大鼠单次全脑照射早期皮层microRNA差异表达的研究

发布时间：2018-06-03 00:55

  本文选题：电离辐射 + 放射性脑损伤　； 参考：《扬州大学》2013年硕士论文

【摘要】：随着医学放射技术的发展,放射技术广泛应用于医学中的诊断和治疗。在疾病诊断中,X射线检查如X线射片、数字减影血管造影(DSA)、X射线计算机断层扫描(CT)和CT血管成像等在医学实践中的应用非常普遍；在疾病治疗中,现代放射治疗是治疗恶性肿瘤的主要手段之一,有时是唯一的手段。然而随着放射技术的不断改进及临床中广泛应用,电离辐射潜在致癌风险及放射性损伤备受关注,因此研究电离辐射致癌及放射性损伤的机理对放射学的发展有重要的意义。 研究发现一类新的非编码RNA—microRNA(miRNA)参与辐射诱导的生物学反应过程,研究表明辐射能在不同的组织和细胞中诱导miRNA的表达改变；miRNA在大脑的发育和功能中有很重要的作用。因此研究miRNA如何参与电离辐射引起的生物效应对揭示辐射的分子生物学机制及潜在的临床应用价值具有重要意义,miRNA有可能成为放射病诊断和治疗的潜在新靶点。 本研究拟探索放射性脑损伤早期miRNA的差异表达,为进一步研究提供依据。而脑组织的放射性损伤主要特征之一是血脑屏障的破坏。脑微血管内皮细胞是血脑屏障的主要组成细胞,因此本实验选择脑微血管内皮细胞模拟体外放射模型。 一、大鼠脑微血管内皮细胞培养的实验研究 目的获取miRNA芯片所需的大鼠脑微血管内皮细胞(BMVECs)。 方法采用两次酶消化法、密度梯度离心法获取BMVECs;应用vWF和GFAP免疫荧光双重标记方法进行BMVECs鉴定。 结果成功在体外培养出BMVECs,免疫荧光标记为vWF标记阳性、GFAP标记阴性。 二、电离辐射后大鼠脑微血管内皮细胞的miRNA表达谱变化的实验研究 目的观察电离辐射后BMVECs中miRNA表达的差异。 方法对融合的BMVECs采用6MV-X线照射,照射剂量为10Gy,用miRNA芯片分析照射组和对照组miRNA表达差异,选取目标miRNA进行RT-qPCR验证。 结果基因芯片结果中显示总共有18个miRNA在照射后发生差异表达,其中7个上调,11个下调；miR-34a上调10.8倍,miR-347上调约17.9倍,miR-106b约下调3.6倍。RT-qPCR验证显示照射组BMVECs的miR-34a、miR-347和miR-106b表达谱改变和芯片结果相符合。 三、大鼠单次全脑照射后早期大脑皮层miRNA表达改变的实验研究 目的分别观察在0、2、10和30Gy大鼠单次全脑照射后,1天和7天时大脑皮层目标miRNA表达的改变。 方法建立0、2、10和30Gy照射剂量大鼠单次全脑照射模型,分别在1天和7天时取大脑皮层,使用RT-qPCR观察miR-106b、miR-34a和miR-34表达谱改变。 结果与OGy照射组比较,照射后1天,2Gy组miR-106b表达无显著差异、miR-34a约上调6.4倍、miR-347约上调2倍；10Gy组miR-106b约下调1.7倍、miR-34a约上调5.3倍、miR-347表达无显著差异；30Gy组miR-106b约下调2.5倍、miR-34a表达无显著差异、miR-347约下调1.6倍。照射后1周,2Gy组miR-106b表达无显著差异、miR-34a约上调1.4倍、miR-347约上调2.3倍；10Gy组miR-106b表达无显著差异、miR-34a约上调2.6倍、miR-347约上调5.3倍；30Gy组miR-106b约下调1.4倍、miR-34a约上调1.8倍、miR-347约上调3.3倍。 结论 1.通过两次酶消化法和密度梯度离心法可获得较理想的BMVECs,经vWF免疫荧光标记阳性、GFAP标记阴性。 2.照射后早期即发生miRNA表达谱的改变,miRNA芯片提示总共有18个miRNA在照射后发生差异表达,其中7个上调,11个下调；应用RT-qPCR对芯片结果进行验证,结果显示照射组BMVECs的]miR-34a、miR-106b和miR-347表达谱改变和芯片结果相符合。 3.使用RT-qPCR观察大鼠大脑皮层放射后不同时间点和不同照射剂量下目标miRNA表达谱的改变,照射后miR-106b表达下调和miR-34a表达上调可能与放射性脑损伤的损伤机制相关；miR-347表达上调可能与放射性脑损伤的保护机制相关。
[Abstract]:With the development of medical radiation technology, radiation technology is widely used in the diagnosis and treatment of medicine. In the diagnosis of disease, X ray examination, such as X-ray radiography, digital subtraction angiography (DSA), X ray computed tomography (CT) and CT angiography, is very common in medical practice; in the treatment of disease, modern radiation therapy is One of the main means for the treatment of malignant tumors is sometimes the only means. However, with the continuous improvement of the radiation technology and the wide application of the clinic, the potential carcinogenic risk and radiation damage of ionizing radiation are concerned. Therefore, it is of great significance to study the mechanism of ionizing radiation carcinogenesis and radioactivity damage to the development of radiology.
A new class of non coded RNA - microRNA (miRNA) has been found to be involved in radiation induced biological reactions. Research shows that radiation can induce changes in the expression of miRNA in different tissues and cells; miRNA plays an important role in the development and function of the brain. Therefore, the study of how miRNA participates in the biological effects caused by ionizing radiation. It is of great significance to reveal the molecular biological mechanism of radiation and its potential clinical value. MiRNA may be a potential new target for the diagnosis and treatment of radiation sickness.
This study is to explore the differential expression of miRNA in the early stage of radiation brain injury and provide a basis for further research. One of the main characteristics of the brain tissue damage is the destruction of the blood brain barrier. The brain microvascular endothelial cells are the main components of the blood brain barrier, so this experiment chooses the model of the brain microvascular endothelial cells to simulate the radiological model in vitro.
