Store-operated channels)。
1986年James Putney提出了库作性钙内流(SOCE)的概念。一组选择性刺激可以诱发细胞的应答,激活细胞表面受体从而诱导了胞膜磷酸肌醇的水解。通过磷脂酶C(PLC)产生活动性钙信号Inositol 1,4,5-trisphosphate[Ins(1,4,5)P3],从内质网(ER)钙库释放钙离子。同时,钙离子的释放伴随着外钙的内流。由于胞外钙离子的内流是由胞内钙库钙离子的释放倾空引起的,所以称为库作性钙内流。SOCE已经发现并提出多年,但其机制一直不甚清楚。最近研究发现可能与内质网膜上分布的感受分子Stiml和胞膜上的Orail等分子相互作用有关。随着人们对SOCE机制认识的逐渐发展,研究SOCE机制的工具药也逐渐受到人们的重视,其中普遍使用的有SK&F96365和2-APB。
中性粒细胞在受到趋化剂刺激后发生极性化的过程中,胞内Ca~(2+)浓度和分布会发生一系列改变。PMNs趋化剂甲酰甲硫氨酰-亮氨酰-苯丙氨酸(fMLP)可以激活内质网膜上IP3受体从而诱导内质网Ca~(2+)库释放钙离子。内质网Ca~(2+)库释放可以激活SOCE通路,促进外钙的内流。但SOCE机制是否参与中性粒细胞极性化过程以及在此过程中起何种作用,阻断SOCE后对中性粒细胞极性化过程以及趋化性有何影响,尚无确切报道。
SOCE在细胞极性化过程中可能起着重要的作用,而且这种作用可能与胞膜上脂筏(Lipid raft)结构存在密切的关系。脂筏的关键功能是它们能够作为信号的平台。为了进一步理解SOCE和脂筏的关系,我们观察了mβCD(甲基-β-环糊精,Methyl-β-cyclodextrin)对中性粒细胞极性的影响。mβCD能够清空中性粒细胞胆固醇,破坏脂筏完整性,从而可能影响到极性的形成过程。
目的
本实验通过研究中性粒细胞极性化过程中胞内Ca~(2+)信号的变化,以及应用工具药作用于SOCE机制后对细胞极性和趋化性的影响,探讨SOCE机制在中性粒细胞极性化过程中的作用。
方法
1.采用重复梯度密度离心的方法(Percoll非连续密度梯度离心法),急性分离并纯化健康成人静脉血中性粒细胞,分离纯化后的中性粒细胞进行4℃温度预处理备用。
2.使用激光共聚焦显微镜,观察不同处理因素下加入趋化剂fMLP时中性粒细胞胞质内Ca~(2+)瞬间动态变化,以确定SOCE机制是否参与中性粒细胞极性化过程。根据处理因素不同分为四组:空钙组(细胞外液使用无钙HEPEs液)、正常钙组(细胞外液使用含有1mM钙离子的HEPEs液)、高钙组(细胞外液使用含有2mM钙离子的HEPEs液)和2-APB组(细胞外液使用含有1mM钙离子的HEPEs液并使用SOCE阻断剂2-APB 100nM孵育15分钟)。
3.使用膜片钳,全细胞模式电压钳下,在不同protocol下记录不同药物预处理后加入趋化剂时全细胞电流的变化。以观测在中性粒细胞极性过程中SOCC通道电流是否参与,从而进一步确定SOCE机制是否参与中性粒细胞极性化过程,以及SOCE机制在中性粒细胞极性化过程中的作用。根据处理方法不同分组,分别为:Control组(对照组,不使用任何药物预处理,其后也不加入趋化剂fMLP)、fMLP组(不使用任何药物预处理,其后和实验组以相同方法加入终浓度为100nM的趋化剂fMLP)、SKF96365组(使用SOCE阻断剂SKF96365 10μM孵育15分钟后,加入终浓度为100nM的趋化剂fMLP)、2-APB组(使用SOCE阻断剂2-APB 100nM孵育15分钟后,加入终浓度为100nM的趋化剂fMLP)和TG组(使用内质网钙库倾空药物TG100nM孵育15分钟后,加入终浓度为100nM的趋化剂fMLP)。
4.采用流式细胞术,观察不同药物处理下加入趋化剂后,中性粒细胞极性化过程中标志性蛋白分子——极性相关骨架蛋白(F-actin)的免疫荧光变化。以观察SOCE阻断剂SKF96365、2-APB和内质网钙库倾空剂TG对细胞极性变化的影响,从而确定SOCE机制在中性粒细胞极性化过程中的作用。根据文献选择在加入趋化剂fMLP后0s(即不加fMLP)、10s、30s、60s、120s和240s这6个时间点内比较各个时间点不同组间的F-actin免疫荧光的变化差异。分组方法基本同膜片钳实验分组方法。
