本文選題：心肌細(xì)胞肥大　切入點：17β雌二醇　出處：《武漢大學(xué)》2005年碩士論文

【摘要】：目的 以體外培養(yǎng)的新生大鼠心肌細(xì)胞為模型,在細(xì)胞和分子水平觀測17β雌二醇(17β-estradiol,E_2)對腎上腺素(phenylephrine,PE)刺激的心肌細(xì)胞肥大的影響并探討其可能的機(jī)制,試圖為心肌肥大的預(yù)防和治療提供新的思路和實驗依據(jù)。 方法 胰蛋白酶和膠原酶聯(lián)合消化法分離心肌細(xì)胞,差速貼壁法和絲裂霉素C純化心肌細(xì)胞,心肌特異性MHCα/β和Tn I抗體免疫細(xì)胞化學(xué)染色鑒定心肌細(xì)胞,建立心肌細(xì)胞模型;心肌細(xì)胞無血清培養(yǎng)24h后分為四組:對照組(不加處理因素),PE組(腎上腺素10~(-5)mol/L),E組(17β雌二醇10~(-8)mol/L),PE+E組(兩者都加);根據(jù)實驗要求在不同的作用時間檢測如下指標(biāo):①計算機(jī)圖像分析軟件測量心肌細(xì)胞表面積,②[~3H]標(biāo)記亮氨酸摻入法測定心肌細(xì)胞蛋白質(zhì)合成速率,③免疫細(xì)胞化學(xué)染色方法檢測心肌細(xì)胞原癌基因c-fos的蛋白表達(dá),④半定量RT-PCR檢測心肌細(xì)胞肥大特征性胚胎型基因β-肌球蛋白重鏈(β-myosin heavy chain,β-MHC)、α-骨骼肌肌動蛋白(α-skeletal-actin,α-skA)和心房肽(atrial natriuretic peptide,ANP)的mRNA表達(dá)。 結(jié)果 1.心肌特異性MHCα/β和Tn I抗體免疫細(xì)胞化學(xué)染色結(jié)果顯示:心肌細(xì)胞純度大于95%,橫紋清晰可見。2.腎上腺素刺激48h后,心肌細(xì)胞表面積明顯增加,較對照組增加約2.6倍(P0.05);17β雌二醇對腎上腺素刺激所致心肌細(xì)胞表面積增加有顯著抑制作用(P0.05)。3.腎上腺素刺激24h后,心肌細(xì)胞蛋白質(zhì)合成速率明顯增加,較對照組增加約3.1倍(P0.05);17β雌二醇對腎上腺素刺激所致心肌細(xì)胞蛋白質(zhì)合成速率增加有顯著抑制作用(P0.05)。4.腎上腺素刺激2h后,c-fos蛋白表達(dá)較強,心肌細(xì)胞胞核的周邊區(qū)域可見許多綠色熒光免疫反應(yīng)陽性產(chǎn)物,呈環(huán)狀排列;17β雌二醇干預(yù)后,c-fos蛋白表達(dá)顯著減弱。5.腎上腺素刺激48h后,與對照組相比,心肌細(xì)胞胚胎型基因β-MHC、α-skA和ANP mRNA表達(dá)水平明顯增加(P0.05);17β雌二醇顯著降低肥大心肌細(xì)胞β-MHC、α-skA的mRNA表達(dá)(P0.05),但進(jìn)一步增加ANP mRNA表達(dá)(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 偽 / 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), 偽 -skeletal muscle actin (偽 -skAn) and atrial natriuretic peptide (ANPs) in cardiomyocyte hypertrophy.Result 1.The results of immunocytochemical staining of myocardial specific MHC 偽 / 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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R329

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本文編號：1714820