Cytogenetic studies

Cytogenetic studies - a set of methods of investigation of the connection between the phenomena of heredity and structure of cells (especially structures of the cell nucleus). Cytogenetic studies play an important role in biomedical works, as with their help, find out genetic features, variability (see), the origin and evolution of living beings.
The object of cytogenetic investigations are first of all the chromosomes (see) human, animal and plants with specific for each type properties (number, size, structure) and forming a characteristic of this organism karyotype. Therefore, the methods of cytogenetic studies are used to construct the natural classification of living organisms.
In cytogenetic studies pay particular attention to the polyploidy is the phenomenon of increased fold increase in the number of chromosomes, accompanied by the appearance of a number of new properties (the increase in the total size, taste qualities of fruits and vegetables, viability of plants and so on). Development problems of polyploidy has practical value in agriculture, breeding of plants and animals.
Using cytogenetic investigations detect changes in the chromosomes that are passed to the offspring and in a specific way affect the characteristics of the organism. Learn harmful chromosomal rearrangements, loss, loss, or add a separate chromosomes or parts of chromosomes. They allow to identify the part of the hereditary factor in the emergence of a range of human diseases (see Hereditary diseases), including developmental, susceptibility to malignant neoplasms, and so on Cytogenetic studies led to the correct understanding of the nature of radiation sickness.
Using cytogenetic research found, for example, that in the nuclei of cells of various tissues and organs, but only in females, are intensively painted with special dyes education, so-called bull Barra or sexual chromatin (see). It turned out that the sex chromatin is found in many animals and humans. Opening of sex chromatin allowed to determine the gender of the person at the cellular level (this is of particular importance for forensic medicine), to diagnose the gender of the embryo in the early stages of pregnancy and solve a number of other issues of medical practice.
Cm. also, Genetics, Heredity.

Cytogenetic studies - microscopic study of special structures of cells that determine the processes of inheritance and development.
Cytogenetic studies receive more and more wide application in clinical medicine. The most simple, fast and affordable method of cytogenetic analysis is the study of sex chromatin.
Sex chromatin is a chromatin body, which is not in individuals males and females is adjacent to the nuclear shell.
Thus, this body can serve as cytological sex, therefore it and has received the name of sex chromatin.
Dimensions Taurus sex chromatin in humans range from 0.7 to 1.2 MK, their form may vary (Fig. 1 - 3). Women sex chromatin is defined in average 40% of the nuclei (Fig. 4). He formed one of the X-chromosomes of female fetuses that are in the inactive spiralizatia condition. Sex chromatin can be defined in the cells of the mucous membrane of the mouth, vagina, and urethra, and in blood cells, bioperegnoy skin, cultured tissue adult, embryonic tissues, nerve cells.
The most simple and convenient method of determining the sex of chromatin in the cells of the mucous membranes of the oral cavity proposed tiree (N. Thiries) and refined by Sanderson (S. Sanderson). To study take a scraping from a mucous membrane of cheeks. The material is transferred to a glass slide, dried in the air and within 10 minutes fix in methyl alcohol. Colouring make a drop svejeprigotovlennogo of atooraya (1 g of synthetic oreina dissolved in 45 ml glacial acetic acid, heated to boiling and after cooling filter to 45 ml filtered solution add 55 ml of distilled water and the mixture is filtered again). When using the microscope immersion objective count the number grammatiktraining cores per 100 cells.
Study of sex chromatin used for cytological sex determination, fast and early diagnosis of diseases associated with aberrations sex chromosomes (in particular, syndromes of Kleinfelter, Shereshevsky-Turner and others), characteristics of a number of physiological processes (in particular, the menstrual cycle), the study of General and local regularities of a number of pathological processes, and first of all malignant neoplasms, to investigate the action of some therapeutic methods and tools (antibiotics, corticosteroids, cytostatic drugs).
To the methods of cytogenetic analysis is the study of the karyotype (see).
It is established that the chromosome set of a person consists of 46 chromosomes (23 pairs), two sex chromosomes (XX - the woman XY is the male), 22 pairs of autosomes (Fig. 5) and is highly consistent in human organism cells.
Depending on the length of chromosomes and the location of their centromar whole chromosomes are divided into 7 groups - a, b, C, D, E, F, G.
For the study of chromosomal person (karyotype) use the methods of cultivation of peripheral blood leukocytes, fibroblasts embryonic tissue cultivation of cages of a skin and a direct method of determining chromosomes in the cells of the bone marrow.
For the first time about the successful cultivation of non-fissile leukocytes informed the Soviet biologist, K. Khrushchev (1935). In 1958 Nowell (P. Nowell) suggested to use to stimulate division of cells substance isolated from legumes - phytohemagglutinin (PHA). Cultivation of cells carried out on modified and advanced methods. 10 ml of venous blood collected in sterile test tube with heparin (part 1 ml heparin diluted 20-fold with a solution Hanks), a place for 30-40 minutes in the refrigerator. Then sterile (in box) in the blood add 0,7 - 1 ml of 10% solution of gelatin to accelerate precipitation of erythrocytes. After settling blood plasma sucked off and placed into a sterile flask. To add plasma medium 199 or Wednesday Needle at the rate of 1.5 ml of the environment on 1 ml of plasma.

