摘要
吸入含游离二氧化硅的生产性粉尘可导致矽肺。至今矽肺发病机制仍不明确，目前普遍认为粉尘诱导的长期持续的肺组织炎症是矽肺的发病基础。肺泡巨噬细胞(Alveolar Macrophage，AM)是粉尘进入支气管肺泡后最主要的靶细胞，其与石英尘之间的相互作用是矽肺发病的关键，认为是石英引起肺部炎性反应的早期关键环节之一。然而AM在石英尘作用下的早期应答机制仍不是很清楚。近年一些研究已经确认巨噬细胞钾离子通道(Voltage dependent potassium channel)与脂多糖作用下的细胞活化及随后的功能应答有关。钾离子通道是细胞膜上的一种跨膜蛋白，不仅受细胞外刺激信号影响，还可通过膜电位改变传递信号至胞内。AM钾离子通道是否可以作为早期跨膜信号参与粉尘诱导的巨噬细胞炎性反应?据此本研究以AM电压依赖性钾通道为研究对象，探讨其在石英致细胞炎性反应中的作用与意义。
第一部分大鼠肺泡巨噬细胞膜电压依赖性钾离子通道膜片钳研究
目的：建立全细胞膜片钳记录模式及穿孔膜片钳记录模式，探讨大鼠肺泡巨噬细胞膜电压依赖性钾通道的电生理特性，并比较这两种记录模式的异同。
方法：SPF级SD大鼠，支气管肺泡灌洗法收集肺泡巨噬细胞，铺种在装有载玻片的24孔板内，于5％CO_2、37℃条件下培养2h后，取出玻片置于细胞池中。全细胞膜片钳玻璃微电极用标准硼硅酸玻璃毛细管分两步拉制而成，充灌内液后入水阻抗在4～6MΩ；穿孔膜片钳玻璃微电极用薄壁硼硅酸玻璃毛细管分两步拉制而成，充灌含制霉菌素的内液后入水阻抗在2～4 MΩ。记录模式建立后，在-160～60 mV范围内给予持续时间500 ms、以20 mV去极化跃迁的方波刺激。使用pClamp 9．0软件进行电流信号分析。钾通道激活曲线用Boltzmann方程：G／G_(max)=1／{1+exp[(V_(0．5)-V_m)／k]}进行拟合。
结果：全细胞膜片钳记录模式和穿孔膜片钳记录模式均可记录到AM膜上电压依赖性的内向整流钾电流及外向延迟钾电流。最大激活电压下，全细胞膜片钳记录模式记录到的外向延迟钾电流密度为8．32±4．24 pA／pF，内向整流钾电流密度为-6．77±3．89pA／pF，相应的半数激活电压分别为-28．69±2．65 mV和-90．04±2．15 mV，斜率因子为28．27±1．27与19．57±2．14；穿孔膜片钳记录模式记录到的AM膜外向钾电流密度为9．42±4．41 pA／pF，内向钾电流密度为-8．49±4．71 pA／pF，同比略大于全细胞记录结果，相应的半数激活电压分别为-15．38±2．30mV和-99．04±2．45mV，斜率因子为15．62±2．89和11．97±2．97，明显不同于全细胞记录模式所得参数，差异有统计学意义(P＜0．05)。全细胞膜片钳记录模式下膜电流在12min开始有衰减现象，20 min内几乎衰减完全，穿孔膜片钳记录模式下膜电流没有明显衰减现象，最长记录时间可接近40 min。
结论：(1)大鼠AM可表达电压依赖性内向整流钾电流和电压依赖性外向延迟钾电流两种。这两种电流的表达在巨噬细胞个体之间不同，综合起来主要有三种类型：以外向延迟钾电流为主，以内向整流钾电流为主，内向与外向钾电流表达皆明显。
(2)两种记录模式均可用于AM电压依赖性钾通道的研究，但全细胞膜片钳更适合短时大量的细胞记录，而穿孔膜片钳适合即时观察毒物或药物的作用。
(3)全细胞膜片钳记录模式实验参数为：电极选用标准壁厚的电极，入水阻抗控制在4～6 MΩ，刺激电压脉冲在-160～60 mV之间，记录应在破膜后10 min内完成；穿孔膜片钳记录模式实验参数为：电极选用薄壁电极，制霉菌素终浓度400μg／ml，电极入水阻抗控制在2～4 MΩ间，刺激电压脉冲在-160～60 mV之间，记录时间不宜超过40 min。
第二部分石英颗粒对大鼠肺泡巨噬细胞电压依赖性钾离子通道的影响
目的：研究石英颗粒对大鼠AM电压依赖性钾离子通道的影响及其动力学变化，探讨AM钾离子通道在石英颗粒即时处理或长时间(24 h)处理后的应答变化。
方法：AM获取与纯化同第一部分，细胞铺种于装有载玻片的24孔板内，浓度为1×105个／ml。向培养孔内加入不同浓度的石英颗粒：0μg／ml、25μg／ml、50μg／ml、100μg／ml、200μg／ml，及无定型石英颗粒100μg／ml。细胞继续培养24 h后取出进行常规全细胞膜片钳实验，记录钾电流变化。
