| Calcium serves as a intracellular second messenger and mediator play a very important role in biological systems. Consequently, the measurement of cytosolic Ca2+has become an important area of investigation in biological and medical research. At present, most measurement of intracellular Ca2+ are accomplished using fluorescent Ca2+ indicators developed by Tsien, such as Quin-2,Indo-l,Fura-2 and Fluo-3. The indicators Quin-2, Indo-land Fura-2 have the advantage of wavelength-shift upon binding Ca2+ but share a common drawback in that the excitation bands for these indicators occur at UV wavelength(340-360nm), where autofluorescence from cells is high, radiation-damage of cellular structures is serious, the transmission of microscope optics is poor, and the availability of aberration-corrected optics for confocal microscopy is limited. Fluo-3, a visible light-excitable Ca2+ indicator, overcomes the limitation of Quin-2, Indo-1 and Fura-2 but no longer display any spectral shifts upon Ca2+ binding and thus ratiometric measurements are not possible. In addition, all of these indicators described above are difficult to target precisely to specific intracellular locations. When they are transfected into cells, they not only distributed in cytosolic compartment but also in the nucleus and this may cause some disaccuracy for real cytosolic Ca2+ measurement.In view of the above-discussed technological backdrop, it is apparent that the field has urgently awaited the innovative development of long-wavelength and ratiometric fluorescent Ca2+ indicators which are concomitantly able to target precisely to specific intracellular locations.In this thesis, the investigations include the following several aspects: 1. Two new fluorescent Ca2+ indicators STDIn and STDBT have been designed and synthesized. The structures and absorption and fluorescence spectral characteristics of these reagents have been thoroughly studied. 2. Confocal microfluoroscope and the new fluorescent Ca2+ indicator STDIn-AM were used to investigate the mechanism of 5-hydroxytryptamino(5-HT) on intracellular calcium dynamics in cultured rat stomachfundus smooth muscle cells comparing with that of fluo-3.The results shown that the new fluorescent Ca2+ indicator, STDIn, developed in our laboratory. Not only can it be excited by visible light but also displays a spectrum-shift upon binding with Ca +. Moreover, it targets precisely into cytosol only, and thereby exhibits great advantage beyond flou-3 and ruro-2. Thus it is possible to use STDIn-AM to measure real-time variation of [Ca2+] in cytosol. 3. 5-HT induced local Ca 2+ transients (Ca2+ sparks) in cultured rat stomach fundus smooth muscle cells(SFSMC) was first observed by using the new fluorescent Ca2+ indicator STDIn and laser scanning confocal microscope. The mechanisms of initiation of Ca2+ sparks and propagating Ca2+ waves and their relation to E-C coupling were also discussed. 4.The effects of reactive oxygen species on the spectral properties of the new fluorescent Ca2+ indicator STDIn were evaluated using four different oxidant-generating systems. The effect of hydrogen peroxide on the intracellular Ca2+ levels of HL-60 cells labeled with STDIn was studied also. 5.The relationship between the structure and the properties of the new fluorescent Ca2+ indicators were studied using quantum-chemical calculations.The thesis includes four parts:1. Measurement of Cell Calcium (Review)Methods for the intracellular Ca2+ measurement such as Aequorin, Metallochromic indicators, Ion-selective Electrodes and Fluorescent Ca2+ indicators, particularly the fluorescent Ca2+ indicators were reviewed in detail.2. Synthesis of new fluorescence Ca2+ indicators and study on their propertiesTow new fluorescent Ca2+ indicators STDIn and STDBT have been designed and synthesized and their structures, spectral and biological properties have been studied thoroughly. The experimental results showed that the visible absorption spectrum of STDIn and STDBT display a dramatic shift in the long wave...
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