Authors: Isaenko O.Yu.
While carrying out series of experiments we supposed that mild receptor interaction between pertussis pathogen antigens and epithelial cells can effect the functional activity of the test-cells, have an impact on the nucleus chromatin structure that would help to use this method as an additional test for evaluating the harmfulness of pertussis vaccine candidate drugs.
The goal of this work was to study the influence of B.pertussis antigenic fractions on heterochromatin structure of interphase nuclei of buccal epithelium.
Obtaining the native antigen was carried out by ultrasound disintegration in different frequency ranges of Bordetella pertussis industrial strains № 267 and № 475: the low-frequency (frequency of 60 kHz, power of 5 W), medium-frequency (frequency of 130 kHz, power of 9 W), high-frequency (frequency 1,6 MHz, power of 3 W), followed by desintegrate centrifugation (18000g), filtration, concentration and fractionation of antigenic complex into separate fractions by gel - chromatography.
Assessment of the heterochromatin stability in the buccal epithelium of nuclei cells after adding each individual B.pertussis antigen was performed as follows. We got buccal epithelium from the inner surface of the healthy adult human cheek and placed it to the buffer solution. We added experimental preparations (1: 1 by volume) to epithelial cell suspension and physiological solution of sodium chloride to the control sample. We kept it for 4 hours, then made preparations on a slide and tinted with 2% orcein for 30-60 minutes. Under the light microscope we counted the number of heterochromatin granulas in 30 epithelial cell nuclei.
Evaluation of heterochromatin stability in interphase nuclei of buccal epithelium during applying pertussis antigen microbes extracted in different frequency ranges of ultrasound (low - 60 kHz, average - 130 kHz, high - 1.6 мHz) showed growth of figures in all experimental groups comparatively to control values. There was not found reliable difference in the amount of heterochromatin in the nuclei of buccal epithelium cells after adding purified antigens with molecular weight ≥ 1000 kDa and ~ 3.0 kDa between each other (all frequency ranges of ultrasound). Reliable increase in the number of heterochromatin comparatively to other fractions was seen after interaction of buccal epithelium with cell-free preparations of molecular weight ~ 8.1 kDa obtained in the medium frequency range of ultrasound (130 kHz). This method is sensitive to adding the antigen and can be used as an additional test for evaluating the harmfulness of candidate drugs of pertussis vaccine.
Key words: buccal epithelium, heterochromatin, antigens, Bordetella pertussis, аnimal.