5.采用Transwell方法,观察不同药物处理后中性粒细胞的趋化性,分析SOCE机制和脂筏在中性粒细胞趋化过程中的作用。小室下室放入含fMLP 100nM的RMPI1640 300μL,上室加入经不同药物处理后的中性粒细胞悬液100μL[分别经TG 100nM(TG组),2-APB 100nM(2-APB组),SKF96365 10μM(SKF96365组)和mβCD 10mM(mβCD组)处理,37℃,5%CO_2孵箱内孵育15分钟],并设fMLP对照组(上室加入DMSO,剂量同溶解上述药物所用量,即fMLP组)。同时设空白对照组(上室加入DMSO,下室加入PBS液,剂量同溶解上述药物所用量,即Control组)。
结果
1.空钙组加入100nM fMLP后中性粒细胞胞内Ca~(2+)荧光值变化标准化比值显著小于正常钙组(n=9,P=0.027)。2-APB组中性粒细胞胞内Ca~(2+)荧光值变化标准化比值显著小于正常钙组(n=9,P=0.005)。
2.正常状态下中性粒细胞,加入终浓度100nM fMLP后,全细胞内向电流开始明显增加。持续一分钟之后缓慢下降并持续很长一段时间。经SKF96365 10μM、2-APB 100nM孵育15分钟后加入终浓度100nM fMLP,全细胞内向电流的增加(以钳制电位为-80mV时为例)显著被抑制(在step protocol下分别为P=0.004和P=0.022;在ramp protocol下分别为P=0.030和P=0.043)。经内质网钙库倾空剂TG 100nM孵育15分钟后,全细胞内向电流显著增大(在step protocol下为P=0.002;在ramp protocol下为P=0.002),加入终浓度100nM fMLP后全细胞内向电流增加不明显。
3.在fMLP作用240秒的时间点内2-APB预孵育组F-actin荧光值显著小于fMLP组(P=0.036),各时间点内SKF96365预孵育组和2-APB预孵育组胞内F-actin荧光值的均值均小于fMLP组。
4.2-APB预孵育组中性粒细胞趋化迁移能力显著小于fMLP组(P=0.034),SKF96365和mβCD预孵育组中性粒细胞趋化迁移率标准化比值均数小于fMLP组。
结论
1.中性粒细胞经趋化剂fMLP处理后,胞内钙离子浓度的升高包含两部分来源,即胞内钙库的钙离子释放和胞外钙离子的流入。fMLP诱导的中性粒细胞极性化过程中胞外钙离子的流入主要是通过SOCC通道完成的。SOCE机制在fMLP诱导的中性粒细胞极性化过程中胞外钙离子的流入过程中起重要作用。
2.中性粒细胞极性化过程中,SOCC通道占有重要地位,SOCE机制起了重要作用。同时可能存在其它钙离子通道和钙离子内流机制参与了中性粒细胞的极性化过程。
3.SOCE参与了中性粒细胞极性化过程,并在其中起关键性作用,阻断SOCE机制可以抑制中性粒细胞的极性化过程。
4.SOCE机制在中性粒细胞趋化迁移过程中也起重要作用,阻断SOCE可以严重影响到中性粒细胞的趋化迁移能力。同时,中性粒细胞的趋化迁移过程与脂筏密切相关,破坏细胞膜脂筏的完整性也可以影响到中性粒细胞的趋化迁移能力。在中性粒细胞趋化迁移过程中,SOCE是否与脂筏密切相关仍需要进一步实验确定。
Background
Polymorphonuclear neutrophils are phagocytic cells which have the function of directional locomotion.They are play an important role of host response in resisting to bacterium and eumycete,which due to their chemiotaxis.Neutrophils can recept signaling molecule and follow the concentration gradient of chemiotaxin to the location of inflamation.And the foundation of this migratory function is the process of neutrophils polarity according to the chemiotaxin.
