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Fluorescent Studies on the Modulation of Cytosolic Calcium Signaling of Lymphocytes by Little Extrinsic Molecules

Author: WangHaiYan
Tutor: ShuangShaoMin；CaiMingFa
School: Shanxi University
Course: Analytical Chemistry
Keywords: Fura-2 Fluo-3 [Ca2] i Acrylamide β- lactam antibiotics
CLC: Q2
Type: PhD thesis
Year: 2008
Downloads: 192
Quote: 1
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Abstract

Intracellular free calcium concentration and changes in the distribution represents the start of some kind of cell function, strengthen and inhibit the formation of Ca ^{2 signal basis. Decipher different types of Ca ^{2 signals represent biological significance, can greatly deepen people's understanding of the regulatory mechanism of lymphocyte function, and exogenous stimulus to further explore the mechanism of immune cell calcium signaling system disturbance . In this paper, two measurements of intracellular free calcium fluorescent probe fura-2, fluo-3, as well as the determination of membrane voltage with a negative charge class cyanine fluorescent dye Bis-oxonol using fluorescence spectrometer with confocal laser scanning microscope in a multi-cell and the two levels of the single-cell studies the impact of exogenous chemical the small molecule penicillin G, the of cefoperazone and acrylamide acute extracted from human peripheral blood lymphocytes intracellular free calcium signal. Select the sarcoplasmic reticulum calcium pump inhibitor (by Thapsigargin in), non-specific calcium channel inhibitor La ^{3 Ni ^{2 , voltage-dependent L-type calcium channel blocker nicardipine ( Nicardipine), voltage-dependent N-type calcium channel inhibitor from Conus toxins (ω-conotoxin) and fura-2 fluorescence quencher Mn ^{2 in exogenous small molecule intracellular free calcium signal disturbance processes, analyzes the mechanism of action of these small molecules Exogenous initiator lymphocyte intracellular calcium levels rise and reduce the phenomenon. Fluorescence spectroscopy method of antibiotic drug safety issues of food safety issues of acrylamide given the underlying data. While establishing acrylamide SPE-HPLC-UV detection method. The first chapter cell calcium signal transduction dynamic brief regulatory pathway for lymphocyte intracellular calcium levels, the progress of the concentration of intracellular calcium detection method. Chapter II of the β-lactam antibiotics penicillin G is not only a wide range of clinical applications, and cultured in vitro to cells in a variety of cell physiological fluid were added 100U/mL penicillin G to inhibit bacterial contamination. Per hectare for agricultural use more than one kg penicillin G residues content in the soil, these penicillin G may enter the food chain and ultimately affect human health. Application fura-2 fluorescent probe of calcium intends physiological state of low doses of penicillin G drug molecule on lymphocytes reduced intracellular calcium signal mechanism. Penicillin G can be activated on the cell membrane calcium pump induced lymphocyte intracellular free calcium efflux. Extracellular sodium, calcium ion concentration change co-lymphocyte intracellular free calcium levels, low sodium and high calcium extracellular environment to strengthen the role of calcium excretion of penicillin G. The experiments confirmed penicillin G can inhibit the ability to promote calcium influx NCX. Penicillin G can weaken cardiac stimulant drug ouabain enhance the efficacy of intracellular calcium levels. Chapter cefoperazone third-generation β-lactam antibiotics in the intended physiological state where calcium row has the same trend with a low dose of penicillin G, strength weaker penicillin G. Cefoperazone also able to inhibit the sodium-calcium exchange proteins promote extracellular calcium influx, low sodium and high calcium extracellular environment can also enhance calcium excretion of cefoperazone. Select the sarcoplasmic reticulum calcium pump inhibitors (Thapsigargin, Tg) and non-specific calcium channel inhibitor La ^{3 Ni ^{2 participate in cefoperazone promote calcium efflux or in the inhibition the calcitonin process calcium influx. The study found cefoperazone has the non-specific calcium channel inhibitors Ni ^{2 similar for the role of the class of calcium channel protein sites. Cefoperazone activated a reservoir can be suppressed Tg manipulating Calcium Channel (SOC) within the flow of the manipulation of the extracellular calcium ions, the competitive binding of the calcium binding sites of the SOC channel, and cefoperazone manipulation of