Saratov JOURNAL of Medical and Scientific Research

Genetics

A novel comprehensive approach for human vascular allografts cryopreservation and radiation sterilization for the tissue engineering industry

Year: 2015, volume 11 Issue: №4 Pages: 624-632
Heading: Genetics Article type: Original article
Authors: Lauk-Dubitsky S.E., Astrelina Т.А., Saprykin V.P., Fediunin A.A., Paklina O.V., Gordeev A.V., Shakurov A.V., Belikov N.V., Khaidukova I.V., Burkov I.A., Antonov E.A., Ponomarev D.E., Kobzeva I.V., Savrasov G.V., Vorobyov G.V., Brumberg V.A., Nikitina V.A., Suchkova Y.B., Dobrovol'skaya E.I., Karaseva T.V., Gubarev K.K., Bushmanov A.Yu., Samoilov A.S.
Organization: State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency, Moscow State Technical University n.a. N.E. Bauman, City Clinical Hospital n.a. S.P. Botkin
Summary:

Aim: to verify new techniques for human cadaveric vascular allografts cryopreservation, thawing and sterilization for the tissue engineering purposes. We use polydimethylsiloxane (PDMS) as a well-known, promising coolant. This allowed us to completely omit any cryoprotective or vitrifying solutions. Using of PDMS also makes possible an applying these allografts directly after freezing and decellularization and also it will also provide an opportunity to develop secure protocols of tissue— engineered vascular conduits cryopreservation. Matherial and methods. After mathematical modeling of cooling process and its validation the experiment for sealed (isolated) freezing at low temperature conditions of 30 femoral arterial segments has been conducted. The segments were at least 10 cm in length and taken from 15 cadaveric donors in the age of 65-85 years. The freezing process was carried out using the abovementioned coolant— PDMS, and then physico-mechanical properties of these allografts were evaluated with the special Instron machine. According to the results obtained, a modeling of their sterilization conditions was conducted (the grafts were freezed). Results. By physico-mechanical properties validation and restricted histological analysis it was shown that there was an accordance between freezed/thawed allografts properties and native vessels. Conclusion. The abovementioned approach for allografts cryopreservation and thawing was efficient enough for further work in this direction.

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Cytogenetic examination in remote period after the stay in the Semipalatinsk nuclear test site area

Summary:

Aim: retrospective cytogenetic indication of doses probably received by individuals during the residence in the region of the Semipalatinsk nuclear test site, at work or service in the Armed Forces on it. Material and methods: the analysis of chromosomal aberrations in peripheral blood lymphocyte cultures of 16 people с using FISH-method. Re-sults. The frequencies of FISH-registered translocations were not significantly different from control values in 7 out of 16 cases; the dose evaluations ranged from 0.20 to 0.75 Sv for the remaining 9 persons. Conclusion. FISH-technique of chromosome aberration analysis allows to make retrospective dose assessment; however it is desirable simultaneously to produce classical analysis also for a more correct interpretation of the results; Detection of single multiaber-rant cells in peripheral blood lymphocyte cultures is characteristic of persons who were in the areas of alpha-emitting radionuclide pollution, what is allowed to talk about radiation damage, but does not allow to make dose assessment.

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Analysis of production of the decellularized scaffolds and their potential use in cardiovascular surgery

Year: 2015, volume 11 Issue: №4 Pages: 615-620
Heading: Genetics Article type: Review
Authors: Brumberg V.A., Lauk-Dubitsky S.E., Astrelina Т.А., Kobzeva I.V., Bushmanov A.Y., Samoilov A.S.
Organization: State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency
Summary:

Data on application of decellularized scaffolds and tissue-engineered vascular conduits in the field of cardiovascular surgery have been analyzed, and also techniques for their procurement have been studied. For finding, selection and synthesis resources of research data from the systems Pubmed, ScienceDirect were used. The preference was given to the most informative, comprehensive and contemporary publications.