Calcium signal plays an important role not only in signal conduction in cells but also in executing function of cells and it participates the whole vital process from fertilization of ovum to apoptosis of cells.Calcium ion can be not only the first messenger but also the second messenger.It can regulate the vital process and itself as well.The concentration of calcium ion in extracellular fluid is more than 10~(-3)M, while it in intracellular fluid is around 10~(-7)M.To maintain the homeostatic of Ca~(2+),it depends on some important region of accommodation to controlling and accommodation.Some proteins can be the ion channels in cell membrane or organelle plasmalemma to permeation Ca~(2+),and they have three groups according to their gating mechanisms:voltage-gating channel,ligand-gating channel and store-operated channel..
James Putney introduced the concept of a store-operated ca2+ entry(SOCE) in 1986.A select group of stimuli initiate cellular responses by acting on cell-surface receptors to increase the cytosolic Ca~(2+) concentration.The stimulation of these receptors induces the hydrolysis of membrane phosphoinositides by phospholipase C (PLC) enzymes yielding the diffusible Ca~(2+)-mobilizing messenger inositol 1,4,5-trisphosphate[Ins(1,4,5)P3],to release Ca~(2+) from non-mitochondrial Ca~(2+) stores.However,Ca~(2+) release is also followed by a stimulated Ca~(2+) entry in such cells and the ultimate reason for the Ca~(2+) influx is a decrease in the Ca~(2+) content of the endoplasmic reticulum(ER).The mechanism by which the ER Ca~(2+) stores communicate with the plasma membrane(PM) and the molecules participating in the Ca~(2+)-influx process remained elusive until a series of key discoveries identified the stromal interaction molecule(STIM) and Orai(also known as calcium-release-activated calcium-modulator[CRACM]) proteins that serve as ER calcium sensors and calcium channels,respectively.Following with the mechanism of SOCE was gradually recognized,the pharmacological means are also highlight to manipulate the functions of SOCE,and SK&F96365 and 2-APB were used widespread.
Neutrophils can be polarity when stimulated by chemotaxin,and in that process the concentration of Ca~(2+) in introcelluar fluid can developed a series of changes as well as its distribution.The chemotaxin fMLP can active the receptor of IP3 in ER and induce releasing Ca~(2+) from Ca~(2+) storage of ER.The releasing Ca~(2+) from Ca~(2+) storage of ER can active SOCE and promote Ca~(2+) inflow from extrocellar fluid.It is still unknown about whether SOCE participates in the process of cell polarity in neutrophils and what SOCE plays in this process.It also is not reported about what influence in the process of cell polarity and chemotaxis in neutrophils by block SOCE.
SOCE may play an important role in the process of cell polarity,and it may has well relationship with lipid raft in membrane.The key function of lipid raft is to be a platform for cell signal.To understand more about the relationship of SOCE with lipid raft,we have studied the influence in cell polarity by mβCD,mβCD can clear the cholesterol of membrane to destroy the integrality of lipid raft.It may influence the process of polarity.
Objective
The experiment is to investigate the role of SOCE in the process of cell polarity in neutrophils by observation the changes of calcium ion in introcellular fluid and the changes in cell polarity and chemotaxis after using the pharmacological means to manipulate the functions of SOCE.
Methods
1.Human peripheral blood neutrophils were obtained from healthy adult volunteers and separated on a discontinuous gradient consisting of percoll solution.The cells were preserved in 4℃.
2.Confocal microscopy was used to observe the changes of the concentration of calcium ion in intracellular fluid of neutrophils after stimulated by chemotaxis fMLP in different groups.According to different disposal,four groups were set: no calcium group(there was not any calcium ions in extracellular fluid),normal calcium group(there was 1mM calcium ions in extracellular fluid),high calcium group(there was 2mM calcium ions in extracellular fluid),and 2-APB group (there was 1mM calcium ions in extracellular fluid and 100nM 2-APB was used to block SOCE).
3.Patch clamp was used to observe the changes of the whole-cell current after stimulated by chemotaxis fMLP in different groups by using different protocol in the whole-cell and voltage clamp mode.According to different disposal,five groups were set:Control group(no drug was used to pretreat the neutrophils and after that fMLP was not used either),fMLP group(no drug was used to pretreat the neutrophils and after that fMLP was used as other experimental groups), SKF96365 group(10μM SKF96365 was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed),2-APB group(100nM 2-APB was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed),TG group(100nM TG was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed).
