【作者】 林瑾仪；

【导师】 葛均波； 邹云增；

【作者基本信息】 复旦大学 ， 心血管内科学， 2009， 博士

【摘要】 促红细胞生成素（Erythropoietin,Epo）是一种低氧诱导的谱系特异性的内分泌激素,可调节红细胞的形成,临床上广泛应用于各种原因引起的贫血,如终末期肾病、获得性免疫缺陷综合征以及需要化疗的非造血系统恶性肿瘤。后来研究发现Epo受体在心血管系统有着广泛的分布,基础研究揭示Epo可保护缺血再灌注心肌和限制心肌梗死的范围。有关Epo对心肌细胞肥大影响的报导在国内外非常有限。本研究以Epo作为干预因素,离体培养新生大鼠心肌细胞作为研究对象,以明确Epo对心肌肥大的影响并初步探讨其肥厚相关的信号通路。第一部分新生大鼠心肌离体培养和Epo刺激后形态学观察目的:1)取得稳定的新生大鼠心肌细胞离体培养模型,cTnI免疫组化染色观察Epo刺激不同时间对于心肌细胞大小的影响;2)观察Epo刺激引起的细胞超微结构改变。方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为2%FBS低血清培液同步化24h。实验分为:空白对照组、AngⅡ组和Epo组。AngⅡ终浓度为1×10^-6M;Epo终浓度为10U/ml。于24h、48h和72h固定各组细胞,cTnI免疫组化染色,光镜下测量心肌细胞面积。电镜观察心肌细胞超微结构所用的低血清培养液含0.1%FBS,实验分组同前。于24h、48h和72h固定各组细胞,电镜下观察各组心肌细胞超微结构。结果:1)干预24h,对照组心肌细胞面积2135.6±524.8μm²,AngⅡ干预组3202.3±661.2μm²,Epo组2586.2±165.3μm²,AngⅡ组相比对照组和Epo组,心肌细胞明显增大,P＜0.05。干预48h,对照组心肌细胞面积1844.5±323.3μm²,AngⅡ干预组3321.0±428.8μm²,Epo组3318.9±321.3μm²,AngⅡ组和Epo组相比对照组,心肌细胞明显增大,P＜0.05;AngⅡ组与Epo组相比,差别无统计学意义。干预72h,对照组心肌细胞面积2213.90±149.5μm²,AngⅡ干预组3222.20±225.3μm²,Epo组3317.20±196.1μm²,AngⅡ组和Epo组相比对照组,心肌细胞明显增大,P＜0.05;AngⅡ组与Epo组相比,差别无统计学意义。Epo干预48h和干预72h相比24h组,细胞面积明显增大,P＜0.05。2)电镜观察,培养72h,对照组心肌细胞肌丝溶解,细胞器大多破坏;AngⅡ组和Epo组细胞核不规则,核膜凹陷,核仁较清晰,染色质浅;线粒体数量增多,肿胀;内质网以及高尔基器等与合成分泌有关的细胞器发达,72小时观察到细胞内出现亚细胞结构的空泡化改变。结论:Epo刺激可使心肌细胞增大,使心肌细胞出现肥厚的亚细胞结构改变。第二部分Epo对心肌细胞蛋白激酶活性及肥大相关基因的影响目的:考察Epo刺激后细胞外信号调节激酶ERK的激活和心肌细胞肥大相关基因ANP、BNP、c-fos mRNA的表达量的变化。方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为0.1%FBS低血清培液同步化24h,加入Epo干预。1)Epo终浓度为10U/ml,分别在0 min（对照组）、10min、15min、30min、60min和120min终止干预,提取心肌细胞总蛋白,Western blot检测心肌细胞磷酸化ERK1/2（p-ERK1/2）含量;2)Epo终浓度为0 U/ml（对照组）、5 U/ml、10 U/ml、100 U/ml,干预30min,提取心肌细胞总蛋白,Western blot检测心肌细胞p-ERK1/2含量;3)实验分两组,对照组和Epo组,Epo10U/ml干预30min,提取心肌细胞总RNA,RT—PCR法测定两组c-fos mRNA表达量;同样Epo和对照组分组干预2h,RT—PCR法测定ANP mRNA和BNP mRNA表达量。结果:1)Epo10U/ml干预心肌细胞后p-ERK2表达随时间出现先升高后下降的变化,30min时达到高峰,与干预前和干预后120min相比,P＜0.05;p-ERK1在Epo干预后也观察到先升高后降低的趋势。2)Epo不同浓度干预心肌细胞,随Epo干预浓度增大,p-ERK2水平升高,10 U/ml及100 U/ml浓度干预组与对照组相比,差别有统计学意义（P＜0.05）。p-ERK1在Epo处理后也观察到随浓度升高而升高的趋势。3)Epo10U/ml干预心肌细胞,RT-PCR结果显示,30min c-fosmRNA表达量与对照组相比差异不明显,2h ANP mRNA和BNP mRNA表达量和对照组相比,明显升高,P＜0.05。结论:1)Epo刺激使心肌细胞p-ERK1/2呈时间相关性和浓度依赖性升高;2)要观察p-ERK1/2变化,可取Epo10U/ml刺激30min做为最佳观察条件;2)Epo刺激促进了心肌肥大相关基因表达。第三部分Epo引起心肌细胞肥大反应信号通路初探目的:1)观察Epo刺激对心肌AGT mRNA表达量的影响;2)初步探讨心肌中Epo-ERK信号通路和RAS系统之间是否存在相互关系。方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为0.1%FBS低血清培养液同步化24h,开始分组干预。1)Epo10U/ml干预细胞,分别在0h（对照组）、1h、2h、6h、12h、24h提取心肌细胞总RNA,RT-PCR法测定并比较干预不同时间后AGT mRNA表达量。2)实验分为6组:a)空白对照组;b)AngⅡ组:AngⅡ1×10^-6M 8min;c)ARB+AngⅡ组:替米沙坦1×10^-6M阻断30min后加入AngⅡ1×10^-6M 8min;d)Epo组:Epo 10U/ml 30min;e)ACEI+Epo组:依那普利1×10^-6M阻断2h后加入Epo 10U/ml 30min;f)ARB+Epo组:替米沙坦1×10^-6M阻断30min后加入Epo 10U/ml 30min。干预结束提取心肌细胞总蛋白,Western blot测定各组心肌细胞p-ERK1/2含量。3)实验分3组:a)空白对照组;b)Epo组:Epo 10U/ml 6h;c)PD98059+Epo组:PD98059浓度50×10^-6M阻断30min后加入Epo 10U/ml 6h。终止干预,提取心肌细胞总RNA,比较三组的AGT mRNA表达量。结果:1)Epo10U/ml干预心肌细胞24h,使AGT mRNA表达量自2h起升高（P＜0.05）,随干预时间延长表现为先上升后下降趋势,6h时AGT mRNA表达量水平最高。2)Epo干预心肌p-ERK1/2的升高可被依那普利和替米沙坦所逆转。3)p-ERK1/2的阻断,并不能逆转Epo对于AGT mRNA表达的促进作用。结论:Epo可上调心肌细胞中AGT基因的转录,Epo激活ERK信号通路与RAS系统存在相互关系。更多还原

