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DOI: http://dx.doi.org/10.20534/ESR-17-3.4-35-38
Isanbaeva Landish Muchamedzakievna, Kadyrova Diloram Abdullaevna, Institute of bioorganic chemistry AS Rebublic of Uzbekistan
E-mail: etoyaek@mail.ru
Association of extracellular DNA concentration with efficacy of treatment of patients with 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 tumor, growing from immature mi-osity of vascular walls 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]. Numerous hypotheses, explaining development and course of different fibroids of uterus, are under discussion and have not been studied enough, that gives difficulties in the designing of new organ-preserving methods of treatment and decreases the efficacy of treatment of this disease [4; 5].
At present, the issue of treatment of uterus myoma is the most difficult and debatable [6]. The choice of treatment method is determined by many factors such as features of pathogenesis, form and grow rate of tumor, age, absence or presence of children in woman and etc. [7].
Till the certain time it has been considered that DNA has been only in cellular structures: especially in nuclei of cells and some amounts in mitochondria, where it performs function of carrier of genetic information. The possibility of using of extracellular DNA in medicine is conditioned by changes of its concentration, changes in structure and molecule sizes of extracellular DNA of blood plasma, appearance of different mutations. The interest to extracellular DNA (ecDNA) immeasurably increased after it was revealed that a number of it might increase in several diseases, which can be taken into account as early sign of corresponding pathologies [8]. It gave the real practical meaning for the studying of circulating nucleic acids.
The aim of this work: To study efficacy of treatment of patients with uterus fibroids according to concentration of extracellular DNA.
Materials and methods of investigation:
As the materials of investigation it was used the blood of women with varies fibroids of uterus. The blood in volume 1 ml was taken with the help of catheter from ulnar vein into vacuum tubes from 0,5 M EDTA. It was studied 40 blood samples of women of different ages.
Extracellular DNA was revealed by method described in previous work [9].
Analysis of molecular mass of extracellular DNA has been done by method of electrophoresis in 2% of agorozny gel. The gel has been painted with ethidium bromide and taken the photo on trans-lyuminator through passing beams of UV. As markers of length it was used gidrolizatrestriktaza of phage DNA A (HindII/EcoRI) and DNA ladder 100.
Results and discussion.
Early and timely diagnostic offibroids ofuterus may completely replace the surgical intervention, to determine tactics of conservative way in treatment and plan the forms of prevention. At present, it was established that the content of ecDNA changed in diabetes, myocardial infarction, system lupus erythematosus, rheumatoid arthritis, glomerulonephritis, hepatitis and other pathological conditions [10,11]. Pathogenic processes, leading to the increasing of ecDNA rate in blood in diseases, almost have not been studied. But it was received preliminary data about presence of correlation between concentration of ecDNA in blood and developing of some pathology. Qualitative and quantitative composition of ecDNA, detected in blood plasma, is system indication which can characterize the course ofpathological processes in organism. The most interest is aroused by dynamics of changes in content of ecDNA, rather than single meaning of their concentration in blood. In order to determine of informational content of qualitative and quantitative changes of ecDNA for estimation of treatment efficacy it was made comparative analysis on change of ecDNA concentration, in the course of treatment one can make conclusion about efficacy of treatment.
It was observed 30 patients of different age category with various forms of fibroids of uterus. Patients were divided into 3 groups: 1st - simple myoma of uterus (8 patients); 2nd - progressive (fast growing) myoma of uterus (10 patients); 3rd - symptomatic myoma - disturbance of menstrual cycle, symptom of bleeding (12 patients). As a control there were healthy donors (10 donors). From plasma samples of peripheral blood of women with fibroids of uterus it was revealed ecDNA. It was decided to get blood plasma, free from cellular elements, with the help of centrifugation of the whole blood on low speeds (3000 sp/min, 800g). Centrifugation ofblood on high speeds leads to destruction of part of cells and hit of nucleic acids in plasma. Besides it, high speed centrifugation may result the distortion of qualitative and quantitative composition of extracellular nucleic acids. On the 1st picture it was given the electrophoresis of revealed ecDNA.
Concentration of ecDNA was determined with spectrophotometry and PCHR method in real time, as the using of this method is thought to give more detailed results. On the 2nd picture it was presented data of PCHR analysis in real time. By data of PCHR analysis the quantity of ecDNA in blood plasma of patients with fibroids of
Section Б. Medical science
uterus increases in comparison with healthy donors. With the help concentration of ecDNA ofblood plasma may serves as effective in-of PCHR to polymorphic marker C3435 T of MDR1 gene it was dication in differentiation of patients with fibroids of uterus and may determined reliable differences in quantity of ecDNA. Analysis of be used for monitoring of treatment.
Picture 1. Electrophoresis of ecDNA in 1,5% of agarose gel
Qualitative changes of ecDNA ofblood plasma as well as quan- changes, i. e. happening of DNA fragmentation and appearing of titative are system indications, capable to characterize the course low molecular nucleosome DNA. Determination of nucleosome in
of pathological processes. In fibroids of uterus it has happened not circulating blood may be used for diagnostics, stage of disease and
only quantitative changes in ecDNA of plasma, but also qualitative monitoring of treatment of various fibroids.