Experimental study on culture of rat brain microvascular endothelial cells
Objective to obtain rat brain microvascular endothelial cells (BMVECs) required for miRNA chip.
Methods BMVECs was obtained by two enzyme digestion and density gradient centrifugation. BMVECs was identified by vWF and GFAP immunofluorescence double labeling.
Results BMVECs was successfully cultured in vitro. Immunofluorescence labeling was positive for vWF and negative for GFAP.
Two, the change of miRNA expression profile in rat brain microvascular endothelial cells after ionizing radiation
Objective To observe the difference of miRNA expression in BMVECs after ionizing radiation.
Methods the fusion BMVECs was irradiated with 6MV- X-ray and the dose of irradiation was 10Gy. The difference of miRNA expression between the irradiated group and the control group was analyzed with miRNA chip, and the target miRNA was selected for RT-qPCR verification.
Results the results showed that 18 miRNA were expressed differently after irradiation, including 7 up-regulated and 11 down-regulation, miR-34a up 10.8 times up, miR-347 up-regulated about 17.9 times, and miR-106b down regulation 3.6 times.RT-qPCR verification that the miR-34a of BMVECs in the irradiated group, miR-347 and miR-106b expression profiles were in accordance with the results of the chip.
Three, the change of miRNA expression in the early cerebral cortex after single whole brain irradiation in rats
Objective To observe the changes of miRNA expression in cerebral cortex of rats in 0,2,10 and 30Gy after 1 days and 7 days respectively.
Methods a single whole brain irradiation model of 0,2,10 and 30Gy irradiated rats was established. The cerebral cortex was taken at 1 days and 7 days respectively. The expression of miR-106b, miR-34a and miR-34 were observed by RT-qPCR.
Results compared with the OGy group, there was no significant difference in miR-106b expression in group 2Gy 1 days after irradiation, miR-34a was up to about up to 6.4 times, miR-347 was about 2 times up, miR-106b in 10Gy group was about 1.7 times down, miR-34a was up to 5.3 times, miR-347 expression was no significant difference, 30Gy miR-106b was about 2.5 times down, miR-34a expression was 1.6 times down. After 1 weeks, there was no significant difference in miR-106b expression in group 2Gy, miR-34a about up 1.4 times, miR-347 up to 2.3 times up, miR-106b expression in 10Gy group was not significantly different, miR-34a was about up 2.6 times, miR-347 was about up 5.3 times, miR-106b of 30Gy group was about 1.4 times down, miR-34a was about 1.8 times up, miR-347 approximately up 3.3 times.
conclusion
1. the ideal BMVECs can be obtained by two enzyme digestion and density gradient centrifugation. VWF is positive by immunofluorescence and negative by GFAP.
The expression profiles of miRNA were changed at the early stage of 2. irradiation. The miRNA chip suggested that 18 miRNA were expressed differently after irradiation, of which 7 were up and 11 down, and RT-qPCR was used to verify the results of the chip. The results showed that the]miR-34a of BMVECs in the irradiated group, miR-106b and miR-347 expression profiles were in accordance with the results of the chip.
3. RT-qPCR was used to observe the changes of target miRNA expression profiles at different time points and different doses of radiation in the cerebral cortex of rats. Down regulation of miR-106b expression and up regulation of miR-34a expression after irradiation may be related to the damage mechanism of radioactive brain injury, and the up-regulation of miR-347 expression may be related to the protective mechanism of radiation brain injury.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R818

【参考文献】

相关期刊论文 前1条

1 陈胜利,黄齐好,朱栋梁,邹蓉珠,黄子诚,陈国东,慕容爱;脑血管介入放射诊治中患者的X射线辐射评价[J];中国辐射卫生;2004年02期

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