另取未经任何处理的AM进行穿孔膜片钳实验。穿孔膜片钳建立以后，通过灌流系统使细胞外液以2 ml／min的速度缓慢流过细胞池，用自制注射加样系统在细胞池入口即时注入25μg／ml、50μg／ml、100μg／ml、200μg／ml的石英颗粒及100μg／ml的无定型石英颗粒测试液，串联刺激模式记录细胞膜钾通道的变化。不间断记录条件下即时加入石英颗粒，记录受体依赖的阳离子或阴离子电流。
细胞分离纯化后铺种在96孔板上，细胞浓度调为5×10~5个／ml，依次加入0μg／ml、25μg／ml、50μg／ml、100μg／ml、200μg／ml的标准石英颗粒及100μg／ml无定型石英颗粒，继续培养24h后，取出检测细胞乳酸脱氢酶(LDH)漏出率及细胞成活率(MTT法)。
结果：(1)100μg／ml石英颗粒24 h处理，可使AM外向延迟钾电流显著增加(P＜0．05)，但内向整流钾电流变化不明显(P＞0．05)。25μg／ml、50μg／ml石英颗粒及100μg／ml的无定型石英颗粒对AM外向与内向钾电流没有显著影响。
(2)与对照组相比，除100μg／ml石英组外，各处理组激活曲线均右移，但没有统计学差异(P＞0．05)，100μg／ml石英组使激活曲线显著左移(P＜0．05)；100μg／ml石英组斜率因子明显小于100μg／ml无定型石英组(P＜0．05)。
(3)即时作用下，随石英颗粒浓度增加，与对照组相比各处理组AM外向延迟钾电流与内向整流钾电流均增加，其中50μg／ml、100μg／ml、200μg／ml组增加明显(P＜0．05)。100μg／ml的无定型石英即时处理对AM外向与内向钾电流均没有显著影响。
(4)即时作用下，与对照组比较，石英各处理组外向延迟钾电流激活曲线均左移，半数激活电压和斜率因子明显小于对照组(P＜0．05)；内向整流钾电流激活曲线均右移，半数激活电压明显大于对照组(P＜0．05)，斜率因子明显小于对照组(P＜0．05)。100μg／ml无定型石英颗粒可使外向钾电流斜率因子显著降低(P＜0．05)，但对内向钾电流激活曲线影响不显著；外向钾电流半数激活电压明显大于相同剂量的石英处理结果(P＜0．05)，内向钾电流半数激活电压明显小于相同剂量的石英处理结果(P＜0．05)。
(5)石英颗粒不能诱发细胞膜阳离子或阴离子电流的出现。
(6)随石英颗粒浓度增加，LDH漏出率明显增加，存活率显著下降，与对照比较有统计学意义(P＜0．05)。100μg／ml的无定形石英颗粒处理，对AM存活率影响不明显，但LDH漏出率却达到46．0％，与等剂量的石英颗粒处理组相比程度较轻(P＜0．05)。
结论：石英颗粒对AM外向延迟钾通道有激活作用，使其开放概率增加，速率增加；对AM内向整流钾通道有激活作用，使其开放概率增加，速率增加，但长时间处理激活作用不明显，石英颗粒不能以受体方式影响AM电生理活性。提示AM电压依赖性钾离子通道可能是石英颗粒诱导细胞活化的早期信号蛋白之一，参与石英诱导的细胞坏死。
第三部分电压依赖性钾通道在石英致肺泡巨噬细胞活化及损伤中的作用
目的：应用电压依赖性钾通道阻断剂和激活剂，研究大鼠AM电压依赖性钾通道活性在石英颗粒诱导的AM活化、损伤和炎性介质分泌等生物效应的作用及意义。
方法：肺泡灌洗法获取SD大鼠AM，调整细胞浓度为5×10~5个／ml或1×10~6个／ml(用于TNF-α检测)铺种于96孔板内，每孔200μl。细胞纯化后，将钾通道阻断剂四乙基铵(TEA，终浓度为2．5 mM、5 mM、10 mM、20mM)、4氨基吡啶(4-AP，终浓度0．625 mM、1．25 mM、2．5 mM、5 mM)和激活剂K~+(终浓度15 mM、30 mM、60 mM、120 mM)分别与100μg／ml石英颗粒同时处理AM，正常培养24 h后测定细胞LDH漏出率、细胞存活率(MTT法)，ELISA法测定培养上清液TNF-α含量。
结果：(1)不同剂量的阻断剂TEA与石英颗粒同时处理AM，细胞膜LDH漏出率是单独石英颗粒处理组的51．6％～69．8％(P＜0．01)。阻断剂4-AP与石英颗粒同时处理AM，细胞膜LDH漏出率是单独石英颗粒处理漏出率的18．1％～39．1％(P＜0．01)。激活剂K~+与石英颗粒同时处理AM，细胞膜LDH漏出率是单独石英颗粒处理漏出率的101．1％～108．3％(P＞0．05)。
