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9. Tamkovich S. N., Litviakov N. V., Bryzgunova O. E., Dobrodeev A.Yu., Rykova E.Yu., Tuzikov S. A., Zav'ialov A. A., Vlassov V. V., Cher-dyntseva N. V., Laktionov P. P. Cell-surface-bound circulating DNA asaprognostic factorinlung cancer.//Ann. N. Y. Acad. Sci. - 2008. -Vol. 1137. - P. 214-217.
10. Kolesnikova E. V., Tamkovich S. N., Bryzgunova O. E., Shelestyuk P. I., Permyakova V. I., Vlassov V. V., Tusikov A. S., Laktionov P. P., Rykova E. Y. Circulating DNA in the blood of gastric cancer patients.//Ann. N. Y. Acad. Sci. - 2008. - Vol. 1137. - P. 226-231.
11. Ambudkar S. V., Dey S., Hrycyna C. A. Biochemical, cellular, and pharmacological aspects of the multidrug transporter//Annu. Rev. Pharmacol. Toxicol. - 1999. - Vol. 39. - P. 361-398.
DOI: http://dx.doi.org/10.20534/ESR-17-3.4-38-41
Isanbaeva Landish Muchamedzakievna, Kadyrova Diloram Abdullaevna, Institute of bioorganic chemistry AS Rebublic of Uzbekistan
E-mail: etoyaek@mail.ru
Studying of influence of patients' genotype on content of extracellular DNA in treatment process of fibroids of uterus
Abstract: Despite considerable progress in studying of various myomas of a uterus, a number of key questions remain insufficiently studied, such as identification of genetic factors and the primary molecular defects leading to development and evolution of changes of a uterus, early diagnostics. It is now established that small amounts of DNA found outside the cells and, especially in the human blood plasma. The quantity of extracellular DNA (ecDNA) can essentially increase at a number of diseases what probably to consider as an early sign of corresponding pathologies. In this article the analysis of quantitative violations of ecDNA, exposure of genotypes of patients, is conducted with different hysteromyomas. The results showed that ecDNA can be used in practical gynecology solutions for early diagnosis of problems, and predicting the course of monitoring the effectiveness of treatment of this disease.
Keywords: extracellular DNA; genetic factors; early diagnostics; prognostication; efficiency of treatment.
Myoma of uterus is benign, well delimited capsulated tumor, which develops from smooth muscle cells of cervix and body of uterus [1]. This pathology is on the first step among benign tumors of genital organs, at the same time each tenth patient of gynecology suffers from myoma of uterus [2; 3]. Studying of genetic factors of developing of uterus myoma in women of all age groups is actual problem of modern medicine. It is due to the myoma of uterus and its complications remain one of the main reasons of radical operations in modern gynecology practice. Myoma of uterus is a cause of infertility, uterine bleeding, and disturbance of functions of pelvic organs [4]. In spite of significant progress in the studying of this pathology, a number of issues such as identification of genetic factors and primary molecular defects, leading to the development and evolution of myoma changes of uterus, and early diagnostics have not been studied enough.
The most difficult problem is treatment efficacy of fibroids of uterus. At present, the issue of treatment of this pathology is most difficult and under discussion [6]. Selection of treatment method is determined by many factors such as features of pathogenesis, forms and grow rate of tumor, age, absence and presence of children in woman and etc. [7].
In spite of significant progress in the studying of fibroids of uterus, the issue of treatment efficacy of this pathology is the most actual. One of the obstacles for successful and effective treatment of fibroids of myoma is developing of drug resistance of myoma cells to the action of medications. Making of genetic test before the therapy, which, with high reliability, will predict the probability of developing and severity of side effects in each patients is the solution of this situation. As fundamental information it will be used the genotype of patients resistance to therapy, which will be determined by us with the help of PCHR methods. For the performing of effective treat-
ment of fibroids of uterus it will be considered individual reaction of organism on the action of medications.
The aim of this work: The studying of influence of individual peculiarities of the organism of patients with fibroids of uterus on the content of excDNA.
Materials and methods of investigation:
As the material it was used the blood of women with different fibroids of uterus. Blood in volume 1 ml was taken with the help of catheter from ulnar vein into vacuum tube with 0,5 M EDTA. It was studied 40 blood samples of women of different age.