【Abstract】 Erythropoietin（Epo）,a hypoxia-induced,lineage-specific hormone,is essential for normal erythropoiesis.Recombinant human Erythropoietin（rh-Epo） is widely used in the treatment of various kinds of anemia,such as end-stage renal disease,Human Immunodeficiency Virus infection,and in patients with non-myeloid malignancies on chemotherapy.Recently it is found out that Epo receptor is widely existed in cardiovascular system.Animal studies demonstrated that Epo can potentially protect myocardium against ischemia-reperfusion-induced injury in and restrict infarcted size. However,little is known about the role of Epo in the development of cardiac hypertrophy.In this study,we examined the in vitro effects of Epo on hypertrophic responses of cultured cardiomyocytes of neonatal rats.PART ONE:Epo-induced morphological changes in cultured cardiomyocytes of neonatal ratsObjective:To observe the morphological changes of cultured cardiomyocytes after addition of Epo for various times.Methods:Primary cardiac myocytes were isolated from neonatal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%fetal bovine serum（FBS） for the first 24 hours,and then in 2%FBS-containing medium for synchronization in the next 24 hours.Three groups were included in these experiments:control group;AngⅡtreated group and Epo treated group.The final concentration of AngⅡwas 1×10^-6M; and the final concentration of Epo was 10U/ml.Culture cells were fixed at 24h,48h and 72h,respectively,and immunohistochemical stained with cTnI.The sizes of the cardiomyocytes were measured with light microscopy.Low serum culture medium containing 0.1%FBS was used for myocardium ultrastructure study with transmission electron microscope.Three groups were included as describe before. Cells were fixed at 24h,48h and 72h,and myocardium ultrastructure was observed by a transmission electron microscope.Results:1) After 24 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 2135.6±524.8μm² for control group,3202.3±661.2μm² for AngⅡtreated group,and 2586.2±165.3μm² for Epo group.Comparing to the control group and the Epo treated group,the mean size of the ceils was significantly increased for AngⅡtreated group（P＜0.05）.After 48 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 1844.5±323.3μm² for control group,3321.0±428.8μm² for AngⅡtreated group,and 3318.9±321.3μm² for Epo treated group.Comparing to the control group,the mean surface area of the cells is significantly increased in AngⅡtreated group and in Epo treated group（P＜0.05）.The difference between AngⅡtreated group and Epo treated group was not significant.After 72 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 2213.90±149.5μm² for control group,3222.20 ±225.3μm² for AngⅡtreated group,and 3317.20±196.1μm² for Epo treated group.Compared to control