Scientific journal
European Journal of Natural History
ISSN 2073-4972

PATHOLOGIC ANATOMY AND MOLECULAR BIOLOGY ON THE BOUNDARY OF MILLENIA

Razin А.P.
The role of pathoanatomy in the development of biomedical sciences is of great value and diversity. A wide introduction of biochemical methods, and in morphology - histological chemistry, allowed studying metabolic and molecular changes. The progress of molecular biology and immunohistochemistry, in situ hybridization became the foundation for creation of a new discipline - molecular pathology studying molecular biology of general pathologic processes and diseases in the level of structure, functional activity and gene expression changes.

The pathoanatomy gradually co-opted current achievements and up-dated engineering solutions of such sciences as anatomy, physiology, chemistry, microbiology, immunology, genetics, cellular and molecular biology. Nowadays it has got an opportunity to study structural and functional imperfections starting with the organismic level and finishing with the molecular-genetic one.

Let us define the main engineering achievements of medical and biological sciences which have given the master pulse to the development of modern pathoanatomy, which nowadays combines elements of classical and molecular pathology, in brief.

The methods based on the immune mechanisms rest on the interaction of human tissue and molecular antigens with specially obtained antibodies bearing various marks on themselves. The light imuunohistochemistry antibody marks can be represented by various fluorochromes, horseradish peroxidase, alkaline phosphatase, peroxidase-antiperoxidase, avidin-biotin-peroxidase and avidin-streptavidin-peroxidase complexes, and also radiogenic substances. In the electronic immunohistochemistry it is preferable to use the marks in the form of colloid silver or gold.

The light immunohistochemistry allows revealing antigens in tissue and cellar levels and evaluating the resultant amount on the fluorescence intensity or tissue coloration. The electronic immunohistochemistry is used to study the subcellular antigen focalization.

The immunohistochemistry serves also for the evaluation of cell-specific gene expression on the corresponding protein products, coded by the given genes, in tissues and cells.

The immunohistochemical cells´ marks coupled with their flow cytophotometry, laser and computer sorting allow detaching cell groups according the availability of definite antigenic determinants that is widely used in the hemopathy diagnosis.

The investigation of disease molecular foundations is associated with the identification of separate products (abnormal proteins, for example), transmission paths of cellular and intercellular signals, and synthesis of definite proteins, glycol- and lipoproteins.

The development of modern DNA analysis became possible after the discovery of series of enzymes (endonucleases, restrictases, polymerases, transcriptases) providing specific manipulations with DNA and RNA. Due to the fact it became possible to obtain specific fragments of the DNA molecule from different cells and tissues, to synthesize amino acid sequences typical of definite proteins, to create new DNA molecules by the recombination of molecule fragments from different sources. The newly synthesized DNA molecules´ fragments are often used as a cloning vector for separate proteins with predetermined properties. The fragments of already existing DNA are transformed with the help of endonucleases to the vectors which are distributed in phages in the nature of a genomic library. The genomic library is necessary for the identification of newly discovered proteins.

The use of molecular analysis current technology allowed beginning the investigation of the expression of separate genes controlling the production of a definite protein. The analysis of gene structure helped understand their transcription mechanisms and identify many factors regulating it. In some cases these factors appeared to be hormones, in others - nucleoproteins responding extracellular cues.

The opportunity to investigate separate genes´ functions appeared after the introduction into practice the method of obtaining transgenic animals and models with a definite gene knockout. Into the egg cell of animals (mice) separate genes responsible for the synthesis of a definite factor are introduced, and, as a result, animals with hyperexpression of this focalized tissue-specifically factor are obtained. The gene knockout technique is particularly widespread nowadays as it allows studying the role of separate factors in various diseases´ pathogenesis.      

The gene expression leads to the intensification of protein synthesis. The proteins can be detected and identified both by immunochemical method - with the help of gel electrophoresis, and by immunohistochemical methods - using high-specific antibodies.

The polymerase chain reaction (PCR) discovery in 1986 became a revolution in practical molecular biology due to the possibility of quick amplification of DNA specific fragments. For this method use it is enough to have one molecule or a DNA or RNA fragment to produce a necessary for the identification amount of DNA copies with the help of gel electrophoresis and Southern-blot- hybridization. This method is widely used for gene structure and expression investigation. For nucleic acids isolation and their conversion into the liquid phase cellular and tissual breakdown is necessary, and it complicates the comparison of amplification results and histopathological picture and also cell counting.

The in situ hybridization method provides an accurate focalization of specific nucleotide sequence in cells. Unfortunately, it possesses a low-grade (compared to PCR) sensitivity, and, to carry out the reaction it is needed not less than 10-20 m-RNA copies per a cell.

The use of molecular technology has allowed combining the high PCR sensibility and cellular focalization of in situ hybridization. This method got the name of in situ PCR.

All the three methods are widely used in pathology. The greatest number of the in situ PCR investigations is focused on the definition of viral or foreign sequence of nucleic acids. The possibility to detect latent viruses in single copies is an important measure on the way to viral disease pathogenesis comprehension.

The in situ PCR method is also used to study endogenic DNA sequences inclusive of single human gene copies, chromosomal translocations and mapping of numerically insignificant copies of genomic sequences in metaphase chromosomes. The possibility of carcinogenesis genetic determinants studying including DNA mutations and chromosomal translocations is extremely important for the comprehension of latent period between DNA damage and the appearance of morphological signs of atypia or malignization.

The use of the complex of molecular-biological, immunological and morphological methods in pathoanatomy has lead to a more thorough understanding of the interconnection between the structure and the function, and to the formation of a new line in the development of pathology - functional morphology, which in XXI century is becoming a guiding approach in studying the human body and various diseases morphogenesis. 

The article is admitted to the International Scientific Conference " Development prospects of higher school science" ; Sochi, Dagomys, September 20-23, 2007.; came to the editorial office on 27.07.07