4.Flow cytometry was used to observe the changs of immunofluorescence of F-actin,the skeleton protein related to polarity,after stimulated by chemotaxis fMLP in different groups of neutrophils.According to reported,six time points (0 second,10 seconds,30 seconds,60 seconds,120 seconds and 240 seconds) after using fMLP was taken to observe the difference among the different groups in each time point.And in all these time points,the group settings are all similar to the setting in the experiment of patch clamp.
5.Transwell was used to observe the changes of chemotaxis of neutrophils in different groups.According to different disposal,six groups were set:Control group(RMPI1640 300μL with PBS was dropped in infra-room and the cell suspension of neutrophils 100μL with DMSO was put in upper-room,the dosage of PBS and DMSO was just as the dosage which dissolve the drugs using in experimental groups),fMLP group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with DMSO was put in upper-room),SKF96365 group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with SKF96365 10μM pretreatment for 15 minutes in 37℃and 5%CO_2 was put in upper-room),2-APB group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with 2-APB 100nM pretreatment for 15 minutes in 37℃and 5%CO_2 was put in upper-room),TG group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with TG 100nM pretreatment for 15 minutes in 37℃and 5%CO_2 was put in upper-room),and mβCD group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with mβCD 10mM pretreatment for 15 minutes in 37℃and 5%CO_2 was put in upper-room).
Results
1.The standard ratio of the change of fluorescence of calcium ion in intracellular fluid of neutrophils in no calcium group is significant minor to which in normal calcium group(n=9,P=0.027),and in 2-APB group it is also significant minor to which in normal calcium group(n=9,P=0.005).
2.The whole cell current of neutrophils began to augment quickly after stimulated by 100 nM fMLP and last about 1 minute around the peak.After that,the current began to decrease slowly but last a few minutes above the base current(more than 5 minutes).In the command potential of-80,the augmentation of the current was significantly inhibited if SKF96365 10μM or 2-APB 100nM was used to pretreat the cells(P=0.004 and 0.022 respectively in step protocol and P=0.030 and 0.043 respectively in ramp protocol).The whole cell current augmented significantly after the cells pretreatment by TG 100nM(P=0.002 in step protocol and P=0.002 in ramp protocol),while after that,the current did not augment significantly any more when stimulated by fMLP.
3.In the time point of 240 seconds,the fluoresence of F-actin in 2-APB group is significant smaller than fMLP group(P=0.036).In all of these time points,the fluoresence of F-actin in SKF96365 group is smaller than fMLP group according to their means.
4.The migration capability of neutrophils in 2-APB group is significant less than which in fMLP group(P=0.034).And The capability in SKF96365 and mβCD groups is both less than which in fMLP group just according to their means.
Conclusion
1.After neutrophils stimulated by fMLP,the augment of calcium ion concentration in cells has two resources:the release of ca~(2+) from ca~(2+) storage in cells and the inflow of ca~(2+) from outside.And the inflow of ca~(2+) from outside due to SOCC. So SOCE plays an important role in the process of cell polarity in neutrophils.
2.According to the experiment of patch clamp,it can be concluded that SOCC play an important role in the process of neutrophils polarity,and SOCE is most important to polarity.At the same time,other ca~(2+) channels may participate this process.
3.According to the experiment of flow cytometry,it suggested that SOCE participates in the process of neutrophils polarity and may play the key role in it. Blocking SOCE can inhibite the process of polarity.
4.SOCE plays an important role in the process of migration of neutrophils,and blocking it can inhibit the process.The process of neutrophils migration also has well relationship with lipid raft.Destroying the integrality of lipid raft can also inhibit the process of migration.Whether SOCE is correlated with lipid raft in this process still needs more experiments.
