| Birth defects problem has become one of important reason affecting the quality of the population. Many factors cause of birth defects, and iatrogenic factors cannot be ignored. The clinical diagnosis methods such as X-ray, CT and certain drugs, such as antitumor drugs have been confirmed with birth defects and abortion, but other methods, such as Magnetic Resonance Imaging (MRI), are not clear whether have an effects on embryonic development.Based on magnetic field, MRI is the imaging technology by gathering the signals produced by nuclear magnetic resonance and rebuilding it to form the image. MRI which was considered to be a safe technology in the beginning of the application, is another significant progress after CT. Ionizing radiation produced by CT can cause atoms conformation irreversibly change, giving rise to biological molecular ionization or covalent bond rupture, but the electromagnetic radiation generated by MRI is just change the position of the atoms, and will not change the structure, composition, properties of biological molecules. In addition, MRI has become one of the most important methods of clinical diagnosis because of its characteristics of flexibility, easy acceptability for patients, evaluating pathological and physiological parameters at the same time, non-invasion and so on, and has greatly promoted the rapid development of medicine, nerve physiology and cognitive neuroscience. MRI obtained the rapid development and has come into use in the field of obstetrics and gynecology since the early 1980s. Now MRI is applied in pregnancy middle-late prenatal diagnosis and has played a increasingly important role for birth defects control. But because of its widespread use, there are more and more pregnant early embryo is inevitably exposed to MRI environment. Therefore, it a time of increasing concerns over MRI safety on embryo development.There are three kinds of security risk to embryo that MRI generated:a strong Static Magnetic Field (SMF), Gradient Magnetic Field (GMF) and Radio Frequency Magnetic Field (RFP). The major harm of SMF is biological effects and the projection effect, GMF is noise, biological effects and peripheral nerve stimulation, RFP is heating effect. So far, the safety research of MRI on embryos mainly focused on low-middle intensity of SMF, the research about GM and RFP is very less, not to mention the combined effects of three kinds of magnetic field. As a consequence, there is lack of comprehensive understanding on biological effect of MRI. With the development of MRI technology, higher intensity of magnetic field will be used to pursue higher spatial resolution and shorter time to scan. So it is urgent to answer the question of whether MRI is safety totally.This paper studied the effects of MRI exposure on embryonic development with the methods of embryo intrauterine growth index assessment and high-throughput proteomics screening based on TMT notation and LC-MS/MS after the model mice of E10.5d exposing to the working status of 1.5T MTR for short time. Next, we further analyzed which part of important biomolecules change the expression level during the embryonic development after MRI exposing. In vitro, we observed cell physiological characteristics change with the methods of proliferation, cycle, migration and subcellular structure after cell exposing to the working status of 1.5T MTR for short time. This paper aims to analyze the hazard of MRI exposure from multiple perspectives and the molecular mechanism of the influence, and provide sufficient scientific evidence for the rational and secure use of MRI techniques for clinical.PART I The analysis of embryo intrauterine growth and proteomics after 1.5 T MRI exposureObjectiveThis paper studied the effects of MRI exposure on embryonic development with the methods of embryo intrauterine growth index assessment and high-throughput proteomics screening based on TMT notation and LC-MS/MS after the model mice of E10.5d exposing to the working status of 1.5T MTR for short time.MethodsBuild 10.5 dpc pregnant mice, exposed to the working status of 1.5 T MRI 15 minutes,30 minutes,60 minutes, dissect pregnant mice to observe embryo intrauterine development situation at 18.5 dpc or dissect pregnant mice to analyze high-throughput proteomics based on TMT notation and LC-MS/MS with extract embryonic total protein at 4 hours and 24hours after MRI exposure. Results1.10.5 d pregnant mice got 15 minutes,30 minutes or 60 minutes of 1.5 T working-status MRI exposure, dissected pregnant mice to observe embryo intrauterine development situation at 18.5 dpc, and found MRI exposure did not affect the total number of implantation, tire nest gross weight, total weight of placenta, embryo survival rate, mortality rate and absorption rate, live births fetal weight, body length and tail length index.2. In the group that collecting embryos at 4 hours after MRI exposure, compared with control group, the number of protein levels increased more than 2 times respectively are 12,15 and 52; the number of protein levels increased more than 1.5 times respectively are 104,82 and 162; the number of protein levels reduced at least 0.5 times respectively are 3,1 and 8; the number of protein levels reduced at least 0.667 times respectively are 43,5 and 124. In the group that collecting embryos at 24 hours after MRI exposure, compared with control group, the number of protein levels increased more than 2 times respectively are 36,30 and 26; the number of protein levels increased more than 1.5 times respectively are 105,128 and 122; the number of protein levels reduced at least 0.5 times respectively are 105,128 and 122; the number of protein levels reduced at least 0.667 times respectively are 82,193 and 309.3. In the group that collecting embryos at 24 hours after MRI exposure, biological process of GO analysis with 122 protein ID that protein level increased more than 1.5 times by proteomics mainly related to RNA splicing, mRNA metabolic process, cardiac contraction, blood circulation, and so on; signaling pathways mainly related to RNA splicing, myocardial contraction, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy. Biological process of GO analysis with 309 protein ID that protein level reduced at least 0.667 times by proteomics mainly related to transport, cell division, metabolism of protein, RNA metabolism, and so on; signaling pathways mainly related to ubiquitin protein degradation mediated, huntington, cell cycle, and so on.Conclusions1.10.5 d pregnant mice got 15 minutes,30 minutes or 60 minutes of 1.5 T