17β雌二醇对培养的新生大鼠心肌细胞肥大的影响

发布时间：2018-04-05 12:55

本文选题：心肌细胞肥大　切入点：17β雌二醇　出处：《武汉大学》2005年硕士论文

【摘要】：目的以体外培养的新生大鼠心肌细胞为模型,在细胞和分子水平观测17β雌二醇(17β-estradiol,E_2)对肾上腺素(phenylephrine,PE)刺激的心肌细胞肥大的影响并探讨其可能的机制,试图为心肌肥大的预防和治疗提供新的思路和实验依据。方法胰蛋白酶和胶原酶联合消化法分离心肌细胞,差速贴壁法和丝裂霉素C纯化心肌细胞,心肌特异性MHCα/β和Tn I抗体免疫细胞化学染色鉴定心肌细胞,建立心肌细胞模型;心肌细胞无血清培养24h后分为四组:对照组(不加处理因素),PE组(肾上腺素10~(-5)mol/L),E组(17β雌二醇10~(-8)mol/L),PE+E组(两者都加);根据实验要求在不同的作用时间检测如下指标:①计算机图像分析软件测量心肌细胞表面积,②[~3H]标记亮氨酸掺入法测定心肌细胞蛋白质合成速率,③免疫细胞化学染色方法检测心肌细胞原癌基因c-fos的蛋白表达,④半定量RT-PCR检测心肌细胞肥大特征性胚胎型基因β-肌球蛋白重链(β-myosin heavy chain,β-MHC)、α-骨骼肌肌动蛋白(α-skeletal-actin,α-skA)和心房肽(atrial natriuretic peptide,ANP)的mRNA表达。结果 1.心肌特异性MHCα/β和Tn I抗体免疫细胞化学染色结果显示:心肌细胞纯度大于95%,横纹清晰可见。2.肾上腺素刺激48h后,心肌细胞表面积明显增加,较对照组增加约2.6倍(P0.05);17β雌二醇对肾上腺素刺激所致心肌细胞表面积增加有显著抑制作用(P0.05)。3.肾上腺素刺激24h后,心肌细胞蛋白质合成速率明显增加,较对照组增加约3.1倍(P0.05);17β雌二醇对肾上腺素刺激所致心肌细胞蛋白质合成速率增加有显著抑制作用(P0.05)。4.肾上腺素刺激2h后,c-fos蛋白表达较强,心肌细胞胞核的周边区域可见许多绿色荧光免疫反应阳性产物,呈环状排列;17β雌二醇干预后,c-fos蛋白表达显著减弱。5.肾上腺素刺激48h后,与对照组相比,心肌细胞胚胎型基因β-MHC、α-skA和ANP mRNA表达水平明显增加(P0.05);17β雌二醇显著降低肥大心肌细胞β-MHC、α-skA的mRNA表达(P0.05),但进一步增加ANP mRNA表达(P0.05)。
[Abstract]:Objective to observe the effects of 17 尾 estradiol 17 尾 -estradiolol on the hypertrophy of cardiomyocytes stimulated by epinephrine (phenylephrinephrine PE) at cellular and molecular levels in neonatal rat cardiomyocytes cultured in vitro, and to explore its possible mechanism.This paper attempts to provide new ideas and experimental evidence for the prevention and treatment of myocardial hypertrophy.Methods Cardiac myocytes were isolated by trypsin and collagenase digestion. Myocardial cells were purified by differential adherent method and mitomycin C. Myocardial specific MHC 伪 / 尾 and TNI antibody immunocytochemical staining were used to identify cardiomyocytes and establish cardiomyocyte model.The surface area of myocardial cells was measured by computer image analysis software.2 the protein expression of proto-oncogene c-fos in cardiomyocytes was detected by immunocytochemical staining method for detecting the protein synthesis rate of cardiomyocytes by leucine incorporation method.4Semi-quantitative RT-PCR was used to detect the mRNA expression of 尾 -myosin heavy chain (尾 -MHCN), 伪 -skeletal muscle actin (伪 -skAn) and atrial natriuretic peptide (ANPs) in cardiomyocyte hypertrophy.Result 1.The results of immunocytochemical staining of myocardial specific MHC 伪 / 尾 and TNI antibodies showed that the purity of cardiomyocytes was greater than 95 and the striation was clearly visible.After 48 h of adrenaline stimulation, the surface area of cardiomyocytes increased significantly, and the surface area of cardiomyocytes was increased by 2.6 times than that of the control group. P0.05 and 17 尾 estradiol significantly inhibited the surface area increase of cardiomyocytes induced by epinephrine stimulation.After 24 hours of adrenaline stimulation, the protein synthesis rate of cardiomyocytes was significantly increased, and increased by about 3.1 times than that of the control group. P0.05 and 17 尾 estradiol significantly inhibited the increase of protein synthesis rate induced by epinephrine.After 2 hours of adrenaline stimulation, the expression of c-fos protein was stronger, and many green fluorescent immunoreactive products could be seen in the peripheral region of the nucleus of cardiac myocytes. The expression of c-fos protein decreased significantly after intervention with 17 尾 estradiol.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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