group,the mean surface area of the cells was significantly increased in AngⅡtreated group and in Epo treated group（P＜0.05）;however,no significant difference was seen between AngⅡtreated group and Epo treated group. Epo intervention for 48h and 72h significantly increased the mean surface area of the cells comparing to the 24h treated group（P＜0.05）.2) Transmission electron microscopy observation showed that in the control group myofilament dissolved gradually,and most of organelle was damaged when exposure to 72hs low-FBS culture medium.Cardiac myocytes in AngⅡtreated group and the Epo treated group had irregular nuclei,distinct nucleolus,faint chromatin,recessed nuclear membrane,lots of swollen mitochondria and prospersous secretion and synthesis related organelles such as endoplasmic reticulum and Golgi body,showed vacuolization occurred within sub-cellular structures when exposed to AngⅡand Epo for 72h.Conclusion:Addition of Epo to culture medium can induce enlargement of cardiomyocytes and ultrastructural changes in cardiomyocytes.PART TWO:Epo-induced upregulation of ERK activation and hypertrophy-related gene expression in cultured cardiomyocytesObjective:To investigate whether Epo upregulates ERK activation and some specific gene expression,such as ANP,BNP and c-fos,in cultured cardiomyocytes.Methods:Primary cardiac myocytes were isolated from natal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%fetal bovine serum（FBS） for the first 24 hours,then in 0.1%FBS-containing medium for synchronization for the next 24 hours, before Epo intervention.1)Epo treated cardiomyocyte with final concentration of Epo 10U/ml,Epo intervention was ended at 0min（control group）,10min,15min,30min, 60min and 120min respectively.Total cellular protein was extracted and Western blot detection was used to quantify the myocardial p-ERK1/2;2) Epo treated cardiomyocyte for 30min,with the final concentration 0（control group）,5 U /ml,10 U/ml and 100 U/ml respectively.Total cellular protein was extracted and western blot detection was used to quantify the myocardial p-ERK1/2 with different Epo concentration treatment;3) Cardiomyocytes were divided into two groups,control group and the Epo treated group（Epo10U/ml treated for 30min）.The total RNA of the cardiomyocytes were extracted and RT-PCR was introduced to semi-quantify c-fos mRNA expression.Similarly,ANP and BNP mRNA was extracted from the cardiomyocytes with Epo treatment or blank control for 2hs and gene expression level was measured by RT-PCR.Results:1) p-ERK2 expression in cardiac myocytes first increased and then decline in 120min after Epo 10U/ml intervention,and the expression level was at peak when harvested from Epo 10U/ml treatment for 30min,comparing to the control group and the 120min-treated group（P＜0.05）.The similar changes of p-ERK1 expression were also seen but the differences were not significant.2) Increasing concentration of Epo intervention resulted in increased p-ERK2 expression levels in cardiac myocytes, p-ERK2 expression was significantly higher in the group of Epo 10 U/ml and 100 U/ ml than that of the control group（P＜0.05）.The p-ERK1 expression was also increased with the increasd Epo concentration,yet the differences were not significant. 