【作者】 王海雁；

【导师】 双少敏； 蔡明发；

【作者基本信息】 山西大学 ， 分析化学， 2008， 博士

【摘要】 细胞内游离钙离子的浓度及其分布的变化代表着某种细胞功能的启动、加强和抑制,是形成Ca²⁺信号的基础。破译不同型式的Ca²⁺信号所代表的生物学意义,可以大大加深人们对淋巴细胞功能调节机制的认识,并进一步探索外源性刺激对免疫细胞钙信号系统的扰动机理。本文运用两种测定胞内游离钙的荧光探针fura-2、fluo-3,以及测定细胞膜电压的带负电荷类菁荧光染料Bis-oxonol,应用荧光光谱仪与激光共聚焦扫描显微镜分别在多细胞和单细胞两个层面研究了外源性化学小分子青霉素G、头孢哌酮和丙烯酰胺对急性提取的人外周血淋巴细胞胞内游离钙离子信号的影响。选择肌浆网钙泵抑制剂（Thapsigargin）、非特异性钙通道抑制剂La³⁺、Ni²⁺、电压依赖L型钙通道抑制剂尼卡地平（Nicardipine）、电压依赖N型钙通道抑制剂芋螺毒素（ω-conotoxin）和fura-2荧光猝灭剂Mn²⁺参与外源性小分子对胞内游离钙离子信号的干扰过程,分析了这些外源小分子引发淋巴细胞胞内钙水平的升高和降低现象的作用机制。以荧光光谱学方法对抗生素的用药安全问题、丙烯酰胺的食品安全问题给出基础数据。同时建立了丙烯酰胺SPE-HPLC-UV的检测方法。第一章阐述了细胞钙离子信号转导的研究动态,简单介绍了淋巴细胞胞内钙离子水平的调控通路,综述了细胞内钙离子浓度检测方法的进展。第二章β-内酰胺抗生素中的青霉素G不仅临床应用广泛,而且体外培养细胞的各种细胞生理液中均被加入100U/mL的青霉素G以抑制杂菌污染。每公顷农业用土壤中青霉素G残留含量也超过一千克,这些青霉素G有可能进入食物链最终影响人体健康。应用fura-2荧光探针测钙技术研究了拟生理状态下低剂量的青霉素G药物小分子对淋巴细胞胞内钙信号的降低机制。青霉素G可以激活细胞膜上的钙泵,诱导淋巴细胞胞内游离钙离子外排。细胞外的钠、钙离子浓度的改变协同影响淋巴细胞内游离钙的水平,低钠和高钙的胞外环境能够加强青霉素G的排钙作用。实验证实了青霉素G能够抑制钠钙交换蛋白的促进外钙内流的能力。青霉素G能够消弱强心剂药物乌本苷提升胞内钙水平的药效。第三章第三代β-内酰胺抗生素头孢哌酮在拟生理状态下与低剂量的青霉素G有着相同的排钙趋势,强度弱于青霉素G。头孢哌酮也能够抑制钠钙交换蛋白促进的外钙内流,同时低钠和高钙的胞外环境也能够加强头孢哌酮的排钙作用。选择肌浆网钙泵抑制剂（Thapsigargin,Tg）和非特异性钙通道抑制剂La³⁺、Ni²⁺参与头孢哌酮的促进内钙外排或抑制外钙内流的降钙过程中。研究发现头孢哌酮有着和非特异性钙通道抑制剂Ni²⁺相似的针对该类钙通道蛋白的作用位点。头孢哌酮能够抑制Tg激活的储池操纵钙通道（SOC）操纵的胞外钙离子的内流,竞争性结合SOC通道的钙结合位点,而且头孢哌酮对SOC操纵的胞外钙离子的内流的抑制作用是可逆转的。实验证实了微量青霉素G和头孢哌酮都能够对静息状态免疫系统开始免疫应答所要求的胞内游离钙水平升高产生抑制,发现血液中微量β-内酰胺抗生素的存在能促进淋巴细胞胞内钙流失,导致淋巴细胞内钙水平降低,延迟淋巴细胞对抗原的应激反应,扰动免疫系统平衡,从而降低淋巴细胞的免疫活力。第四章近年来高温烘烤煎炸淀粉类食物中所含相应剂量的丙烯酰胺的毒性研究成为全球热点。丙烯酰胺对多细胞的钙离子信号的干扰试验表明丙烯酰胺对胞内钙离子浓度变化的扰动表现为低浓度促进内钙增加,高浓度抑制内钙增加。虽然钙超载与钙缺乏相互对立,但均由同一刺激引起,只是表现在剂量上、时间上的不同。随着丙烯酰胺的作用剂量加大,作用时间延长,淋巴细胞钙离子的跨膜转运被抑制,细胞内游离的总钙量减少,该作用与细胞内钠离子和钾离子协同相关,最终导致静息钙水平低于正常。单细胞层面重点论证了淀粉类食品中广泛存在的低于54μg/mL以下的丙烯酰胺使淋巴细胞[Ca²⁺]_i迅速升高的作用机制。低于54μg/mL以下的丙烯酰胺使细胞[Ca²⁺]_i迅速升高并在400秒之内形成较高水平钙浓度平台,若无其他干扰,升高的内钙可以在一小时之内回复至初始状态。胞外无钙的外环境使丙烯酰胺无法诱发内钙升高。丙烯酰胺不能刺激胞内内质网钙库和线粒体上的钙池释钙,表明受体协调的钙进入在此条件下没有作为。1.0 mM Ni²⁺的加入将丙烯酰胺诱导的[Ca²⁺]_i升高降低了35%,丙烯酰胺能够诱导Mn²⁺进入淋巴细胞导致的fura-2荧光促灭,说明丙烯酰胺刺激了非选择性离子通道的钙进入。电压依赖钙通道（VGCC）L型抑制剂尼卡地平对丙烯酰胺的升钙作用没有作为,而电压依赖N型钙通道抑制剂芋螺毒素使内钙升高明显降低30%,该结果表明,丙烯酰胺通过对非选择性离子通道以及某些N型VGCC的刺激,诱发内钙升高。丙烯酰胺对淋巴细胞的[Ca²⁺]_i的干扰与细胞内含量丰富的K⁺、Na⁺和Ca²⁺的协同作用相关联。另一方面,实验证实生理水平存在的维生素C、E能显著降低丙烯酰胺导致的内钙升高,表明饮食中的抗氧化剂可以降低丙烯酰胺导致的细胞损伤。第五章建立了丙烯酰胺SPE-HPLC-UV的检测方法。样品中丙烯酰胺的提取和纯化选择在低温（0℃）高速离心（13000×g）的条件下。离心过程中通过硬化油脂层并伴随微孔滤膜过滤,简单高效的降低了脂类杂质的干扰。测试过程中选择4.0%v/v的乙腈水作为流动相,梯度洗脱程序既能改善色谱柱,又可以将丙烯酰胺从疏水杂质中分离出来。工作曲线系列标准溶液的浓度限定在50-2000μg/L之间,检测210 nm处吸收强度,校正曲线线性良好,线性系数R＞0.999。在100μg/kg浓度水平的加标回收率为78%～107%。检出限和定量限分别为6和23μg/kg。比LC-MS/MS方法的相应数值稍高,但低于国家监控食品中丙烯酰胺的标准。本方法的室内精度在2.1-10.9%之间。更多还原

【Abstract】 Chapter 1 The modulation of cytosolic calcium signaling of lymphocytes was studied by little extrinsic molecules,useing fura-2 and fluo-3 as fluorescence probe.Ca²⁺ as a universal intracellular messenger and regulates a broad of physiological functions.The specific and coordinated activation of many cellular processes by stimulus-induced changes in the intracellular Ca²⁺ concentration[Ca²⁺]_i requires appropriate temporal and spatial coding patterns.As a result,low agonist concentrations cause Ca²⁺ oscillations or repetitive Ca²⁺ spikes arising from cyclical Ca²⁺ release from,and Ca²⁺ reuptake into,the