Get Adobe Flash player

THE STUDY OF HETEROCHTOMATIN IN NUCLEI OF BUCCAL EPITELIAL CELLS AFTER THE LOADING OF B.PERTUSSIS ANTIGEN, TAKEN IN DEFFERENT ULTRASOUND FREQUENCY

Authors: Isaenko O.Yu.

Pages: 181-187

Abstract


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.

This email address is being protected from spambots. You need JavaScript enabled to view it.

The full text

To view the full text

References

  1. [Provedenie opyton na zhyvotnykh]. Animal Experimentation. Retrieved from: http://www.animalmosaic.org/Images/Animal%20Experimentation_Russian_tcm46-28242.
  2. Vaughan Monamy Animal Experimentation: A Student Guide to Balancing the IssuesAustralian and New Zealand Council for the Care of Animals in Research and Teaching. English 1996:56 p.
  3. Bernard ER [Animal Rights and Human Morality]. Prometheus Books. English, 2006.400 р.
  4. [2005 Report on Enforcement of the Animal Welfare Act], U.S. Department of Agriculture. Retrieved from: http://www.all-creatures.org/saen/articles-2005enf-c.html.
  5. Reznikov A
  6. Hajime Kojima JaCVAM: An organization supporting the validation and peer review of new alternatives to animal testing. World Congress on Alternatives & Animal Use in the Life Sciences. Tokyo, Japan. 2007:august 21-25. Retrieved from: http://altweb.jhsph.edu/wc6/paper483.pdf.
  7. Shkorbatov YuG, Sutiushev TA, Rolupaeva TV [Izmienieniie sostoianiia khromatina i eliektrootritsatielnosti iadier kletok bukkalnoho epiteliia pri fizicheskikh nahruzkakh u donorov raznoho vozrastay]. Biolohicheskie miechanizmy starieniia: materialy VII simpoziuma. Kharkiv, 2006, pp. 32–34. (In Ukrainian).
  8. Cheshun VF [Kilkist mikroiadier i osoblyvosti struktury khromatinu v kletynach bukkalnoho epiteliia ta riven gomotsysteinu u plazmi krovi zhinok z puchlynamy molochnoi zalozy]. Onkologiia. 2011;9(4):311-315.
  9. Maianskii AN, Zaslavskaia MI, Zielienova Ieg [Adgezivnyie reaktsii bukkalnyh epiteliotsytov v indikatsii narushenii miestnogo i obshchego gomieostaza]. Nizhegorodskiimeditsinzkyizhurnal. 2005;1:158–161.
  10. Abadzhydi MA, Machrova TV, Maianskii MI, Zaslavskaia YuYu, Maianskii AN [Bukkalnyh epiteliotsyty kak instrument kliniko-laboratornych issledovanii]. Retrieved from: http://www.medicum.nnov.ru/nmj/2003/3-4/23.php
  11. Shachbazov VG, Kolupaeva TV, Nabokov AL [Novyi metod opredeleniia biologicgeskogo vozrasta cheloveka]. Laboratornoedelo. 1986;7:404-406.
  12. Shkorbatov YuG, Shachbazov VG [Eliektrokineticheskiesvoistvakletochnykhiadiersviazssostoianiem kletkii]. Tpudy po fyndamentalnoi i prikladnoi genetiki. Kharkiv, 2003(2), pp. 71–92. (In Ukrainian).