组蛋白H3K9三甲基化（H3K9me3）与神经系统高糖代谢记忆现象的相关性研究

发布时间：2018-06-09 04:43

本文选题：高血糖 + 代谢记忆　；参考：《宁夏医科大学》2016年硕士论文

【摘要】：目的探究神经系统中H3K9me3的表达与高糖代谢记忆现象的相关性。方法一、高剂量STZ造模1型糖尿病小鼠,利用胰岛素控制血糖的方法制备高糖代谢记忆模型,利用Western blot检测脑皮质和海马组织中H3K9me3以及甲基化转移酶SUV39H1的表达。二、在三种神经细胞模型中(神经前体细胞、神经元、HT22细胞系),给予高糖刺激不同时间,利用Western blot检测H3K9me3以及甲基化转移酶SUV39H1对高糖刺激的敏感性。三、在三种神经细胞模型中,给予高糖刺激不同时间后,撤除高糖刺激后制备代谢记忆模型,检测Western blot检测H3K9me3以及甲基化转移酶SUV39H1的表达并利用免疫荧光的方法,检测H3K9me3在HT22的表达。四、体外培养的HT22细胞中,研究高糖代谢记忆与异染色质结构改变的相关性。(1)利用Western blot检测高糖刺激以及撤除高糖刺激后的H3K9me3和异染色质蛋白HP1的表达。(2)利用Real-Time PCR法检测高糖刺激以及撤除高糖刺激后,大卫星DNA的相对表达量。(3)利用qChIP法检测高糖刺激以及撤除高糖刺激后,大卫星DNA区域中H3K9me3的相对占有率。五、在HT22细胞高糖代谢记忆模型中,利用XFe/XF 24分析仪和细胞线粒体压力试剂盒检测细胞能量代谢水平(OCR值),分析高糖代谢记忆与细胞能量代谢的关系。结果(1)糖尿病小鼠大脑中H3K9me3表达升高,在胰岛素控制高糖血症一周后的代谢记忆组H3K9me3的表达量仍高于正常对照组,并且与糖尿病组H3K9me3的表达相近;(2)在三种神经细胞模型中,H3K9me3的表达均对高糖刺激敏感,在短时间内H3K9me3表达量迅速升高至峰值;撤除高糖刺激后,H3K9me3的高表达现象维持一定的时间,即记忆现象。(3)在HT22细胞高糖代谢记忆模型中,H3K9me3持续高水平表达伴随异染色质蛋白HP1的持续高表达,异染色质松散程度的标志之一——大卫星DNA(major-SAT)区域H3K9me3维持高水平;(4)在HT22细胞高糖代谢记忆模型中,撤除高糖刺激后,线粒体中ATP产量可以迅速恢复到正常对照组的水平,即不表现代谢记忆现象。
[Abstract]:Objective to investigate the relationship between the expression of H 3K 9me3 and hyperglycemic memory in nervous system. Methods 1. High dose STZ was used to model type 1 diabetic mice. The hyperglycemic memory model was established by insulin control. The expression of H3K9me3 and SUV39H1 in cerebral cortex and hippocampus were detected by Western blot. Secondly, the sensitivity of H3K9me3 and SUV39H1 to hyperglycemic stimulation was detected by Western blot in three neural cell models (neural precursor cells, neuronal HT22 cell lines) treated with high glucose for different time. 3. In three nerve cell models, after high glucose stimulation for different time, the metabolic memory model was prepared after high glucose stimulation. The expression of H3K9me3 and methyltransferase SUV39H1 was detected by Western blot and immunofluorescence was used to detect the expression of H3K9me3 and SUV39H1. The expression of H3K9me3 in HT22 was detected. Fourth, in vitro culture of HT22 cells, To study the correlation between hyperglycemic memory and structural changes of heterochromatin. (1) Western blot was used to detect the expression of H3K9me3 and HP1 after high glucose stimulation, and to detect the expression of H3K9me3 and HP1 by Real-Time PCR. QChIP method was used to detect the relative share of H3K9me3 in the large satellite DNA region after high glucose stimulation and removal of high sugar stimulation. 5. In HT22 cell model of high glucose metabolism memory, the energy metabolism level of HT22 cells was measured by XFe / XF24 analyzer and mitochondrial pressure kit, and the relationship between high glucose metabolism memory and cell energy metabolism was analyzed. Results 1) the expression of H3K9me3 in the brain of diabetic mice was increased, and the expression of H3K9me3 in the metabolic memory group was still higher than that in the control group after one week of hyperglycemia controlled by insulin. And the expression of H3K9me3 was similar to that of diabetic group. The expression of H3K9me3 was sensitive to high glucose stimulation in the three nerve cell models, and the expression of H3K9me3 increased rapidly to its peak in a short time, and the overexpression of H3K9me3 was maintained for a certain time after the removal of high glucose stimulation. In the hyperglycemic memory model of HT22 cells, the high expression of H3K9me3 was associated with the continued high expression of heterochromatin protein HP1. One of the markers of the loose degree of heterochromatin, H3K9me3 maintained a high level of H3K9me3 in the large satellite DNA major-SAT region. In the hyperglycemic memory model of HT22 cells, ATP production in mitochondria recovered rapidly to the level of normal control after high glucose stimulation. That is, not showing the phenomenon of metabolic memory.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1

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