endoplasmic reticulum,whereby agonist concentration and in the frequency of Ca²⁺ osillations arecorrelated.Thus,the Ca²⁺ signal is frequency-modulated.The aim of this study was to investigate the molecular mechanisms responsible for the generation of these amplitude-modulated Ca²⁺ signal.Chapter 2 The effect of penicillin on intracellular free calcium concentration was studied by monitoring the fluorescence of human peripheral lymphocytes loaded with fura-2.High internal Na⁺ was expected to increase the driving force for Ca²⁺ influx via Na⁺/Ca²⁺ exchanger,and under the effect of penicillin high internal Na⁺ was also able to increase the driving force for Ca²⁺ efflux.Furthermore,when penicillin acted on the cells, a substantial role for the Ca²⁺ efflux through a Na⁺-dependent mode in Ca_i²⁺ homeostasis was indicated.Decreased temperature significantly slowed the Ca²⁺ elevation in Na⁺-PSS solution,it seemed that penicillin had little influence on the increasing of[Ca²⁺]_i induced by higher temperature.The effects of penicillin G were retained in the presence of nikadipine,a potent inhibitor of voltage-dependent calcium influx pathways.These results showed that penicillin G stimulated Ca²⁺ extrusion following Na⁺-dependent transporters. Both an increase in cytosolic[Ca²⁺]and reversal of the Na⁺ gradient are necessary to demonstrate enhanced effect of penicillin on Ca²⁺ homeostasis.The more[Ca²⁺]_i increased, the more penicillin effected.In conclusion,penicillin-induced decreases in intracellular free calcium concentrations and stimulated Ca²⁺ extrusion following Na⁺-dependent transporters.It is evident that penicillin can induce the Ca²⁺ release,which can be produced independent of ouabain.The results support the conclusion of the penicillin effect studies and indicate that there is involment of the Na⁺/Ca²⁺ antiporter system and/or calcium channels in the Ca²⁺ mobilization induced by reversal Na⁺ gradient in lymphocytes.The general increase trend of[Ca²⁺]_i can be diminished by penicillin.Chapter 3 Cefoperazone is widely distributed into most body fluids and tissues reaching conc- entrations higher than the minimum concentrations of susceptible bacteria.We first investigated the effect of cefoperazone on the resting[Ca²⁺]_i of human lymphocytes. With the increasing concentration of cefoperazone,the remarkable decreases of[Ca²⁺]_i were observed.The basal[Ca²⁺]_i in resting human peripheral lymphocytes was 100±10nM.In a Ca²⁺-containing solution,addition of 200μg/mL cefoperazone to the extracellular medium produced a cytosolic calcium decrease of 55±11 nmol/L. However,this calcium decrease was observed as 20±11 nmol/L in a Ca²⁺-free solution, suggesting that,it might be due to the inhibition of calcium influx from the external medium³.The more[Ca²⁺]_i increased,the more cefoperazone effected.In a Na⁺-free and Ca²⁺-free medium,200μg/mL cefoperazone significantly reduced a 30%decrease of [Ca²⁺]_i on control samples.These further reveal that cefoperazone induced the decline of Ca²⁺ rise in human lymphocytes by the inhibition of Na⁺-dependent Ca²⁺ entry. Preincubation