發(fā)布時(shí)間：2018-06-09 04:43

  本文選題：高血糖 + 代謝記憶　； 參考：《寧夏醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的探究神經(jīng)系統(tǒng)中H3K9me3的表達(dá)與高糖代謝記憶現(xiàn)象的相關(guān)性。方法一、高劑量STZ造模1型糖尿病小鼠,利用胰島素控制血糖的方法制備高糖代謝記憶模型,利用Western blot檢測腦皮質(zhì)和海馬組織中H3K9me3以及甲基化轉(zhuǎn)移酶SUV39H1的表達(dá)。二、在三種神經(jīng)細(xì)胞模型中(神經(jīng)前體細(xì)胞、神經(jīng)元、HT22細(xì)胞系),給予高糖刺激不同時(shí)間,利用Western blot檢測H3K9me3以及甲基化轉(zhuǎn)移酶SUV39H1對(duì)高糖刺激的敏感性。三、在三種神經(jīng)細(xì)胞模型中,給予高糖刺激不同時(shí)間后,撤除高糖刺激后制備代謝記憶模型,檢測Western blot檢測H3K9me3以及甲基化轉(zhuǎn)移酶SUV39H1的表達(dá)并利用免疫熒光的方法,檢測H3K9me3在HT22的表達(dá)。四、體外培養(yǎng)的HT22細(xì)胞中,研究高糖代謝記憶與異染色質(zhì)結(jié)構(gòu)改變的相關(guān)性。(1)利用Western blot檢測高糖刺激以及撤除高糖刺激后的H3K9me3和異染色質(zhì)蛋白HP1的表達(dá)。(2)利用Real-Time PCR法檢測高糖刺激以及撤除高糖刺激后,大衛(wèi)星DNA的相對(duì)表達(dá)量。(3)利用qChIP法檢測高糖刺激以及撤除高糖刺激后,大衛(wèi)星DNA區(qū)域中H3K9me3的相對(duì)占有率。五、在HT22細(xì)胞高糖代謝記憶模型中,利用XFe/XF 24分析儀和細(xì)胞線粒體壓力試劑盒檢測細(xì)胞能量代謝水平(OCR值),分析高糖代謝記憶與細(xì)胞能量代謝的關(guān)系。結(jié)果(1)糖尿病小鼠大腦中H3K9me3表達(dá)升高,在胰島素控制高糖血癥一周后的代謝記憶組H3K9me3的表達(dá)量仍高于正常對(duì)照組,并且與糖尿病組H3K9me3的表達(dá)相近;(2)在三種神經(jīng)細(xì)胞模型中,H3K9me3的表達(dá)均對(duì)高糖刺激敏感,在短時(shí)間內(nèi)H3K9me3表達(dá)量迅速升高至峰值;撤除高糖刺激后,H3K9me3的高表達(dá)現(xiàn)象維持一定的時(shí)間,即記憶現(xiàn)象。(3)在HT22細(xì)胞高糖代謝記憶模型中,H3K9me3持續(xù)高水平表達(dá)伴隨異染色質(zhì)蛋白HP1的持續(xù)高表達(dá),異染色質(zhì)松散程度的標(biāo)志之一——大衛(wèi)星DNA(major-SAT)區(qū)域H3K9me3維持高水平;(4)在HT22細(xì)胞高糖代謝記憶模型中,撤除高糖刺激后,線粒體中ATP產(chǎn)量可以迅速恢復(fù)到正常對(duì)照組的水平,即不表現(xiàn)代謝記憶現(xiàn)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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.1

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