嗜中性粒细胞(PMNs)是具有定向移动功能的吞噬细胞,在抵抗细菌和真菌感染的宿主反应中起着重要的作用,这种功能是依靠趋化能力来实现的,趋化性是细胞感受信号分子从而具有的定向移动的能力,中性粒细胞能够沿着趋化物浓度梯度定向迁移到急性炎症的位置,这种迁移是中性粒细胞的核心功能。中性粒细胞能够迁移的基础在于其具有接受趋化刺激时具有形成极性化形状的能力。
Ca~(2+)信号在细胞的信号传导和执行功能等各个方面都发挥着重要的作用,影响着从卵子受精到细胞凋亡的整个生命过程。Ca~(2+)的独特性质在于其既能充当第一信使也能充当第二信使;既能调控细胞的生命过程,又能进行自身的调控。胞外Ca~(2+)浓度常超过10~(-3)M,而胞内至少低于胞外四个数量级之多(10~(-7)M),细胞这种Ca~(2+)稳态的维持依靠一些重要的调节区域通过一系列精妙的方式进行严密的控制和调节。在细胞膜或细胞器膜上,一些蛋白质形成的离子通道,其主要功能是通透Ca~(2+),这些离子通道依据门控机制分属于三组:电压门控性通道、配体门控性通道和库作性钙离子通道(Store-operated channels)。
1986年James Putney提出了库作性钙内流(SOCE)的概念。一组选择性刺激可以诱发细胞的应答,激活细胞表面受体从而诱导了胞膜磷酸肌醇的水解。通过磷脂酶C(PLC)产生活动性钙信号Inositol 1,4,5-trisphosphate[Ins(1,4,5)P3],从内质网(ER)钙库释放钙离子。同时,钙离子的释放伴随着外钙的内流。由于胞外钙离子的内流是由胞内钙库钙离子的释放倾空引起的,所以称为库作性钙内流。SOCE已经发现并提出多年,但其机制一直不甚清楚。最近研究发现可能与内质网膜上分布的感受分子Stiml和胞膜上的Orail等分子相互作用有关。随着人们对SOCE机制认识的逐渐发展,研究SOCE机制的工具药也逐渐受到人们的重视,其中普遍使用的有SK&F96365和2-APB。
中性粒细胞在受到趋化剂刺激后发生极性化的过程中,胞内Ca~(2+)浓度和分布会发生一系列改变。PMNs趋化剂甲酰甲硫氨酰-亮氨酰-苯丙氨酸(fMLP)可以激活内质网膜上IP3受体从而诱导内质网Ca~(2+)库释放钙离子。内质网Ca~(2+)库释放可以激活SOCE通路,促进外钙的内流。但SOCE机制是否参与中性粒细胞极性化过程以及在此过程中起何种作用,阻断SOCE后对中性粒细胞极性化过程以及趋化性有何影响,尚无确切报道。
SOCE在细胞极性化过程中可能起着重要的作用,而且这种作用可能与胞膜上脂筏(Lipid raft)结构存在密切的关系。脂筏的关键功能是它们能够作为信号的平台。为了进一步理解SOCE和脂筏的关系,我们观察了mβCD(甲基-β-环糊精,Methyl-β-cyclodextrin)对中性粒细胞极性的影响。mβCD能够清空中性粒细胞胆固醇,破坏脂筏完整性,从而可能影响到极性的形成过程。
目的
本实验通过研究中性粒细胞极性化过程中胞内Ca~(2+)信号的变化,以及应用工具药作用于SOCE机制后对细胞极性和趋化性的影响,探讨SOCE机制在中性粒细胞极性化过程中的作用。
方法
1.采用重复梯度密度离心的方法(Percoll非连续密度梯度离心法),急性分离并纯化健康成人静脉血中性粒细胞,分离纯化后的中性粒细胞进行4℃温度预处理备用。
2.使用激光共聚焦显微镜,观察不同处理因素下加入趋化剂fMLP时中性粒细胞胞质内Ca~(2+)瞬间动态变化,以确定SOCE机制是否参与中性粒细胞极性化过程。根据处理因素不同分为四组:空钙组(细胞外液使用无钙HEPEs液)、正常钙组(细胞外液使用含有1mM钙离子的HEPEs液)、高钙组(细胞外液使用含有2mM钙离子的HEPEs液)和2-APB组(细胞外液使用含有1mM钙离子的HEPEs液并使用SOCE阻断剂2-APB 100nM孵育15分钟)。
3.使用膜片钳,全细胞模式电压钳下,在不同protocol下记录不同药物预处理后加入趋化剂时全细胞电流的变化。以观测在中性粒细胞极性过程中SOCC通道电流是否参与,从而进一步确定SOCE机制是否参与中性粒细胞极性化过程,以及SOCE机制在中性粒细胞极性化过程中的作用。根据处理方法不同分组,分别为:Control组(对照组,不使用任何药物预处理,其后也不加入趋化剂fMLP)、fMLP组(不使用任何药物预处理,其后和实验组以相同方法加入终浓度为100nM的趋化剂fMLP)、SKF96365组(使用SOCE阻断剂SKF96365 10μM孵育15分钟后,加入终浓度为100nM的趋化剂fMLP)、2-APB组(使用SOCE阻断剂2-APB 100nM孵育15分钟后,加入终浓度为100nM的趋化剂fMLP)和TG组(使用内质网钙库倾空药物TG100nM孵育15分钟后,加入终浓度为100nM的趋化剂fMLP)。