(2)阻断剂TEA与石英颗粒同时处理AM，细胞成活率是单独石英颗粒处理的152．5％～213．2％(P＜0．01)。阻断剂4-AP与石英颗粒同时处理AM，只有1．25 mM组细胞成活率高于单独石英颗粒处理组结果，其他剂量组成活率改变不明显。与单独石英颗粒处理比较，激活剂K~+(60 mM和120 mM)的使用可使细胞成活率降低(P＜0．05)。
(3)阻断剂TEA与石英颗粒同时处理AM，细胞TNF-α释放量是单独石英颗粒处理的47．6％～79．3％(P＜0．01)。阻断剂4-AP与石英颗粒同时处理AM，细胞TNF-α释放量是单独石英颗粒处理的44．1％～56．9％(P＜0．01)。激活剂K~+与石英颗粒同时处理AM，细胞TNF-α释放量是单独石英颗粒处理的132．8％～217．2％(P＜0．01)。
结论：AM钾离子通道与石英颗粒诱导的细胞膜损伤及死亡有关，对石英颗粒诱导的AMTNF-α释放有调节作用，可能是石英颗粒作用下的早期信号蛋白之一。
第四部分石英作用下肺泡巨噬细胞钾离子通道与胞内钙的关系
目的：应用电压依赖性钾通道阻断剂和激活剂，研究石英颗粒诱导下大鼠肺泡巨噬细胞电压依赖性钾通道与胞内钙离子浓度的关系，探讨粉尘诱导的细胞活化损伤信号机制。
方法：SD大鼠肺泡巨噬细胞获取与培养同第一部分。细胞浓度为1×10~5个／ml，均匀铺种在载有玻片的6孔板中，每孔2 ml。细胞纯化后，继续培养2 h，用Fluo-3／AM负载细胞30min后，置于激光扫描共聚焦显微镜上，激发波长488 nn，放射波长526 nm，记录细胞荧光变化，扫描频率为0．5 Hz。记录基线平稳后(约100 s)，即时加入各处理组(100μg／ml石英颗粒组，100μg／ml无定型石英颗粒组，100μg／ml石英颗粒与20 mM TEA组，100μg／ml石英颗粒与5 mM 4-AP组，100μg／ml石英颗粒与120 mM K~+组)，持续扫描记录20 min。分析比较各组荧光强度变化。以未加样时的钙离子荧光强度作为基础值，将加样后的钙离子荧光强度与之做比，得到标化的钙离子信号。
结果：在5个处理组中以石英颗粒处理胞内钙离子荧光强度增幅最高，达到2倍以上，且记录时间内信号强度变化一直维持在较高水平，荧光波动范围为2371．98±378．55，明显大于无定型石英颗粒处理组(P＜0．05)；无定型石英颗粒处理后钙离子浓度没有明显变化，荧光波动范围为447．62±164．36；K~+与石英颗粒共同处理后钙信号增幅小于单独石英颗粒处理结果，但差异没有统计学意义(P＞0．05)，荧光波动范围略大于单独石英颗粒作用，为2455．27±536．39，钙离子荧光强度在150 s～300 s有很明显的波峰出现；钾通道阻断剂4-AP与TEA分别与石英颗粒同时处理巨噬细胞，胞内钙离子浓度变化程度很小，与无定型石英颗粒处理持平，信号波动频繁，但范围明显小于石英颗粒处理结果(P＜0．05)。
结论：石英颗粒可致AM迅速启动钙信号过程，而无定型石英颗粒没有此作用，提示胞内钙信号是AM炎性应答的重要机制之一。钾离子通道阻断剂可明显降低石英颗粒致AM的升钙作用，提示AM细胞钾离子通道参与胞内钙信号的调节。
Silicosis is a lung disease that develops subsequent to inhalation of silica dust.Amechanism of action to explain these consequences of silica exposure is not established.It isgenerally accepted that persistent lung inflammation is the basis of silicosis.Alveolarmacrophages (AM) are main target cells after dust flow into the bronchoalveolar lavage,andthe interaction between AM and dust is a key process to silicosis,which is an early event ininflammatory response induced by silica.The mechanism of the early response of AM tosilica,however,is still not clear.Recently some research has confirmed that potassium ionchannels