Valeriya Maryakhina, Sergey Letuta.
Orenburg State University
Optical techniques of research are often used in different fields of biology and medicine for a design of alternative diagnostic’s methods of biotissue pathologies. State of biotissues or cells is estimated by intensive of light scattering as well as fluorescence spectrum of endogenous and exogenous fluorophores. As alternative, registration of triplet states of dye can be used because this technique has high sensitivity to molecular oxygen. It is most important when oxygen-depend processes are registered. At a time, molecular oxygen concentration can be different during all stages of development of tumor growth.
In the work, influence of oxygen concentration in cancer and normal cells to delayed fluorescence (DF) kinetics of xanthenes dyes was demonstrated as dependence from pathological process stage.
The objects of research were cells obtained from normal and cancer mammary gland of female mice (BYRB line with spontaneous cancer tumor). Fragments of the mammary gland tissue were taken into collagenase’s solution (0.5 mg/ml) in a phosphate buffer (pH = 7.4) and were incubated at 370С. After fermentation dissociation the obtained mammary cell suspension was placed on the nutrient medium (biomaterial “Hyamatrix”) and has been incubated for 24 hours at 370C and atmosphere pressure. Cancer cells were taken from mice’s spontaneously tumor with diameter 1÷3.5 cm. Oxygen-penetrating of biomaterial and cells was researched by DF technique changing air pressure above samples. Xanthene dyes (erythrosine, bengal rose and eosine) were used as molecular probes for kinetic measurements. The setup for study of DF kinetics of dye molecules was based on YAG:Nd laser. Molecules were excited by second harmonic of the laser (λex = 532 nm). Impulse’s duration was 10 ns, him energy was 10-50 mJ. Xanthene dyes concentrations were 10-3M and 10-4M.
The results of experiments (C=10-3M) were shown that kinetic curves have two-exponential type for registration signals during 400 µs. The time of The triplet-triplet annihilation of dye molecules is characterized by the first exponent (t1), the time of E type fluorescence is characterized by the second exponent (t2). The curves characterizing dyes DF in biomaterial have largest mean t2. At a time, curves for dye in cancer cells have smallest mean t2 for rise of the tumor diameter before 3 cm. The air pressure change above the biomaterial without cells from 150 torr to the atmosphere pressure doesn’t influence to the dyes DF kinetics. Quite another character of DF function is observed at exogenous fluorophores luminescence in cells received from biotissues. DF kinetics of xanthene dyes into cells has a hump-shaped type. This is evidence of interaction of dye molecules with molecular oxygen. The dye molecules in culture of normal mammary cells, however, has higher sensitive to air pressure above samples then in cancer cells. Authors obtained that stationary concentration of the molecular oxygen and him lifetime in normal cells is larger then in cancer cells. Received results can be connected with hypoxia of cancer cells. These differences in the experimental data will observe if we have alive cells only.
In the above described data, start part of curves (0÷50 µs) is not resolute. After his resolution (for registration 0÷50 µs), we had soon dependence between accumulations of triplet states of dyes in the two cell types. In the case of normal cells the maximum of DF accumulation is shifted toward shorter times at 3±1 µs indicating to rise of the oxygen concentration in it. With development of the pathological process (rise of the tumor diameter), the influence of the cancer cells rise as a result of the influence of tumor hypoxia, probably.
Decrease of the dye concentration (C=10-4M) leads to an increase of sensitivity curves of air pressure changes above the samples.
Thus, in this work the influence of the oxygen concentration into cells to the DF kinetics of xanthene dyes is presented.
Dr. Valeriya Sergeevna Maryakhina
Orenburg State University, senior researcher
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