Detection and study of the formation and repair of DNA double-strand breaks after irradiation

Transcription

1 Laboratory of Radiation Biology (LRB) Detection and study of the formation and repair of DNA double-strand breaks after irradiation Students: Ioana-Cezara Bucataru Bianca-Gabriela Zota Katarína Žirová Dan Reimitz JINR Student Practice Dubna, Russia 2016 Supervisors: Tatiana Bulanova Elena Kruglyakova

2 Joint Institute for Nuclear Research Laboratory of Radiation Biology Supervisors: Tatiana Bulanova Elena Kruglyakova Students: Bucataru Ioana Cezara and Zota Bianca Al. I. Cuza University of Iasi Katarína Žirová Comenius University in Bratislava Dan Reimitz The Czech Technical University in Prague

3 1. Introduction 2. Aims 3. Methodology 4. Results 5. Conclusion Outline

4 Introduction Genetic information Damaging agents Endogenous Exogenous Radiation exposure DNA is a critical target of ionizing radiation. DNA damage

5 Introduction Differences between the character of DNA damage caused by high-let and low-let radiation High-LET radiation Low-LET radiation

6 Introduction DNA damage Misrepair Repair Unrepair Mutation Mutated cell Cell survival Cell death

7 Aim of this project Research of the dosedependence and kinetic of formation and elimination of ionizing radiation-induced foci on normal human fibroblasts exposed to γ-rays of 60 Co. Applications of the investigation of DNA DSBs repair : 1. Diagnostics 2. Nuclear medicine 3. Space radiobiology

8 Methodology Preparation Deposition of cells from suspension onto glass slide in Petri dish Irradiation Fixation of cells at different times post-irradiation Visualization of induced DSBs co-immunostaining of Acquisition of images fluorescent microscope Detailed 3D analysis, quantitative evaluation Normal human skin fibroblasts

9 Methodology Preparation Radiation Doses (Gy) Irradiation Fixation of cells at different times post-irradiation γ-rays of 60 Co Dose Rate (Gy/min) LET (kev/μm) 0,745 0,3 Visualization of induced DSBs co-immunostaining of Acquisition of images fluorescent microscope Detailed 3D analysis, quantitative evaluation Rocus-M The laboratory of Nuclear Problems

10 Methodology Preparation Time post-irradiation: 1h, 4h, 24h Irradiation Fixation of cells at different times post-irradiation Visualization of induced DSBs co-immunostaining of Acquisition of images fluorescent microscope Detailed 3D analysis, quantitative evaluation

11 Methodology Preparation Irradiation Immunostaining use of an antibody-based method to detect a specific protein in a sample Fixation of cells at different times post-irradiation gh2ax (FITC - green) 53BP1 (Texas red) Visualization of induced DSBs co-immunostaining of Acquisition of images fluorescent microscope Nuclei (DAPI - blue) Merged Detailed 3D analysis, quantitative evaluation

12 Visualization of induced DSBs co-immunostaining of Secondary antibody Secondary antibody conjugated to fluorescent label Primary antibody H2AX H2AX 53BP1 53BP1 DNA DSB

13 Methodology Irradiation Fixation of cells at different times post-irradiation Visualization of induced DSBs co-immunostaining of Axio Imager.M2 Acquisition of images fluorescent microscope Detailed 3D analysis, quantitative evaluation

14 Methodology Preparation Irradiation Merged 3D image of fibroblast nuclei 1h after irradiation with 3 Gy Fixation of cells at different times post-irradiation Visualization of induced DSBs co-immunostaining of Acquisition of images fluorescent microscope Detailed 3D analysis, quantitative evaluation

15 of per cell after 1Gy irradiation Results Kinetics of the repair of double-strand breaks time post irradiation [h] Control

16 of nuclei Results Dose response Distribution of foci per cell No. of γh2ax/53bp1 per cell 1Gy 3Gy 5Gy

17 Conclusion The maximum number of was observed 1 h after γ-irradiation with 1 Gy. The number of DSBs decreased with time and after 24 hours number of foci reach the control value. The obtained curve of dose response has the dependence close to linear. It was observed the efficiently DSBs repair in human fibroblasts after irradiation with 1 Gy of gamma rays of 60 Co. 1h, 1Gy 24h, 1Gy

18 Thank you for your attention!