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Picture 2. Quantitative estimation of ecDNA concentration in blood plasma of patients with fibroids of uterus by polymorphic marker С3435 Т of MDR1 gene, with the help of PCHR method in real time
In the table 1 it was given data of quantitative and qualitative composition of ecDNA in blood plasma ofwomen in different fibroids of uterus.
Table 1. - Content of ecDNA in blood serum in fibroids of uterus
№ Group Pathology ecDNA, ng/ml Size of fragments
1 n = 5 Control healthy donors 29,00 ± 1,07 20-23
2 n = 5 Simple myoma 85,0 ± 0,91 10; 6; 3,5; 1,5; 0,8
3 n = 9 Progressive myoma (fast growing) 148,6 ± 1,0 4,2; 1,2; 0,9; 0,8; 0,7; 0,6; 0,5;0,4
4 n = 5 Symptomatic myoma (DMC, bleeding) 65,8 ± 1,55 21; 6; 4,2;0,2
P < 0,05 in comparison with control
Presented results have showed that reliable increasing of concentration of ecDNA in blood plasma of women happens in different fibroids of uterus. It should be noted that the greatest emission of ecDNA in blood plasma happens in progressive myoma of uterus (148,6 ng/ml). Increasing of concentration of ecDNA allows making a conclusion about progressing of disease and about possibility
ofusing of this factor as biomarker of treatment efficacy. The data of table show that in healthy donors ecDNA has been presented with high molecular fragments of DNA, and in fibroids of uterus with fragments of degraded DNA.
For determination of treatment efficacy of patients with fibroids of uterus it was performed comparative analysis of concentration
of ecDNA in blood plasma of patients in dynamics. We presented that increased concentration of ecDNA of blood plasma was associated with efficacy of treatment and unfavorable outcome, which showed progressing of disease and potential importance of ecDNA as biomarker of forecast of clinical course ofdisease. After performing of conservative treatment in some patients with fibroids there have been decreasing of concentration of ecDNA, which shows the efficacy of making treatment. Rising of concentration of ecDNA indicates on clinical manifestations of progressing of disease and non-effective treatment, i. e. there are not results ofperforming therapy in patients. On the 3rd picture it was presented data about
changes of concentration of ecDNA in patients in dynamics. This picture shows that patients with symptomatic myoma have good results in the treatment (1, 2, 3). In patients with fast growing progressing myoma ofuterus the concentration of ecDNA does not decrease after the treatment, i. e. the treatment is not effective (6-11). On the basis of received data we came to conclusion that the change of concentration of ecDNA in patients with fibroids of uterus may be used as marker of efficacy of treatment. Estimation of content of extracellular DNA in plasma of patients in fibroids of uterus may help for the prediction of disease course and in the monitoring of efficacy of treatment.
Picture 3. Dependence of treatment efficacy of patients with fibroids of uterus on concentration of ecDNA
Using of ecDNA, taken from plasma samples or serums of patients' blood with fibroids of uterus, may have important role in the improvement of methods of diagnostics, monitoring and forecast of disease course, increasing of treatment efficacy in patients with fibroids of uterus.
On the basis of received data one may make a conclusion that increased content of ecDNA in blood plasma of patients with fibroids ofuterus is associated with the stage of disease, unfavorable outcome, inefficacy of treatment and genotype of patient.
Conclusions:
1. It has been established the correlation between concentration of ecDNA of blood plasma and developing of different forms of fibroids of uterus.
2. It has been shown that analysis of ecDNA concentration of blood plasma of patients with fibroids of uterus may be used as biomarker of efficacy of performed treatment in the observing of patients in dynamics and early diagnostics.
References:
1. Lanchinskiy V. I., Ishenko A. I. Modern views about etiology and pathogenesis of myoma of uterus//Issues of gynecology, obstetrics and perinatology. - 2003. - T. 2, - No. 5-6. - P. 64-69.
2. Saviskiy G. A. Myoma of uterus. The problems of pathogenesis and pathogenetic therapy//Issues of gynecology, obstetrics and perinatology. - 2010. - T. 1, - No. 2-3. - P. 44-48.
3. Tihomirov A. L., Lubnin D. M. Modern views about etiology and pathogenesis of myoma of uterus//Issues of gynecology, obstetrics and perinatology. - 2004. - T. 3. - No. 6. - P. 62-68.
4. Sotnikova N.YU., Malishkina A. I., Posiseeva L. V., Ansiferova YU. S., Suvorkina E. E., Arevadze I. E. Immunological markers of grow rate of myoma of uterus//Medical immunology. - 2002. - No. 3-4. - P. 283-284.
5. Sidorova I. S., Levakov S. A., Mamedbekova R. B. Clinical morphological peculiarities of simple and proliferative fibroids of uterus//Rus-sian vestnik. Obstetrics and gynecology. - 2001. - T. 3. - No 5. - P. 19-24.
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Section 5. Medical science
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.
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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-
