Synthesis And Application Of Targetable Intracellular Fluorescence Ca～(2+) Probe

Calcium acts as an intracellular second messenger and plays a very important role in biological systems. Consequently, the concentration and spatio-temporal distribution of intracellular Ca2+ have become an important area of investigation in biological and medical research. At present, most measurements of intracellular Ca2+ are accomplished using fluorescent Ca2+ indicatorsand fluorescent imaging. So far there aren't the indicators which are able to target to nucleus and cytosol simultaneously.In this thesis, the investigations include the following several aspects: 1. A new membrane-permeated intracellular fluorescent Ca2+ indicator STDBT-AM has been synthesized. 2.The absorption and fluorescence spectral characteristics and the anti-oxidation ability of the new fluorescent Ca2+ indicator STDBT have been thoroughly studied. 3. Confocal microfluoroscope and the new fluorescent Ca2+ indicator STDBT-AM have been used to investigate the nuclear and cytosoliccalcium dynamics of several types of cells comparing with that of Fluo-3. The thesis includes three parts:1. ReviewMethods for the intracellular Ca2+ measurement were reviewed in detail.2. Synthesis of a new fluorescence Ca2+ indicator and study on its propertiesA new membrane-permeated fluorescent intracellular Ca2+ indicator STDBT-AM has been produced from STDBT. The optical spectral and biological properties have been studied thoroughly. The experimental results showed that the visible absorption spectrum of STDBT displays a dramatic shift in the long wavelength maximal from 512nm to 412nm upon Ca 2+ binding. The emission maximum centered at 600nm is well shifted from the absorption. These features made the STDBT not only can be used in visible light regions to avoid the auto-fluorescence and other problems associated with the use of UV excitation wavelengths but also can be used as a ratiometric indicator. Further, the results also showed that, in contrast with Fluo-3, when STDBT-AM was transfected into cells, itdistributes both in the cytosol and the nucleus, but displays a very clear boundary between the cytosol and the nucleus, which made the STDBT a double targetable Ca2+ probe and can reflect changes of cytoplasmic Ca2+ ([Ca2+]c)and nuclear Ca2+ ([Ca2+]n) at the same time. In addition, the effects of oxidants on properties of STDBT are less than Fura-2 and Fluo-3. 3. The applications of the new fluorescence Ca2+ indicator STDBT3.1. Dual-wavelength ratiometric determination of free-calcium with new Ca2+ indicator STDBTAccording to that the new Ca2+ indicator STDBT can form stable complex with Ca2+ and the absorption spectrum of STDBT upon Ca2+ binding displays a dramatic shift in pH 7.2 of buffer medium. A novel method for the determination of free-calcium in serum under physiology condition was established by using STDBT .3.2. The mechanisms of adrenalin-induced cytoplasmic Ca + and nuclear Ca2+ dynamic in different athletic burthen mice's cardiac muscle cells.The new Ca2+ indicator STDBT-AM and confocal microscopy were used to investigate adrenalin-induced cytoplasmic Ca2+ and nucleus Ca2+ dynamic mechanisms in cardiac muscle cells. The effects of different athletic burthen on [Ca2+] in mice's cardiac muscle cells were studied. The experimental results showed that in contrast with Fluo-3, STDBT targets to cytosol and nucleus, the boundary between cytosol and nucleus is very clear. Thus it is possible to use STDBT-AM to measure cytosolic Ca2+ and nuclear Ca2+ simultaneously. The results also showed that adrenalin cause Ca2+ release through T-type calcium channels.3.3.Study of the effects of slim material on the intracellular Ca2+ of growth hormone cells with the new Ca2+ indicator STDBT-AM.Confocal microfluorometry and the new Ca2+ indicator STDBT-AM were used to study the effects of slim material on intracellular calcium signaling in growth hormone cells comparing with that of Fluo-3. The results showed that STDBT can target to nucleus but can not enter nucleolus. And also, as a Ca2+ indicator it responds to the slim material...