For stimulation of mitotic activity of cells in a mix add 0.2 ml PHA. The cell suspension is placed in a thermostat at the temperature 37 deg for 72 hours. 2-3 hours before fixing on each vial (suspension for cultivation is bottled in 1.5-2 ml sterile vials type penicillin) add 0,5-0,75 g of colchicine (working solution colchicine: 10 mg in 1 ml of distilled water) and continue cultivation. In the future, culture centrifuged for 5 minutes at 800 rpm Supernatant liquid is poured to it add 3-5 ml of 0.95% solution of sodium citrate, heated to the temperature 37C, which causes swelling of the cells. In hypotonic solution cells are from 15 to 30 minutes, after which the supernatant fluid is drained off to the draught of carefully type latch (3 hours absolute alcohol + 1 h glacial acetic acid), put in the fridge for 15 minutes, then centrifuged and change the lock. At low fat slides put 1-2 drops of cell suspension and dried over a flame or fire latch ("burnt" drugs). Drugs polychrome painted blue Uns, acetoarsenite or Romanovsky. Chromosomes are studied with the help of immersion microscopy 100 metaphase plates.
For the study of chromosomes also use a direct method of determining chromosomes in the cells of bone marrow: 1 ml svezheokrashennoj punctate bone marrow is placed in a flask with 30 ml of medium 199 and 3 ml solution colchicine (10 mg in 1 ml). The contents of the flask gently shaken to distribute the cells, and then centrifuged. The supernatant fluid is drained and to draft add 10 ml of 0.95% solution of sodium citrate, heated to a temperature of 37 degrees. Cells carefully resuspension and placed in a thermostat at the temperature 37 deg 40-45 minutes then again spend centrifugation, the supernatant fluid is drained and added to the precipitate freshly prepared the lock, consisting of 3 hours of methyl alcohol and 1 ch. concentrated acetic acid. After 10 minutes sediment resuspension and leave release another 20 minutes at room temperature, then centrifuged for 10 minutes, again changing the lock and prepare drugs in the same way as when you commit culture of blood leukocytes.
Karyotype study can be successfully used for the diagnosis of chromosomal diseases. Recently dedicated a whole group of chromosomal diseases associated with pathology as sexual and autosomal chromosomes (see Hereditary diseases). In addition to changes in the number of chromosomes, may be in breach of their morphology. So, chronic myelocytic leukemia was observed unusually small acrocentric chromosome from 21 pairs. The emergence of aneuploidy (increase or decrease in the number of chromosomes, nonmultiple haploid number of chromosomes) may serve as a predictive test for end-stage leukemia.
Cytogenetic studies closer merge with cancer. It is possible that the chromosomal changes in cancerous processes can be used for early diagnosis. For cytogenetic studies using methods of short-term tissue cultures: the method of the plasma bunch and subsequent study of subcultures and primary method tripinsurance suspension cultures. Preference should be given to the first method, because the second requires a large amount of tissue to obtain suspension of cells, capable of reproduction.
To create the most favorable conditions of use metabolism placental serum person, non-toxicity, 50% embryonic extract aborted fetuses person, which is prepared on the environment Needle. To fasten pieces of glass and attach a greater number of cells in the application of the suspension cultures use dry human plasma, group IV, diluted before use, the environment Needle and placental serum 1:1; after making Explant add embryonic extract. The cultivation is carried out in vials Carell (see tissue Culture).

Fig. 1. Sex chromatin in the form of an oval (H).
Fig. 2. Sex chromatin in the form of a triangle (XI100).
Fig. 3. Sex chromatin as thickening of the nuclear envelope (H).
Fig. 4. Chromaticities the core of the woman (H).
Fig. 5. Normal female karyotype (x1100).