Extracellular DNA was revealed from 1 ml ofperipheral blood, taken from ulnar vein and transferred in plastic tubes deposited with EDTA - Na2 Blood was centrifuged in 4 ° C consistently 1,5 thousands revolutions per minute (rpm) - 10 minutes, in 3 thousands rpm - 15 minutes, in 5 thousands rpm - 15minutes. After centrifuge, 400mcl of blood serum were taken from tubes in new sterile tubes. It was added previously 100mkg/ml of RNase A to the serum and incubated during 1 hour in 37 ° C, then it was processed with proteinase K (50 mkg/ml), incubation was 1 hour in 37 ° C. After enzymatic treatment, it was added to yhe blood serum 200mcl of lysis buffer [100mM TrisH-Cl, pH 8,0; 25mM EDTA, pH 8,0; 0,15 M NaCl; 0,7 M |-mer-captoethanol; SDS (final concentration 2%). Lysis was performed on the cold 3 minutes (over ice). Then aliquots were deprotein-ized 15 minutes in 1,5ml of mixture of phenol/chloroform [1:2] with followed by centrifugation in 5 thousands rpm, 15 minutes in 4 ° C. Supernatant was transferred into new tubes. It was completed 1/10 volume of 3 M of sodium acetate, pH 5,2 and 2,5 volume of cooled 96% ethanol, then the tubes were left on the night in -20 ° C. Denatured preparations of excDNA were centrifuged in 5 thousands rpm - 30 minutes, 4 ° C. Sediment of excDNA preparations was washed in 1ml of cooled 70% ethanol with followed by centri-
Studying of influence of patients' genotype on content of extracellular DNA in treatment process of fibroids of uterus
fuge in 13 thousands rpm during 15 minutes in 4 ° C. Preparations of excDNA were kept in -20 ° C. They were dissolved in 100mcl of DNA - binding fluorescent dye (DBFD), comprising 10mM trisHCl, pH 8,0; 1mM EDTA, pH 8,0; ethidium bromide 0,5mkg/ml. Solutions of excDNA were measured on spectrophotometer at wavelength 350 nm. The intensity of fluorescence was increasing linearly with concentration of excDNA.
Genotyping on polymorphic marker С3435 Т of MDR1 gene was performed by method of PCHR-PLRF with the using follow markers:
Primers:
Primer MDR1-C3435 T for: 5' - GAT CTG TGA ACT CTT GTT TCT A - 3'
Primer MDR1-C3435 T rev: 5' -GAA GAG AGA CTT ACA TTA GGC - 3'
Primer, control: IS711: 5' - TGCCGATCACTTAAGGGC-CTTCAT - 3'
Performing of PCHR
At work it was used primers for MDR1 gene encoding protein of drug resistance P-glycoprotein. As control it was used primers of reference gene GAPDH. PCHR was performed on the cycler «BioRad» USA. Before performing RCHR, products of PCHR were kept in - 20 ° C.
Electrophoresis of PCHR products was performed in 2% agarose in TAE buffer, 100 B, 1h. Then agarose gel was painted during 15 minutes in the solution of ethidium bromide (0,5mkg/ml). Results of electrophoretic analysis in gel were observed visually
Tracks: 2, 3, 4, 5, 7, 10, 12, 13 - TT genotype; tracs:6,8,9,11,15 -CC genotype PCHR product was split by restriction enzyme Sau-3AI. Electrophoretic separation of genotypes of PCHR products was performed in 2,0% agarose gel 2,5% agarose, 100V, 3h. M -marker of DNA ladder, 100kb.
Polymorphic marker C3435T of MDR1 gene, representing itself replacement in nucleotide sequence in the position of 3435 cytosine to thymine, is most clinically informative. Therefore, we made investigation using only this marker. Genotypes were determined on the criterion of presence of restriction site in the position 3435. On the 1st picture it was presented electrophoregram of genotypes on polymorphic marker C3435 T of MDR1 gene. It was revealed genotypes TT - resistance and CC - sensitive to the action of medications. Results of genotyping have shown that heterozygous genotype CT - medium sensitivity to the action of medications has not been revealed among patients of this group. It is known from literature data that in carriers of TT - genotype the disturbance of expression of MDR1 gene has been noted at the level of transcription, which leads to increasing of amount of glycoprotein-P and
through passing beams of UV on cycler «Bio-Rad». Agarose gel has been scanned after electrophoresis on densitometer, which analyzes intensity of glow of PCHR-products. Received bands image was processed with the help of computer densitometer (Gel-Pro-Analizer 4.0).