3) With Epo10U/ml intervention in cardiac myocytes,c-fos mRNA expression was not increased significantly,yet ANP and BNP mRNA expression were significantly higher than that of control group（P＜0.05）.Conclusion:1) Epo induces ERK1/2 activation in a time- and concentration-dependent manner;2) Treatment with Epo at 10U/ml for 30min can significantly increase ERK1/2 phosphorylation;3) Epo can stimulate the expression of ANP and BNP genes,and prone to increase c-fos gene expression.PART THREE:Involvement of angiotensinⅡin Epo-induced hypertrophic responses of cardiomyocytesObjective:1) To examine the impact of Epo on angiotensinogen（AGT） mRNA expression in cardiomyocytes;2) To examine whether RAS is involved in Epo-induced ERK activation in cardiomyocytes.Methods:Primary cardiac myocytes were isolated from natal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%FBS for the first 24h,and then in 0.1% FBS-containing medium for synchronization for the next 24h.1) Epo treatment with final concentration of 10U/ml was terminated at 0min（control group）,1h,2h,6h,12h,24h respectively.Total cellular RNA were extracted and RT-PCR was introduced to quantify the AGT mRNA expression.2) 6 groups were included in this study:a) Control group;b) AngⅡgroup:AngⅡ1×10^-6M for 8min;c) ARB+AngⅡgroup:Telmisartan 1×10^-6M blockage for 30min then AngⅡ1×10^-6M for 8min;d) Epo Group:Epo 10U/ml for 30min;e) ACEI+Epo group:Enalapril 1×10^-6M blockage for 2h then Epo 10U/ml for 30min;f) ARB+Epo groups:Telmisartan 1×10^-6M blockage for 30min then Epo 10U/ml for 30min.Total cellular protein was extracted and western blot detection was used to quantify the myocardial p-ERK1/2 from the groups described above.3) The cardiomyocytes were divided into 3 groups:a) Control group; b) Epo Group:Epo 10U/ml for 6h;c) PD98059+Epo group:PD98059 50×10^-6M blockage for 30min then Epo 10U/ml for 6h.Total cellular RNA was extracted and RT-PCR was introduced to quantify the AGT mRNA expression.Results:1) Cardiomyocytes AGT mRNA expression increased after 2 hours（2h,6h,12h,24h Vs control,P＜0.05） treatment of Epo 10U/ml with the highest expression level at 6hrs treatment.After that,its expression was inclined to decrease.2) The increase of p-ERK1/2 caused by Epo treatment could be reversed by Enalapril or Telmisartan treatment.3) p-ERK1/2 pathway blockage with PD98059 could not stop the increase of AGT mRNA expression on the treatment of Epo.Conclusion:1) Epo upregulates AGT gene expression in cultured cardiomyocytes.2) RAS may be involved in Epo-induced ERK activation.更多还原