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Expression of mature micro-RNA involved in the functioning of p53-dependent system of maintaining the genome stability of the individuals exposed to radiation at clinically relevant doses

Year: 2014, volume 10 Issue: №4 Pages: 749-753
Heading: Genetics Article type: Original article
Authors: Shulenina L.V., Galstyan I.A., Nadezhina N.M., Mikhailov V.F., Raeva N.F.
Organization: State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency
Summary:

Purpose: to explore the content of mature micro-RNA involved in the functioning of p53-dependent system of maintaining the genome stability in the blood of patients in distant time after irradiation at clinically relevant doses and to compare these micro-RNA with the development of malignant tumors in the period of late consequences of radiation injury. Materials and methods. We used the blood samples of patients with acute radiation syndrome (ARS), acute radiation syndrome with the development of local radiation injury (ARS+LRI) and local radiation injury (LRI) obtained through 1-51 year after radiation injury. The mature mir34a, mir21, mir145, mir16, mir125b, Iet7a which contained in the common fractions of RNA were reverse transcribed by using specific "stem-loop" — primers. The relative amount of micro-RNA in blood of patients by real-time PCR. Statistical analysis of the results was carried out using the non-parametric Mann —Whitney test. Data are presented as median and quartiles, normalized to median of control group accepted for 1. Results. We found a significant reduction of content of mir34a, mir21 in the blood of patients with a diagnosis ARS and the increase of content of mir145 in patients with LRI. Analysis of the individual values of micro-RNA expression in the blood of patients whose cancer was detected, except for patients with a bazalioma, showed consistency of changes with risk of carcinogenesis. Conclusion. For the first time was investigated the functional activity of p53-dependent system of maintaining the genome stability by measuring of micro-RNA in the blood of patients after many years post radiation injury. We found a significant reduction of content of mir34a, mir21 in blood of patients with ARS, and increased mir145 in patients with LRI. Our results suggest that further research with groups of patients, and analysis the dynamics of micro-RNA content would allow for use the micro-RNA as indicators of risk of late consequences after ARS and LRI.

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Immunogenetic markers of Crohn's disease in adults population of the Moscow region

Year: 2014, volume 10 Issue: №4 Pages: 743-749
Heading: Genetics Article type: Original article
Authors: Stavtsev D.S., Astrelina Т.А., Azova M.M., Lebedeva L.L., Pukhlikova T.V., Chumak A.A., Knyazev O.V., Eremin I.I., Gilmutdinova I.R., Kotenko K.V.
Organization: State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency, Peoples' Friendship University of Russia, Blood transfusion station of the Department of Health of the City of Moscow
Summary:

Aim: to study immunogenetic markers of predisposition to the development and protection for Crohn's disease in adults population of the Moscow region. Material and methods. The study included 53 samples of peripheral blood of patients with Crohn's disease in the Moscow region. The control group was represented by 1,700 samples of umbilical cord blood is healthy newborns. Revealing HLA antigens at low level performed by SSO method on DynalRELI 48 processor. The results received with ambiguous interpretation was using PCR-SSP method (Ivitrogen). Results. Were found the positive and negative associations of groups of HLA alleles with clinical form, the course of Crohn's disease and response to steroid treatment, in particular revealed that, predisposition to the development for Crohn's disease in women and with sensitivity to steroid treatment in this disease associated allele group C*12, to the characteristic restricting markers such as Crohn's disease include the В 38 and A*11 markers nonrestricting, nonpenetrating noninflammatory type groups are alleles B*56 and C*14 and C*14 is also associated with the risk of Crohn's disease in men, characteristic markers of protection to the development of the disease crown with chronic relapsing and severe clinical course are DQB1*02 and DQB1*03, respectively. Conclusion. These results demonstrate the need for studies of gene polymorphism HLA-system, not only in relation to the disease in general, but in selected patients with clinical groups.