Results and their discussions
In spite of significant progress in the studying of fibroids of uterus, the issue of treatment efficacy of this pathology today is the most actual. One of the obstacles for successful and effective therapy of fibroids of uterus is developing of drug resistance of myoma cells to the action of medications. Making genetic test before the therapy, which, with high reliability, will predict the probability of developing and severity of side effects in each patients is the solution of this situation. As fundamental information it will be used the genotype of patients resistance to therapy, which will be determined by us with the help of PCHR methods. For the performing of effective treatment of fibroids of uterus it will be considered individual reaction of organism on the action of medications.
It was carried out the determination of genotype of patients with fibroids of uterus. With the help of PCHR method it was synthesized excDNA with using of specific primers for MDR1 gene. Synthesizing of excDNA was processed by restriction enzyme Sau3A. After amplification and restriction it was performed electrophoresis in 2% agarose gel with the adding of ethidium bromide. Restriction fragments were visualized in ultraviolet translyuminator. Genotyping was made on the base of analysis of polymorphism of restriction fragments length.
rapid removal of medications from organism. As a result, the carriers of TT - genotype have probably significant decreasing of concentrations of medicines in blood, which leads to the developing of undesirable drug reactions, side effects and decreasing of treatment effect [11, 12].
It was interesting to study how genotype of patient influenced on concentration of DNA. On the 2nd picture it was presented data about changes of concentration of excDNA depending on the genotype of patient in progressing myoma of uterus. From the data, it is seen that in patient with genotype CC (curve 2), i. e. sensitivity to the treatment the concentration of excDNA decreases and gains normal level. In patient, who has homozygous genotype (curve 1), i. e. resistance to therapy the concentration of excDNA does not decrease and after 3 courses of treatment stays almost on the same level as before the treatment. On the basis of received data one may come to conclusion that identification of genetic features in patients on polymorphic marker C3435 T of MDR1 gene allows to predict the character pharmacological response. Genotyping, i. e. identification of individual responsible reaction of patient organism on the
Picture 1. Electrophoregram of genotypes by polymorphic marker С3435 Т of MDR1 gene
action of medications gives possibility to increase efficacy and safety replacement of medicines and real opportunity to individualization of applying of medications dose, multiplicity of the introduction, of pharmacotherapy in patients with fibroids of uterus.
Picture 2. Concentration of excDNA depending on the genotype of patient in progressing myoma of uterus
Thus, our studies, aimed to the analysis of quantitative disorders of excDNA and identification of genotype of patients with different fibroids of uterus, may be used in gynecology practice for the solving
of problem of early diagnostics, prediction of course and monitoring of treatment efficacy of this disease.
References:
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6. Arslan A. A., Gold L. I., Mittal K., Suen T. C., Belitskaya-Levy I., Tang M. S., Toniolo P. Gene expression studies provide clues to the pathogenesis of uterine leiomyoma: new evidence and a systematic review//Hum Reprod. - 2009. - Vol. 20. - No. 4. - P. 852-863.
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8. Tamkovich S. N., Vlasov V. V., Laktionov P. P. Circulating deoxyribonucleic acids of blood and their using in medical diagnostic/Molecular Biology. - 2008. - T. 42. - No. 1. - P. 12-23.
9. Tamkovich S. N., Litviakov N. V., Bryzgunova O. E., DobrodeevA.Yu., Rykova E.Yu., Tuzikov S. A., Zav'ialov A. A., Vlassov V. V., Cher-dyntseva N. V., Laktionov P. P. Cell-surface-bound circulating DNA asaprognostic factorinlung cancer.//Ann. N. Y. Acad. Sci. - 2008. -V. 1137. - P. 214-217.