in macrophages associated with cells activation and functional response under LPStreatment.Potassium ion channel is a transmembrane proteins stimulated by extracellularsignal,which also can be conversed to the intracellular signal through membrane potentialchanges.This suggested that potassium ion channels in alveolar macrophage may beinvolved in dust-induced activation and response,which maybe an early transmembranesignal in the interaction between AM and dust.According to this hypnosis the voltage-gatedpotassium ion channels in alveolar macrophages was choused in this study so as to exploreits' roles in silica induced lung inflammation.
Part One Patch clamp study on voltage dependent potassium ionchannel in AM
Objective:To explore voltage-gated potassium channel electrophysiological characteristicsin rat alveolar macrophage cell by method of the whole-cell patch clamp recording mode, perforated-patch clamp recording mode,and compare the differences among those variousrecord modes
Methods:Alveolar macrophages were isolated from Sprague-Dawley rats bybronchoalveolar lavage,and seed at the 24-well plate with cover-slip.After culture 2h underthe conditions of 5% CO2,37℃,cover-slips were removed to the cell pool.Standardborosilicate glass electrode were used as microelectrode for whole-cell patch clampfabricated by two steps,and the resistance were 4-6MΩafter filling with inner solution;thin-walled borosilicate glass electrode were used as microelectrode for perforatedpatch-clamp also fabricated by two-steps,the impedance were 2-4 MΩafter filling withinner solution.A square pulse with duration of 500ms and range of-160-60 mV in step of20mV depolarization were given after recording mode was established.The current signalwas analysis using pClamp 9.0 software.Potassium channel activation curve were fitted byBoltzmann equation:G/G_(max)=1/{ 1+ exp[(V_(0.5)-V_m)/k]}.