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Characteristics of changes in the number of yH2AX and Rad51 protein foci in human skin fibroblasts after prolonged exposure to low-dose rate X-ray radiation

Year: 2014, volume 10 Issue: №4 Pages: 739-743
Heading: Genetics Article type: Original article
Authors: Ozerov I.V., Eremin P.S., Osipov A.N., Eremin I.I., Tsvetkova A.D., Guseva S.S., Ivanova K.Yu., Gavrilenko 0.I., Pustovalova M.V., Smetanina N.M., Grekhova A.K., Lazareva N.L., Pullin A.A., Maksimova О.A., Gordeev A.V., Bushmanov A.Yu., Kotenko K.V.
Organization: Lomonosov Moscow State University, State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency
Summary:

Aim: to compare the repair process of DNA double-strand breaks in mammalian cells after acute versus prolonged exposure to X-ray irradiation with different dose rates. Material and methods. Studies were performed on primary human fibroblasts isolated from skin biopsies of healthy volunteers (women, 29 and 30 years). Cells were irradiated using an X-ray machine RUB RUST-M1 (JSC "Ruselectronics", Moscow, Russia) at 37°C temperature with a dose rate of 400 mGy/min (200 kV, 2*2.4 mA, a filter of 1.5mm AI) or 4 mGy/min (50 kV, 2*0.4 mA, a filter of 1.5 mm AI). Immuno-cytochemical protein staining was utilized for yH2AX and Rad51 foci analysis. Results. Phosphorylated histone H2AX (yH2AX) and the key protein of homologous recombination Rad51 foci formation and disappearance kinetics were investigated simultaneously in primary human dermal fibroblasts after acute and prolonged exposure to X-ray radiation at a same dose. It was shown that the relative yield of yH2AX foci per dose reduces with decrease in dose rate, while the relative yield of Rad51 foci conversely increases. Conclusion. Our findings suggest the fundamental differences in the ratio of non-homologous end joining and homologous recombination DNA repair in acute versus prolonged irradiated cells.

Effect of incubation in sodium chloride hypertonic solutions on y-radiation and hydrogen peroxide induced DNA damage in human peripheral blood lymphocytes

Year: 2014, volume 10 Issue: №4 Pages: 735-739
Heading: Genetics Article type: Original article
Authors: Vorobyeva N.Yu., Arkhangelskaya E.Yu., Bushmanov A.Yu., Osipov A.N.
Organization: State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency
Summary:

Aim: to study the effect incubation in NaCI hypertonic solutions on the yield of DNA single-strand breaks (SSB) and alkali-labile sites (ALS) induced by y-radiation and hydrogen peroxide in human peripheral blood lymphocytes in vitro. Material and methods. The lymphocytes isolated from the peripheral blood of healthy women donors aged 21-26 years were used for the study. Cell irradiation at doses of 1, 2, 3 and 5 Gy was performed using the "AGAT P1" unit (Russia, y-radiation source — 60Co, a dose rate of 1 Gy/min) at 4°C. In case of hydrogen peroxide exposure (25, 50 and 100 uM) the cells were incubated for 15 minutes at 4°C. Analysis of the DNA SSB and ALS was performed using the alkaline DNA comet assay. Results. Incubation of cells in 0.35 M NaCI solution (non-histone proteins dissociation) increases the yield of DNA SSB and ALS induced by v-radiation and hydrogen peroxide at ~ 1.3 times. Increasing the NaCI concentration to 0.6 M (linker histone H1 dissociation) leading to almost double increase in the DNA damage yield (~ 1.7-1.8 times). Further increasing the NaCI concentration leading to disruption of the structure of nucleosomes (1.2 M) and almost complete chromatin deproteinization (2 M) increased the yield of DNA damage not such pronounced as expected (~ 2.2-fold at 2 M concentration). Conclusion. The results showed that incubation in NaCI hypertonic solutions significantly increases the sensitivity of lymphocytes to DNA damaging action of free radicals.