working-status MRI exposure, dissected pregnant mice to observe embryo intrauterine development situation at 18.5 dpc, and found MRI exposure did not affect the total number of implantation, tire nest gross weight, total weight of placenta, embryo survival rate, mortality rate and absorption rate, live births fetal weight, body length and tail length index.2. There are so many protein ID that were identified by proteomics that differentially expressed after MRI exposure, and signaling pathways that these differentially expressed proteins involved mainly related to RNA splicing, ubiquitin protein degradation pathway mediated, and so on. These signaling pathways have important roles in embryonic development, function building and function maintenance.PART Ⅱ Expression analysis of mouse embryos calmodulin and splicing factors after 1.5 T MRI exposureObjectiveCalmodulin and differential expression splicing factors selected by Proteomics were verified with traditional molecular biology method to further illustrate molecular function in embryonic short-term exposure in the working status of 1.5 T MRI.MethodsBuild 10.5 dpc pregnant mice, and exposed to the working status of 1.5 T MRI for 15 minutes,30 minutes,60 minutes, dissect pregnant mice to obtain embryonic tissues after 4 hours and 24 hours exposure, and analyze Calmodulin and splicing factors expression situation by fluorescence quantitative PCR and Western blot.Results1.10.5 dpc pregnant mice were exposed to the working status of 1.5 T MRI for 15 minutes,30 minutes,60 minutes and dissect pregnant mice to obtain embryonic tissues after 24 hours exposure, fluorescence quantitative PCR results showed that the expression of calmodulin was significantly raised at the level of gene transcription.2.10.5 dpc pregnant mice were exposed to the working status of 1.5 T MRI for 15 minutes,30 minutes,60 minutes and dissected pregnant mice to obtain embryonic tissues after 4hours and 24 hours exposure. Proteomics identified 59 protein IDs of splicing factors from those embryonic tissue. Those proteins from 24 hours were more significantly differentially expressed than from 4 hours through the analysis of the heat map.25 out of 59 proteins from 24 hours embryonic tissues expressed increased in more than 1.2 times.3. SCAF11, SR3B6, SRSF1, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7 were selected to carry out fluorescence quantitative PCR, and results showed that the expression of those genes were all raised to certain degree at the level of gene transcription, whereby 30/60 minutes groups of Sf3b6 and Srsf7,15 minutes groups of Srsf5 and Srsf7 were significantly raised at the level of gene transcription.4. SRSF1, SRSF3, SRSF4 and SRSF6 were selected to carry out western blot, and results showed that the expression level of those proteins was significantly reduced after 24 hours exposure.5. Western blot was carried out to test protein levels of SRSF1, SRSF3, SRSF4 and SRSF6 in the different developmental stage of mouse embryos, and results showed that protein levels are highly dynamic.6.10.5 dpc pregnant mice were exposed to the working status of 1.5 T MRI for 60 minutes, and dissect pregnant mice to obtain embryonic tissues after 12/24/48/72 hours exposure, Western blot results showed that protein levels of SRSF1, SRSF3, SRSF4, SRSF6 could returned to normal level in a certain time.Conclusions1. Exposure of working status of 1.5 T MRI for 15 minutes,30 minutes,60 minutes to embryos from 10.5 dpc pregnant mice caused calmodulin gene transcription level increase which hints that calmodulin genes may mediate Ca2+ signal transduction pathways during MRI exposure affecting embryonic development..2. Exposure of working status of 1.5 T MRI for 15 minutes,30 minutes,60 minutes to embryos from 10.5 dpc pregnant mice caused part of splicing factors gene transcription level increase which hints that MRI exposure may affect embryonic development throught change splicing factors gene transcription levels.3. Western blot results that the expression level of SRSF1, SRSF3, SRSF4 and SRSF6 were significantly reduced after 24 hours exposure and could be recoverd after MRI exposure within a certain time, which hints that MRI short-time exposure may have an effect on embryonic development and the influence of MRI exposure on protein levels were reversible.4. During different periods of embryonic development, the protein levels of SRSF1, SRSF3, SRSF4 and SRSF6 are highly dynamic, which hints those protein levels are strictly controlled according to the growth of embryo, and alter of protein level at certain stage could affect the normal development of the embryo.PART Ⅲ 1.5 T MRI exposure effects on cellular physiological characteristicsObjectiveFor further research of the working-status 1.5 T MRI biological effects, we researched the change of cell physiological characteristics by detecting cell proliferation, cycle, migration and subcellular structure changes at the cellular level to provide theoretical basis for further research of MRI safety.MethodsUsing homemade incubator for short-term exposure to the working status of 1.5 T MRI, we detect cell proliferation, cycle, migration and subcellular structure changes at the cellular level by flow cytometry, the WST-1 method, wound scratch assay and transmission electron microscopy.Results1. WST-1 results showed that cell proliferation rate of Swan 71 reduced after 60 minutes exposure of 1.5 T MRI compared with control group, and the difference begun to emerge after 24 hours of MRI exposure, proliferation rate have significant differences after 48 hours of MRI exposure (P=0.0056346).2. Flow cytometry results showed that cell cycle distribution of Swan 71 wasn’t affected after 60 minutes exposure of 1.5 T MRI (P> 0.5).3. Wound scratch assay results showed that migration speed of SiHa was significantly affected after 60 minutes exposure of 1.5 T MRI compared with control group, after 24 Hours the average migration distance of control was 65.27 um, but the average migration distance of MRI exposure group was 45.94μm (P< 0.001).4. Transmission electron microscopy results showed that nucleus of Swan71 was pyknotic, Mitochondrial was swelling significantly after 60 minutes exposure of 1.5 T MRI.Conclusions1.1.5T MRI exposure with 60 minutes reduced the speed of Swan 71 proliferation, but didn’t affect the cell cycle distribution.2.1.5T MRI exposure with 60 minutes reduced SiHa migration velocity.3.1.5T MRI exposure with 60 minutes caused Swan 71 mitochondria swelling. |
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