Results:AM voltage-dependent inward rectifier potassium current and delayed outwardpotassium current can be recorded both in whole-cell patch clamp recording mode and inperforated-patch clamp recording mode,both recorded successfully eight cells respectively.At the condition of the largest stimulating voltage,current density of delayed outwardpotassium in whole-cell patch clamp recording mode was 8.32.24 pA/pF,current densityof inward rectifier potassium was-6.77±3.89 pA/pF respectively;half activation voltage of-28.69±2.65 mV and-90.04 2.15 mV were found respectively;slope factor was 28.27±1.27 and 19.57±2.14 respectively.Current density of outward potassium inperforated-patch clamp recording mode was 9.42±4.41 pA/pF,and the current density ofinward potassium was-8.49±4.71 pA/ pF,which is slightly larger than the one ofwhole-cell recording;the corresponding half of the activation voltage was-15.38±2.30mVand-99.04±2.45mV respectively,the slope factor was 15.62±2.89 and 11.97±2.97respectively,which was significantly different from the whole cell recording mode,thedifference was statistically significant (P＜0.05).Membrane current in whole-cell patchclamp recording mode decay beginning at 12min,and the current was decay almostcompletely in 20min.There was no significant attenuation of membrane current inperforated-patch clamp recording mode during recording period,and the longest recordingtime was close to 40 min.
Conclusions:1.Rat AM can express voltage-dependent inward rectifier potassium currentand voltage-dependent outward potassium currents.There were different among individualmacrophages.In a summary,there are three types of potassium expressing mode:the oneexpress mainly the delayed K~+ currents,the one express mainly the rectifier potassiumcurrent,and the one express both inward and outward potassium current.
2.Both models can be used to record voltage-gated potassium channel activity,butwhole-cellpatch clamp mode is more suitable for records of a large number of cells inshort-term,and perforated-patch clamp mode is suitable for the observation of the effect oftoxins or drugs on channel activity in real-time.
3.The experimental Parameters of whole-cell patch clamp recording mode are:standardwall thickness of the electrodes,impedance is controlled in the range of 4-6 MΩ,range ofvoltage stimulate pulse is-160-60 mV,and more importantly the record should becompleted in 10 min;The experimental parameters perforated-patch clamp recording modeare:thin-walled electrode,final concentration of nystatin is 400μg/ml,electrode impedanceis controlled in the range of 2-4 MΩ,range of voltage stimulating pulse is-160～60 mV,and record time should not be more than 40min.
Part two The effect of silica particles on voltage-gated potassiumion channel in AM
Objective:To study the effect and its dynamic changes of silica particles onvoltage-dependent potassium channel in AM,and explore the response mechanism ofpotassium channel in AM to silica particles after immediate or long (24 h)treatment.
Methods:The collection and purification of AM was same to the first part.cells wereplated 24-well plate with cover-slip,cell concentration was 1×10~5/ml.Differentconcentrations of silica particles:0μg/ml,25μg/ml,50μg/ml,100μg/ml,200μg/ml,amorphous silica particles and 100μg/ml were added to plate.The cover-slips were used forwhole-cell patch clamp experiments after 24h culture.
AMs without any treatment were used for perforated-patch clamp experiments. extracellular fluid flow slowly through cell pool at 2 ml/min by ALA perfusion system afterestablishment of perforated patch-clamp,silica particles of 25μg/ml,50μg/ml,100μg/ml,200μg/ml and 100μg/ml amorphous silica particles were injected to cell pool immediateusing self made tool,and thechange of potassium channel were recorded.Cation or anionoperated currents were tested by means of continuous recording mode after injection ofsilica particles.
For another excrement,cells were plated at 96-well plates,cell concentration adjustedto 5×10~5/ml,then adding 0μg/ml,25μg/ml,50μg/ml,100μg/ml,200μg/ml of standardquartz particles and 100μg/ml amorphous silica particles to each well,cell lactatedehydrogenase (LDH) leakage rate and cell survival (MTT method) were detected after 24hculture.
Results:1.24 h treatment of 100μg/ml silica particles enabled the AM outward potassiumcurrent increased significantly (P＜0.05),but the inward rectifier potassium current did notchange significantly (P＞0.05).25μg/ml,50μg/ml silica particles and 100μg/mlamorphous silica particles had no effect on AM outward and inward potassium currents.itwas not recorded successfully in 200μg/ml silica group,but the same results could beobserved after shorten the treating time to 2h.