4.采用流式细胞术,观察不同药物处理下加入趋化剂后,中性粒细胞极性化过程中标志性蛋白分子——极性相关骨架蛋白(F-actin)的免疫荧光变化。以观察SOCE阻断剂SKF96365、2-APB和内质网钙库倾空剂TG对细胞极性变化的影响,从而确定SOCE机制在中性粒细胞极性化过程中的作用。根据文献选择在加入趋化剂fMLP后0s(即不加fMLP)、10s、30s、60s、120s和240s这6个时间点内比较各个时间点不同组间的F-actin免疫荧光的变化差异。分组方法基本同膜片钳实验分组方法。
5.采用Transwell方法,观察不同药物处理后中性粒细胞的趋化性,分析SOCE机制和脂筏在中性粒细胞趋化过程中的作用。小室下室放入含fMLP 100nM的RMPI1640 300μL,上室加入经不同药物处理后的中性粒细胞悬液100μL[分别经TG 100nM(TG组),2-APB 100nM(2-APB组),SKF96365 10μM(SKF96365组)和mβCD 10mM(mβCD组)处理,37℃,5%CO_2孵箱内孵育15分钟],并设fMLP对照组(上室加入DMSO,剂量同溶解上述药物所用量,即fMLP组)。同时设空白对照组(上室加入DMSO,下室加入PBS液,剂量同溶解上述药物所用量,即Control组)。
结果
1.空钙组加入100nM fMLP后中性粒细胞胞内Ca~(2+)荧光值变化标准化比值显著小于正常钙组(n=9,P=0.027)。2-APB组中性粒细胞胞内Ca~(2+)荧光值变化标准化比值显著小于正常钙组(n=9,P=0.005)。
2.正常状态下中性粒细胞,加入终浓度100nM fMLP后,全细胞内向电流开始明显增加。持续一分钟之后缓慢下降并持续很长一段时间。经SKF96365 10μM、2-APB 100nM孵育15分钟后加入终浓度100nM fMLP,全细胞内向电流的增加(以钳制电位为-80mV时为例)显著被抑制(在step protocol下分别为P=0.004和P=0.022;在ramp protocol下分别为P=0.030和P=0.043)。经内质网钙库倾空剂TG 100nM孵育15分钟后,全细胞内向电流显著增大(在step protocol下为P=0.002;在ramp protocol下为P=0.002),加入终浓度100nM fMLP后全细胞内向电流增加不明显。
3.在fMLP作用240秒的时间点内2-APB预孵育组F-actin荧光值显著小于fMLP组(P=0.036),各时间点内SKF96365预孵育组和2-APB预孵育组胞内F-actin荧光值的均值均小于fMLP组。
4.2-APB预孵育组中性粒细胞趋化迁移能力显著小于fMLP组(P=0.034),SKF96365和mβCD预孵育组中性粒细胞趋化迁移率标准化比值均数小于fMLP组。
结论
1.中性粒细胞经趋化剂fMLP处理后,胞内钙离子浓度的升高包含两部分来源,即胞内钙库的钙离子释放和胞外钙离子的流入。fMLP诱导的中性粒细胞极性化过程中胞外钙离子的流入主要是通过SOCC通道完成的。SOCE机制在fMLP诱导的中性粒细胞极性化过程中胞外钙离子的流入过程中起重要作用。
2.中性粒细胞极性化过程中,SOCC通道占有重要地位,SOCE机制起了重要作用。同时可能存在其它钙离子通道和钙离子内流机制参与了中性粒细胞的极性化过程。
3.SOCE参与了中性粒细胞极性化过程,并在其中起关键性作用,阻断SOCE机制可以抑制中性粒细胞的极性化过程。
4.SOCE机制在中性粒细胞趋化迁移过程中也起重要作用,阻断SOCE可以严重影响到中性粒细胞的趋化迁移能力。同时,中性粒细胞的趋化迁移过程与脂筏密切相关,破坏细胞膜脂筏的完整性也可以影响到中性粒细胞的趋化迁移能力。在中性粒细胞趋化迁移过程中,SOCE是否与脂筏密切相关仍需要进一步实验确定。
Background
Polymorphonuclear neutrophils are phagocytic cells which have the function of directional locomotion.They are play an important role of host response in resisting to bacterium and eumycete,which due to their chemiotaxis.Neutrophils can recept signaling molecule and follow the concentration gradient of chemiotaxin to the location of inflamation.And the foundation of this migratory function is the process of neutrophils polarity according to the chemiotaxin.