10. Kolesnikova E. V., Tamkovich S. N., Bryzgunova O. E., Shelestyuk P. I., Permyakova V. I., Vlassov V. V., Tusikov A. S., Laktionov P. P., Rykova E. Y. Circulating DNA in the blood of gastric cancer patients.//Ann. N. Y. Acad. Sci. - 2008. - Vol. 1137. - P. 226-231.
Microflora of biotopes as a leading etiological factor at oral diseases in children
11. Ambudkar S. V., Dey S., Hrycyna C. A. Biochemical, cellular, and pharmacological aspects of the multidrug transporter//Annu. Rev. Pharmacol. Toxicol. - 1999. - Vol. 39. - P. 361-398.
12. Drescher S., Schaeffeler E., Hitzl M. MDR1 gene polymorphisms and disposition of the P-glycoprotein substrate fexofenadine/^. J. Clin. Pharmacol. - 2002. - Vol. 53. - P. 526-534.
DOI: http://dx.doi.org/10.20534/ESR-17-3.4-41-44
Kazakova Larisa Nikolaevna, Saratov State Medical University n. a. V. I. Razumovsky, epartment of Children Stomatology and Orthodontics,
PhD in Medical Science, E-mail: kazakova-lor@yandex.ru Zholdasova Nurgul Zhanabayevna, Candidate of Medical Sciences, Department of Surgery and Children Stomatology, West Kazakhstan Marat Ospanov State Medical University,
E-mail: n.zholdasova@mail.ru Suetenkov Dmitry Evgenievich, Saratov State Medical University n. a. V. I. Razumovsky, Department of Children Stomatology and Orthodontics, Assistant Professor, PhD in Medical Science, E-mail: kazakova-lor@yandex.ru. Kaikan Azamat Islambekuly, Master of Medical Sciences, Department of Surgery and Children Stomatology, West Kazakhstan Marat Ospanov state medical university, assistant,
E-mail: kaikan@inbox.ru Mahonova Ekaterina Vladimirovna, Saratov State Medical University n. a. V. I. Razumovsky, Department of Children's Stomatology and Orthodontics, Assistant,
E-mail: kazakova-lor@yandex.ru.
Microflora of biotopes as a leading etiological factor at oral diseases in children
Abstract: Being a source of foreign genetic information, microbes and viruses regardless of the exact place of the invasion during their life damage tissues' integrity and as a result provoke the development of disease of various severity. Oral cavity stands on the second place by the number of microbes with different level of virulence. Dysbiotic abnormalities cause biofilms formation in various oral biotopes. The associated links within biofilms facilitate the increase of microflora virulence. Poor oral hygiene stimulates microbes' growth and reproduction. Oral cavity is not homogeneous by the number of microbes' settlements. Most of microbes concentrate in areas with special anatomical features. Stomatitis, dental caries, parodontitis and periodontitis are the most frequent clinical manifestations of high active microflora. Monitoring of oral biocenoses allows to perform the analysis of their variety and structural adjustments, which is vital for optimal treatment and prevention of oral diseases. Keywords: Microbes, Anaerobe, dental caries, oral hygiene, children, stomatitis.
The most frequently spread somatic diseases are stomatological ones. Every child knows what stomatitis or dental caries is. The main factor, which causes stomatitis in children is the oral bacterial flora [1]. Normally the inflammatory response is prevented by specific and nonspecific factors of antimicrobial resistance of oral mucous membrane, as well as biochemical and immunological features of saliva. The forming of oral micro-biocenosis is a multilevel process, closely linked with the regularities of dento-facial system development. Transient microbes constantly getting into the children's mouth by dirty hands, toys and kisses of relatives damage micro-ecological and adaptive microbes' interrelations with both environment and each other.
Clinical picture of inflammation in the particular patient depends on the factor, causing it as well as on the individual conditions of the patient, complementing this pathological process [2].
Understanding of the oral pathological processes in children requires knowledge of topographic, anatomic, histological and his-tochemical processes, often sharply changing depending on age.
There are distinguished 3 periods of age, which significantly differ by constitution and characterize the dynamic of main mucous structures development: the infants - a period of age from 10 days to 1 year, early childhood - the children from 1 to 3 years old and childhood - primary (4-7 years old children) and postprimary (812 years old children). With age the factors of local mucous protec-