2.In addition to 100μg/ml silica group,the activation curve of the other treatment groupswere shifted to right compared with the control group (P＞0.05) after co-cultured 24 h.Activation curve in 100μg/ml silica group shifted significant to left (P＜0.05);and slopefactor in 100μg/ml silica group was less significantly than that in 100μg/ml amorphoussilica group (P＜0.05).
3.With the increase of silica particles concentration,AM delayed outward potassiumcurrent and inward rectifier potassium current increased compared with the control groupunder real-time treatment,and the current increased significantly in 50μg/ml,100μg/ml,200μg/ml group (P＜0.05).The treatment of 100μg/ml amorphous silica to AM have nosignificant effect on outward and inward potassium currents.
4.The activation curve of delayed outward potassium current in all treatment group shiftto the left at the immediate treatment method,and half activation voltage was significantlyless than that of the control group (P＜0.05),and slope factor was also significantly lessthan the control group(P＜0.05);Activation curve of inward rectifier potassium current in all treatment group shifted to right,and half activation voltage was greater significantlythan that of the control group,the difference was statistically (P＜0.05),slope factor wassignificantly less than that of the control group (P＜0.05).100μg/ml amorphous silicaparticles treatment decreased statistically slope factor of outward potassiumcurrentcompared with the control group (P＜0.05),but activation curve of inward potassiumcurrent was not significantly affected.Half activation voltage of outward potassium currentin amorphous silica particles group was greater than that of the same dose of silicatreatment(P＜0.05),but the half activation voltage of inward potassium current inamorphous silica particles group were less significantly than the same dose of silicatreatment (P＜0.05).
5.Cationic or anionic membrane current in AM could not be induced by silica particles.
6.With the increase of silica particles concentration the AM LDH leakage was significantlyincreased,and the viability decreased significantly compared with the control (P＜0.05).100μg/ml amorphous silica particles treatment had no significant effect on the viability of AM,but the LDH leakage rate reached 46.0%,which was less than the result of same dosetreatment of silica (P＜0.05).
Conclusions:Silica particles have activation effect on AM delayed outward potassiumchannel and inward rectifier potassium channel,with the main kinetics of high probabilityand flow rate.Silica particles can not affect AM electrophysiological activity in method ofreceptor operation.These resultes suggested that AM membrane voltage-dependentpotassium channels may be one of the early signaling proteins in cell activation and damageinduced by silica particles.
Part three Roles of voltage-dependent K~+ channel in AM activationand injury induced by silica
Objective:Blocker or agonist of voltage-gated potassium channel were used in this studyto observe voltage-gated potassium channel activity in activation,damage and secretion ofinflammatory mediators of AM of rat treated by silica particles.
Methods:SD rat AM obtained by lavage,cells with concentration were adjusted to 5×10~5/ml or 1×10~6/ml (for detection of TNF-α) platedin 96-weli plates,each well fill with200μl.Purified cells were co-treated by potassium channel blocker tetraethyl ammonium(TEA,final concentration of 2.5mM,5mM,10mM,20mM),4 aminopyridine (4-AP,thefinal concentration 0.625mM,1.25mM,2.SmM,5mM),and agonist K~+ (final concentration15 mM,30 mM,60 mM,120 mM) with 100μg/ml silica particles at the same time,cellswere cultured 24 h and the culture supernatants were used for determination of the rate ofLDH leakage,cell viability (MTT method),TNF-αcontent of culture supernatantsdetermined by ELISA.
Results:1.The leakage rate of LDH in groups of blockers TEA and silica particles wereabout 51.6%-69.8% of that of silica particles dealing with alone (P＜0.01).The leakagerate of LDH in groups of blocker 4-AP and silica particles were about 18.1%-39.1% ofthat of silica particles dealing with alone (P＜0.01).The leakage rate of LDH in groups ofagonist K~+ and silica particles were about 101.1%-108.3% of that of silica particles dealingwith alone (P＞0.05).