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Imperfect conformation of experimental and epidemiological data for frequency of RET/РТС gene rearrangements in papillary thyroid carcinoma for the Chernobyl accident

Year: 2013, volume 9 Issue: №4 Pages: 795-801
Heading: Genetics Article type: Review
Authors: Ushenkova L.N., Koterov A.N., Biryukov А.P.
Organization: State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency
Summary:

In an overview and analytical study of the epidemiological data on the frequency of RET/РТС gene rearrangements in sporadic and radiogenic (patients after radiotherapy, residents of contaminated after the Chernobyl disaster areas, victims after the atomic bombings, etc.) carcinomas of the thyroid gland were examined. In general, the observed epidemiological laws were confirmed in radiobiology experiments by irradiation of different cultures of thyroid cells and ex vivo with the exception of Chernobyl cohorts. Induction of RET/РТС gene rearrangements by 131l exposure in children carcinomas of Chernobyl residents in mice did not observe too. It is concluded that the situation with the frequency of RET/РТС rearrangements in thyroid carcinoma in Chernobyl cohorts once again confirms the multifactorial nature of the induction and development of these tumors with a contribution of radiation and non-radiation factors (iodine deficiency and different stresses).

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Comparative study of the amount of DNA single-strand breaks and alkali-labile sites in human peripheral blood lymphocytes exposed to 365 nm UV versus X-ray radiation

Year: 2013, volume 9 Issue: №4 Pages: 791-795
Heading: Genetics Article type: Original article
Authors: Smetanina N.M., Pustovalova М.V., Bushmanov A.Yu., Osipov A.N.
Organization: State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency
Summary:

Aim: to compare the amount of DNA single-strand breaks (SSB) and alkali-labile sites (ALS) induced by DNA 365 nm ultraviolet (UV) versus X-rays radiation in human peripheral blood lymphocytes at in vitro conditions. Material and methods. Blood lymphocytes were isolated in a Ficoll-verografin density gradient. Irradiation of cells was performed using BLX-365 UV-unit (wavelength 365 ± 10nm)and RUSTM1 biological X-ray unit. Analysis of DNA SSB and ALS was carried out using comet assay at alkaline conditions. Results. For the first time it was demonstrated that the amount of SSB and ALS induced by 365 nm UV radiation at a dose of 10 kJ/m2 in human lymphocytes corresponds to the effect of X-rays at a dose of- 1 Gy. Conclusion. Exposure of human peripheral blood lymphocytes to 365 nm UV radiation at a dose of 1 kJ/m2 induces - 100 DNA SSB per cell.

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Induction and reparation of double-strand DNA breaks in V79 cells continuously exposed to low dose-rate Y-radiation

Year: 2013, volume 9 Issue: №4 Pages: 787-791
Heading: Genetics Article type: Original article
Authors: Ozerov I.V., Bushmanov A.Yu., Anchishkina N.A., Guryev D.V., Pustovalova M.V., Smetanina N.M., Arkhangelskaya E.Yu., Vorobyova N.Y., Osipov A.N.
Organization: State Scientific Research Center n.a. A.I. Burnasyan — Federal Medical Biophysical Center of Federal Medical Biological Agency
Summary:

Aim: to study the patterns of changes in the number of DNA double-strand breaks (DSB) in mammalian cells continuously exposed to low dose-rate y- radiation. Material and methods. Chinese hamster lung fibroblasts (V79) were used in this study. The y- irradiation of cells at a dose rate of 0.1 mGy/min was performed using the «Gamma-Panorama» unit (Cs-137). The fluorescence immunoassay of the phosphorylated H2AX-histone (y-H2AX) foci was used to investigate the DNA DSBs formation. Frequency of apoptotic cells was evaluated using «DNA halo» assay. 5 (6) — chloromethyl-2,7-dichlorodihydrofluorescein diacetate was used to estimate the reactive oxygen species (ROS) production. Results, it was showed that continuous low dose-rate irradiation of Chinese hamster V79 cells induces an increase of the y-H2AX foci number and ROS production rate at the early stages of exposure time (6-24 h, doses 3.6-14.4 cGy), while increasing exposition time and, therefore, the radiation dose (48-72 h, 28.8-43.2 cGy) caused a decrease in these endpoints to almost the control level. There was observed no significant changes in the frequency of apoptotic cells. Conclusion. It is assumed that the processes causing the DSB amount changes in mammalian cells continuously exposed to low dose-rate y-radiation are associated with the development of oxidative stress and subsequent activation of cellular antioxidant defense systems.

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