Early Differentially Express Of MicroRNA In Cortex Of Rats After Single Dose Whole-brain Irradiation

With the development of radiology, it is widely used in diagnosis and treatment in medical science. During the process of diagnosis of disease, the imaging tests with X-ray such as x-ray, digital subtraction angiography(DSA) and computed tomography(CT) are currently used very commonly; during the treatment of disease, radiotherapy is one of the most important method to cure malignancy, sometimes it is the only key. However, with the improvements of radiologic technology and its widely use in clinical practice, the risk of potentially carcinogenic and injury induced by ionzing radiation were concerned, and the pathogenetic researches on them would be of significant importance for the development of radiology.Recent investigation discovered that a new class of noncoding RNAs-microRNA (miRNA) participated the biological process of radiation. Researches showed that radiation can induce differential expression of miRNAs in tissue and cells and miRNAs play an important role in the development and function of brain.Therefore, the study on the role of miRNA in ionizing radiation is significant to reveal the molecular biological mechanisms of radiation and has potential clinical value, miRNA will probably become the potential target of the diagnose and treatment of radiation syndrome.The aim of this research is to explore the differently expressed miRNA during the early stage of radiation-induced brain injury. The damage of blood brain barrier(BBB) is one of the main characteristics of radiation-induced brain injury, furthermore, cerebral vascular injury is followed by degenerative structural changes in white matter(WM). The brain microvascular endothelial cells(BMVECs) are important components of BBB. Therefore, the BMVECs are the target cells of radication.Part I The method for culture of rat’s brain microvascular endothelial cellsObjective To obtain the rat’s brain microvascular endothelial cells(BMVECs) for subsequent miRNA microarray research. Methods The rat’s BMVECs were obtained by the methods of enzymatic digestion twice and density gradient centrifugation; immunofluorescence labeling for cell-specific antigens vWF and GFAP had been done to characterize BMVECs.Results The BMVECs were culture in vitro successfully, the cells were positive for vWF staining, whereas negative for GFAP.Part II The differentially express of miRNA in the rat’s BMVECs after exposure to ionizing radiationsObjective To observe the differentially express of miRNA in the rat’s BMVECs after exposure to ionizing radiations.Methods Fusion BMVECs were exposure to6MV X-ray irradiation at lOGy, and the miRNA microarray were used to analysis the differentially express of miRNA between irradiation group and control group. The real time quantitative PCR(RT-qPCR) were used to verify the result of the miRNA microarray.Results The miRNA microarray shows that18miRNAs were differentially expressed after exposure to ionizing radiations, with7miRNAs up-regulated and11miRNAs down-regulated more than2times. Among these, the miR-34a was up-regulated about10.8times, the miR-106b was down-regulated about3.6times and the miR-347were up-regulate about17.9times. The expression profiles of miR-34a, miR-106b and miR-347verified with RT-qPCR were in good agreement with result of the miRNA microarray.Part Ⅲ Early differentially express of microRNA in cortex of rats after single dose whole-brain irradiationObjective To observe the differentially express of microRNA in cortex of rats after0,2,10and30Gy single dose whole-brain irradiation at the first day and the7th day.Methods To establish the rats0,2,10and30Gy single dose whole-brain irradiation model, and the cortex were taken at the first day and the7th day. RT-qPCR was used to observe the differentially express of miR-34a, miR-106b and miR-347.Results Compare with OGy control group, one day after irradiation, in2Gy irradiation group, the expression profiles of miR-106b were of no significant difference, miR-34a were up-regulated about6.4times and miR-347were up-regulated about2times; in10Gy irradiation group, the expression profiles of miR-106b were down-regulated about1.7times, miR-34a were up-regulated about5.3times and miR-347were of no significant difference; in30Gy irradiation group, the expression profiles of miR-106b were down-regulated about2.5times, miR-34a were of no significant difference and miR-347were down-regulated about1.6times. One week after irradiation, in2Gy irradiation group, the expression profiles of miR-106b were of no significant difference, miR-34a were up-regulated about1.4times and miR-347were up-regulated about2.3times; in10Gy irradiation group, the expression profiles of miR-106b were of no significant difference, miR-34a were up-regulated about2.6times and miR-347were up-regulated about5.3times; in30Gy irradiation group, the expression profiles of miR-106b were down-regulated about1.4times, miR-34a were up-regulated about1.8times and miR-347were up-regulated about3.3times.Conclusions1. The rat’s BMVECs were obtained by the methods of enzymatic digestion twice and density gradient centrifugation, the cells were positive for vWF staining, whereas negative for GFAP.2. The expression profiles of microRNA were changed early after exposure to ionizing radiations. The miRNA microarray shows that18miRNAs were differentially expressed after exposure to ionizing radiations, with7miRNAs up-regulated and11miRNAs down-regulated more than2times. The expression profiles of miR-34a, miR-106b and miR-347verified with RT-qPCR were in good agreement with result of the miRNA microarray.3. The RT-qPCR was used to observe the differentially express of target miRNAs after different doses of6MV X-ray radiotherapy at two different points in time, the down-regulated express of miR-106b and the up-regulated express of miR-34a might be relate to the damage functions of radiation-induced brain injury; the up-regulated express of miR-347might be relate to the protective and reparative functions.