Calcium signal plays an important role not only in signal conduction in cells but also in executing function of cells and it participates the whole vital process from fertilization of ovum to apoptosis of cells.Calcium ion can be not only the first messenger but also the second messenger.It can regulate the vital process and itself as well.The concentration of calcium ion in extracellular fluid is more than 10~(-3)M, while it in intracellular fluid is around 10~(-7)M.To maintain the homeostatic of Ca~(2+),it depends on some important region of accommodation to controlling and accommodation.Some proteins can be the ion channels in cell membrane or organelle plasmalemma to permeation Ca~(2+),and they have three groups according to their gating mechanisms:voltage-gating channel,ligand-gating channel and store-operated channel..
James Putney introduced the concept of a store-operated ca2+ entry(SOCE) in 1986.A select group of stimuli initiate cellular responses by acting on cell-surface receptors to increase the cytosolic Ca~(2+) concentration.The stimulation of these receptors induces the hydrolysis of membrane phosphoinositides by phospholipase C (PLC) enzymes yielding the diffusible Ca~(2+)-mobilizing messenger inositol 1,4,5-trisphosphate[Ins(1,4,5)P3],to release Ca~(2+) from non-mitochondrial Ca~(2+) stores.However,Ca~(2+) release is also followed by a stimulated Ca~(2+) entry in such cells and the ultimate reason for the Ca~(2+) influx is a decrease in the Ca~(2+) content of the endoplasmic reticulum(ER).The mechanism by which the ER Ca~(2+) stores communicate with the plasma membrane(PM) and the molecules participating in the Ca~(2+)-influx process remained elusive until a series of key discoveries identified the stromal interaction molecule(STIM) and Orai(also known as calcium-release-activated calcium-modulator[CRACM]) proteins that serve as ER calcium sensors and calcium channels,respectively.Following with the mechanism of SOCE was gradually recognized,the pharmacological means are also highlight to manipulate the functions of SOCE,and SK&F96365 and 2-APB were used widespread.
Neutrophils can be polarity when stimulated by chemotaxin,and in that process the concentration of Ca~(2+) in introcelluar fluid can developed a series of changes as well as its distribution.The chemotaxin fMLP can active the receptor of IP3 in ER and induce releasing Ca~(2+) from Ca~(2+) storage of ER.The releasing Ca~(2+) from Ca~(2+) storage of ER can active SOCE and promote Ca~(2+) inflow from extrocellar fluid.It is still unknown about whether SOCE participates in the process of cell polarity in neutrophils and what SOCE plays in this process.It also is not reported about what influence in the process of cell polarity and chemotaxis in neutrophils by block SOCE.
SOCE may play an important role in the process of cell polarity,and it may has well relationship with lipid raft in membrane.The key function of lipid raft is to be a platform for cell signal.To understand more about the relationship of SOCE with lipid raft,we have studied the influence in cell polarity by mβCD,mβCD can clear the cholesterol of membrane to destroy the integrality of lipid raft.It may influence the process of polarity.
Objective
The experiment is to investigate the role of SOCE in the process of cell polarity in neutrophils by observation the changes of calcium ion in introcellular fluid and the changes in cell polarity and chemotaxis after using the pharmacological means to manipulate the functions of SOCE.
Methods
1.Human peripheral blood neutrophils were obtained from healthy adult volunteers and separated on a discontinuous gradient consisting of percoll solution.The cells were preserved in 4℃.
2.Confocal microscopy was used to observe the changes of the concentration of calcium ion in intracellular fluid of neutrophils after stimulated by chemotaxis fMLP in different groups.According to different disposal,four groups were set: no calcium group(there was not any calcium ions in extracellular fluid),normal calcium group(there was 1mM calcium ions in extracellular fluid),high calcium group(there was 2mM calcium ions in extracellular fluid),and 2-APB group (there was 1mM calcium ions in extracellular fluid and 100nM 2-APB was used to block SOCE).