2.Cell viability in group of blockers TEA and silica particles were about 152.5%-213.2%of that of group silica alone (P＜0.01).Cell viability in group of blockers 4-AP and silicaparticles were about 152.5%-213.2% of that of group silica alone (P＜0.01).Cell viabilityonly in group of 1.25 mM 4-AP with silica particles was higher than that of group of silicaparticles,the viability change in other groups were not obvious.Cell viability in group ofagonist of K~+ and silica particles (60mM and 120mM)were significantly less than theresults of individual quartz treatment (P＜0.05).
3.TNF-αrelease in group of blocker TEA and silica were 47.6%-79.3% of that of silicatreatment alone (P＜0.01).TNF-αrelease in group of blocker 4-AP and silica were 44.1%-56.9% of that of silica treatment alone (P＜0.01).TNF-αrelease in group of K~+ and silicawere 132.8%-217.2% of that of silica treatment alone (P＜0.01).
Conclusions:Voltage gated potassium ion channel activity in AM may be related tomembrane damage and cell necrosis of AM induced by silica particles,and can modulate therelease of TNF-αinduced by silica particles.Outward and inward potassium channel in AM may be an early signaling protein in response to silica treatment.
Part four Relationship between potassium ion channels inmembrane of AM and intracellular calcium induced by silica
Objective:Blocker and agonist of voltage-gated potassium channel were used to studyrelationship between potassium ion channels in membrane of AM and intracellular calciumsignal induced by silica,to explore signaling mechanism of cell activation and injuryinduced by silica particles.
Methods:Collecting and pre-treatment of alveolar macrophage were same to the first part.Cell concentration were adjusted to 1×10~5/ml and plated to 6-well plate containing glass slip,each well contain 2 ml median.Purified cells continued to culture for 2h,and Fluo-3/AMwas used to load cells for 30min,remove to laser scanning confocal microscope,then usingexcitation wavelength 488 nm and radiation wavelength of 526 nm to record fluorescencechanges in cell,scanning frequency was 0.5 Hz.After fluorescence changes was in a stablebaseline (about 100 s),immediately injected different treatment liquid containing:100μg/ml silica particles,100μg/ml amorphous silica particles,100μg/ml silica particles and20 mM TEA,100μg/ml silica particles and 5mM 4-AP,100μg/ml silica particles and 120mM K~+,then continuous scanning for 20 min.Fluorescence intensity changes in each groupwere analyzed and compared.Standardized changes in calcium signal were calculated byratio of calcium ion fluorescence intensity in excremental group to calcium ion fluorescenceintensity in control group were as the basis of value.
Results:Intracellular calcium fluorescence intensity were increased most in silica group inthese five testing group,reaching more than 2 times,and signal intensity has beenmaintained at a higher level during recording period,range of fluorescence fluctuationswere 2371.98±378.55,which was significantly large than that of amorphous silica particlestreatment (P＜0.05);amorphous silica particles treatment had no effect on calciumconcentration,which even was in a trend of lower,range of fluorescence fluctuations was447.62±164.36;K~+ and silica particles co-treatment increased calcium signal,the amplitude was less than the results of silica particles along,but the difference was notstatistic significant(P＞0.05),range of fluorescence fluctuations was 2455.27±536.39,which was slightly larger than silica particles treatment alone,and there are obvious calciumfluorescence intensity peaks appear in 150 s-300 s;potassium channel blockers 4-AP andTEA co-treatment with silica respectively have no obvious effect on intracellular calciumconcentration changes,which was similar to amorphous silica particles treatment,butfrequent of signal fluctuations was high with the scope significantly less than silica particlestreatment (P＜0.05).
Conclusion:Silica particles have the effect to induce AM initiated calcium signal quikly,suggesting calcium signal is one of most important mechanism in inflammtory response tosilica particles.Blockers of K~+ channel can significantly reduce the increase of calciumconcentration induced by silica particles,which suggested that outward and inwardpotassium channel potassium channels in membranes of cell may be involved in rugulationof intracellar calcium signal.
引文
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