with 0.1 mmol/L ouabain can operate the Na⁺/Ca²⁺ exchanger in the reverse mode by which outer Ca²⁺ can quickly enter the lymphocytes,but it had almost no effect on the cefoperazone-induced calcium flowing out.It indicated that ouabain-induced calcium entry by the reverse mode of Na⁺/Ca²⁺ exchanger was inhibited.The sustained Ca²⁺ signal relies on the operation of the ion channels in lymphocytes,hence the Ca²⁺ channels for Ca²⁺ influx are to serve as targets for this cephalosporins antibiotic,and further interactions lead to the losing of Ca²⁺ reserve in the lymphocytes.Chapter 4 Acrylamide is a small,water soluble,organic molecule being a vinyl monomer formed from the hydration of acrylonitrile.Acrylamide can increase cytosolic free Ca²⁺ concentration,but no studies have investigated the mechanism underlying acrylamide-induced increase in[Ca²⁺]_i in immune cells.In view of this,we investigated the effect of acrylamide（5-50μM） on[Ca²⁺]_i in human lymphocytes using a fluorescence Ca²⁺ indicator,fluo-3.Acrylamide caused sustained increases in[Ca²⁺]_i,in a concentration-dependent manner.The acrylamide-induced increase in[Ca²⁺]_i was abolished by the omission of extracellular Ca²⁺,suggesting that acrylamide induce a calcium influx by the opening of Ca²⁺ channels.We further employed an unspecific inhibitor of plasma membrane Ca²⁺ channels.We observed that Ni²⁺ curtailed significantly the calcium rise.The omission of extracellular Na⁺ failed to inhibit the acrylamide-induced increases in[Ca²⁺]_i.Furthermore,nicardipine had no effect on the increases in[Ca²⁺]i.but this increases was partially inhibited byω-conotoxin.Our results confirmed that acrylamide induced plasma membrane depolarization,known to be involved in the opening of the voltage-gated calcium channels.Acrylamide also induced Mn²⁺ influx into lymphocytes.These results suggest that acrylamide transiently increases [Ca²⁺]_i in lymphocytes by stimulating Ca²⁺ entry via modulating the permeability of calcium channels.Some dietary antioxidants such as Vitamin C and E can be considered as protective agents against interference action of acrylamide.Chapter 5 A fast and cost-effective method using HPLC/UV has been developed for determination of acrylamide in deep-fried flour-based leaven dough foods available in Hong Kong.The samples were purified by a simple solid-phase extraction method which combined Oasis HLB and Bond Elut-Accucat cartridges.The aqueous sample solution was centrifuged at 13000×g and 0℃for 15 rain to successfully remove the fat in the samples.A gradient elution program and a mobile phase of 4.0%（v/v） acetonitrile in water allowed sufficient retention and well resolved acrylamide from the food matrices in the sample extracts.Acrylamide was detected at UV wavelengths of 210 and 225 nm.The amounts of acrylamide in eight food samples were 27-198μg/kg when 1-g samples were analyzed.The recoveries of acrylamide were larger than 78.0%and the precisions were 2.1-10.9%（n=3）,respectively.Our proposed method is especially relevant for analyzing acrylamide in those oily food matrices.更多还原