3.Patch clamp was used to observe the changes of the whole-cell current after stimulated by chemotaxis fMLP in different groups by using different protocol in the whole-cell and voltage clamp mode.According to different disposal,five groups were set:Control group(no drug was used to pretreat the neutrophils and after that fMLP was not used either),fMLP group(no drug was used to pretreat the neutrophils and after that fMLP was used as other experimental groups), SKF96365 group(10μM SKF96365 was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed),2-APB group(100nM 2-APB was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed),TG group(100nM TG was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed).
4.Flow cytometry was used to observe the changs of immunofluorescence of F-actin,the skeleton protein related to polarity,after stimulated by chemotaxis fMLP in different groups of neutrophils.According to reported,six time points (0 second,10 seconds,30 seconds,60 seconds,120 seconds and 240 seconds) after using fMLP was taken to observe the difference among the different groups in each time point.And in all these time points,the group settings are all similar to the setting in the experiment of patch clamp.
5.Transwell was used to observe the changes of chemotaxis of neutrophils in different groups.According to different disposal,six groups were set:Control group(RMPI1640 300μL with PBS was dropped in infra-room and the cell suspension of neutrophils 100μL with DMSO was put in upper-room,the dosage of PBS and DMSO was just as the dosage which dissolve the drugs using in experimental groups),fMLP group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with DMSO was put in upper-room),SKF96365 group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with SKF96365 10μM pretreatment for 15 minutes in 37℃and 5%CO_2 was put in upper-room),2-APB group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with 2-APB 100nM pretreatment for 15 minutes in 37℃and 5%CO_2 was put in upper-room),TG group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with TG 100nM pretreatment for 15 minutes in 37℃and 5%CO_2 was put in upper-room),and mβCD group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with mβCD 10mM pretreatment for 15 minutes in 37℃and 5%CO_2 was put in upper-room).
Results
1.The standard ratio of the change of fluorescence of calcium ion in intracellular fluid of neutrophils in no calcium group is significant minor to which in normal calcium group(n=9,P=0.027),and in 2-APB group it is also significant minor to which in normal calcium group(n=9,P=0.005).
2.The whole cell current of neutrophils began to augment quickly after stimulated by 100 nM fMLP and last about 1 minute around the peak.After that,the current began to decrease slowly but last a few minutes above the base current(more than 5 minutes).In the command potential of-80,the augmentation of the current was significantly inhibited if SKF96365 10μM or 2-APB 100nM was used to pretreat the cells(P=0.004 and 0.022 respectively in step protocol and P=0.030 and 0.043 respectively in ramp protocol).The whole cell current augmented significantly after the cells pretreatment by TG 100nM(P=0.002 in step protocol and P=0.002 in ramp protocol),while after that,the current did not augment significantly any more when stimulated by fMLP.
3.In the time point of 240 seconds,the fluoresence of F-actin in 2-APB group is significant smaller than fMLP group(P=0.036).In all of these time points,the fluoresence of F-actin in SKF96365 group is smaller than fMLP group according to their means.
4.The migration capability of neutrophils in 2-APB group is significant less than which in fMLP group(P=0.034).And The capability in SKF96365 and mβCD groups is both less than which in fMLP group just according to their means.
Conclusion
1.After neutrophils stimulated by fMLP,the augment of calcium ion concentration in cells has two resources:the release of ca~(2+) from ca~(2+) storage in cells and the inflow of ca~(2+) from outside.And the inflow of ca~(2+) from outside due to SOCC. So SOCE plays an important role in the process of cell polarity in neutrophils.
2.According to the experiment of patch clamp,it can be concluded that SOCC play an important role in the process of neutrophils polarity,and SOCE is most important to polarity.At the same time,other ca~(2+) channels may participate this process.
3.According to the experiment of flow cytometry,it suggested that SOCE participates in the process of neutrophils polarity and may play the key role in it. Blocking SOCE can inhibite the process of polarity.
4.SOCE plays an important role in the process of migration of neutrophils,and blocking it can inhibit the process.The process of neutrophils migration also has well relationship with lipid raft.Destroying the integrality of lipid raft can also inhibit the process of migration.Whether SOCE is correlated with lipid raft in this process still needs more experiments.
引文
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