【摘要】：本實驗室之前的研究發(fā)現(xiàn),缺氧/復(fù)氧(H/R)誘導(dǎo)原代培養(yǎng)心肌細(xì)胞核轉(zhuǎn)錄因子NF-κB p65入核,導(dǎo)致下游兩種粘附分子ICAM-1,VCAM-1表達(dá)量增加,引起心肌細(xì)胞炎癥反應(yīng)。而雌激素(E2)通過抑制p65入核來降低ICAM-1,VCAM-1表達(dá)量,從而實現(xiàn)心肌保護作用。IκBα是NF-κB抑制蛋白IκB蛋白家族的主要成員,在調(diào)節(jié)NF-κB信號通路的激活和失活中起重要作用。因此,我們設(shè)計了這個實驗用于檢驗雌激素對原代培養(yǎng)心肌細(xì)胞IκBα的影響。[實驗?zāi)康腯研究雌激素對缺氧/復(fù)氧后心肌細(xì)胞IκBα蛋白磷酸化、表達(dá)以及細(xì)胞內(nèi)定位的影響,從而檢驗雌激素是否是通過調(diào)節(jié)細(xì)胞內(nèi)IκBα來發(fā)揮抗炎作用。[實驗方法]利用原代培養(yǎng)心肌細(xì)胞和已經(jīng)檢驗建立成功的缺氧/復(fù)氧模型模擬心臟缺血再灌注,設(shè)立雌激素(5μM)孵育時間梯度(2h,4h,6h和8h),應(yīng)用RT-PCR技術(shù)檢測IκBαmRNA表達(dá)水平;western blot檢測IκBα蛋白磷酸化(p-IκBα)水平、IκBα蛋白表達(dá)水平以及IκBα蛋白在胞漿和胞核中的量。[實驗結(jié)果]與對照組比較,缺氧/復(fù)氧后心肌細(xì)胞內(nèi)IκBα磷酸化水平顯著增加,mRNA和蛋白表達(dá)水平以及胞核中IκBα蛋白水平顯著降低。與缺氧/復(fù)氧組比較,缺氧/復(fù)氧后孵育雌激素,細(xì)胞內(nèi)IκBα磷酸化水平顯著降低,而IκBαmRNA和蛋白表達(dá)水平以及胞核中IκBα蛋白水平顯著增加。[結(jié)論]雌激素通過抑制缺氧/復(fù)氧誘導(dǎo)的心肌細(xì)胞內(nèi)IκBα蛋白磷酸化,促進(jìn)IκBαmRNA和蛋白表達(dá)使細(xì)胞中IκBα蛋白保持一個相對穩(wěn)定且較高的水平;同時雌激素還可以通過促進(jìn)IκBα蛋白入核,使細(xì)胞核中活化的NF-κB失活,從而促進(jìn)NF-κB由細(xì)胞核中輸出,終止炎性因子轉(zhuǎn)錄反應(yīng),保護心肌細(xì)胞免受炎癥反應(yīng)的傷害,發(fā)揮起抗炎作用。
[Abstract]:Previous studies in our laboratory showed that hypoxia / reoxygenation (H / R) induced primary nuclear transcription factor NF- 魏 B p65 into the nucleus, resulting in an increase in the expression of ICAM-1 and VCAM-1, resulting in the inflammatory response of cardiomyocytes. Estrogen (E2) inhibits the expression of ICAM-1 and VCAM-1 by inhibiting p65 entry into the nucleus, and I 魏 B 偽 is a major member of the I 魏 B family of NF- 魏 B inhibitors, which plays an important role in regulating the activation and inactivation of NF- 魏 B signaling pathway. Therefore, we designed this experiment to test the effect of estrogen on I 魏 B 偽 in primary cultured cardiomyocytes. [objective] to study the effects of estrogen on the phosphorylation, expression and intracellular localization of I 魏 B 偽 protein in cardiomyocytes after hypoxia / reoxygenation, and to determine whether estrogen plays an anti-inflammatory role by regulating the intracellular I 魏 B 偽. [methods] using primary cultured cardiomyocytes and established a successful hypoxia / reoxygenation model to simulate myocardial ischemia reperfusion. Estradiol (5 渭 M) was incubated for 6 h and 8 h respectively. I 魏 B 偽 mRNA expression level was detected by RT-PCR and I 魏 B 偽 protein phosphorylation (p-I 魏 B 偽) level and I 魏 B 偽 protein in cytoplasm and nucleus were detected by western blot. [results] compared with the control group, I 魏 B 偽 phosphorylation in cardiomyocytes after hypoxia / reoxygenation significantly increased the expression of I 魏 B 偽 mRNA and protein, and significantly decreased the level of I 魏 B 偽 protein in the nucleus. Compared with hypoxia / reoxygenation group, the phosphorylation level of I 魏 B 偽 decreased significantly after hypoxia / reoxygenation, but I 魏 B 偽 mRNA and protein expression and I 魏 B 偽 protein in nucleus increased significantly. [conclusion] estrogen inhibits the phosphorylation of I 魏 B 偽 protein in cardiomyocytes induced by hypoxia / reoxygenation, and promotes the expression of I 魏 B 偽 mRNA and protein to maintain a relatively stable and high level of I 魏 B 偽 protein. At the same time, estrogen can inactivate the activated NF- 魏 B in the nucleus by promoting I 魏 B 偽 protein into the nucleus, thus promoting the output of NF- 魏 B from the nucleus, terminating the transcription of inflammatory factors, and protecting cardiomyocytes from inflammatory reaction. Play an anti-inflammatory effect.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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相關(guān)碩士學(xué)位論文 前2條

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本文編號：2132918