extracellular calcium ions on SOC within the flow of the inhibition is reversible. The experiments confirmed trace penicillin G and cefoperazone resting state of the immune system to start the immune response to the requirements of the intracellular free calcium levels produce inhibition found blood trace the presence of β-lactam antibiotics can promote lymphocyte cell loss of intracellular calcium, leading to lower calcium levels in lymphocytes and delayed stress response of lymphocyte antigen, the disturbance of the balance of the immune system, which reduces immune activity of lymphocytes. Chapter acrylamide in fried starchy foods contained in the corresponding dose toxicity studies in recent years, high-temperature baking to become the world's hot spots. The acrylamide multi-cellular calcium ion signal interference test showed that acrylamide on changes in intracellular calcium ion concentration disturbance performance increase in intracellular calcium promote low concentration, high concentrations inhibit intracellular calcium increase. Although the calcium overloaded with calcium lack of opposition to one another, but caused by the same stimulus, but the performance in the dose, different time. With the increasing effect dose of acrylamide, the action time, the lymphocyte transmembrane transport of calcium ions is suppressed, and decrease in total intracellular free calcium, this effect is associated in coordination with the intracellular sodium ion and potassium ion, and ultimately lead to the static interest rate lower than normal levels of calcium. The focus of the single-cell level demonstrated widespread in starchy foods below below 54μg/mL acrylamide lymphocytes rapidly increased [Ca ^{2 ] _i mechanism . Lower than 54μg/mL acrylamide rapid rise and a higher level of calcium concentration platform within 400 seconds, in the absence of other cell [Ca ^{2 ] _i interference, increased intracellular calcium can revert to its original state within one hour. The extracellular calcium outside environment acrylamide can not be induced intracellular calcium increase. Acrylamide can not stimulate the release of endoplasmic reticulum intracellular calcium stores and mitochondrial calcium pool calcium, indicating that the receptor coordination of calcium into this condition as. 1.0 mM Ni ^{2 adding acrylamide induced of [Ca ^{, 2 ] _i increased to reduce by 35% acrylamide able to induce Mn < sup> 2 enter lymphocytes due to fura-2 fluorescence promote off, acrylamide stimulation of non-selective ion channel calcium entry. The voltage-dependent calcium channel (VGCC) l calcium role of L-type inhibitor nicardipine acrylamide as no voltage-dependent N-type calcium channel inhibitors conotoxins intracellular calcium increase was significantly reduced by 30%, the results show that, acrylamide by non-selective ion channels, as well as some of the N-type VGCC stimulation induced intracellular calcium increase. Acrylamide the lymphocytes [Ca ^{2 , _{interference with abundant intracellular K ^{Na ^{associated synergies and Ca ^{2 . On the other hand, the experiments confirmed that physiological levels of the presence of vitamin C, E can significantly reduce acrylamide result of intracellular calcium increase, showed that the antioxidants in the diet can be reduced acrylamide causes cell damage. Chapter acrylamide SPE-HPLC-UV detection method. The sample in the extraction and purification of acrylamide is selected under the conditions of low temperature (0 ℃) high speed centrifugation (13000 × g). Centrifuge process, accompanied by hardening grease layer and the filter membrane lipid impurities is simple and efficient to reduce interference. The testing process of selection 4.0% v / V aqueous acetonitrile as the mobile phase gradient elution procedure can improve the column, and acrylamide can be separated from hydrophobic impurities. Limit the concentration of the working curve series of standard solutions in 50-2000μg / L detection at 210 nm absorption intensity, good linearity of the calibration curve, linear coefficient R> 0.999. Concentration levels in 100μg/kg spiked recoveries of 78% to 107%. The limit of detection and quantification limits were 6 and 23μg/kg. Countries, but lower than the corresponding value is slightly higher than the LC-MS/MS method to monitor the standard of acrylamide in food. Indoor accuracy of the present method is in the range of 2.1-10.9%.}}}}}}}}}}}}}}}}}

Fluorescent Studies on the Modulation of Cytosolic Calcium Signaling of Lymphocytes by Little Extrinsic Molecules

Abstract

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