【作者】 石永英；

【导师】 陈敏生；

【作者基本信息】 广州医学院 ， 心血管内科学， 2008， 博士

【摘要】 左室肥厚(Left Ventricular Hypertrophy,LVH)是高血压主要的靶器官损害之一,约有30%的高血压并发左室肥厚。众多研究表明LVH是发生心血管事件的独立危险因素。因此,逆转LVH是高血压治疗的主要目标之一。心肌细胞肥大是左室肥厚的主要病理变化,其发生、发展的分子机制正越来越受到重视。对心肌细胞肥大病理生理过程中细胞内信号转导机制的研究将有助于进一步探讨左室肥厚的发病机制,并为采取积极有效的药物治疗寻找新途径。转化生长因子β1(Transforming growth factor beta 1,TGF-β1)是一种调控细胞生长的重要细胞因子,参与免疫调节、创伤修复、胚胎发生、细胞增殖与凋亡、骨髓造血等调控。研究表明TGF-β1参与了高血压心肌肥厚、冠状动脉粥样硬化、心肌梗死后心力衰竭及心肌病等多种心血管疾病的发生、发展过程。近年研究发现TGF-β1由心肌成纤维细胞在血管紧张素II受体(Angiotensin II Receptor,ATR II)的刺激下,通过内分泌及旁分泌的方式产生,参与心肌细胞肥大、细胞外基质增生及心肌成纤维细胞肥大和增殖的病理生理过程,但其具体机制尚不明确。TGF/Smad (Drosophlia mothers against dpp and Celegans Sma,Smad)通路参与了心肌细胞肥大的过程,其具体作用复杂,且与其他信号通路交错表达,仍有待进一步研究。对TGF-β1在心肌细胞肥大中的作用特点、TGF-β1诱导心肌肥厚时调控核转录的下游信号分子及针对TGF-β1致肥大效应的干预手段等也研究较少。本研究以TGF-β1诱导的新生大鼠心肌细胞体外原代培养为实验模型,从观察药物刺激后心肌细胞的肥大和凋亡效应,并观察细胞内信号转导通路Smads的作用、癌基因p15及c-myc的表达、以及葛根素干预等的影响,旨在从多个角度探讨TGF-β1对心肌细胞肥大中的作用,为左室肥厚的发病机制和临床药物防治提供依据。实验内容主要包括以下三部分:第一部分新生SD大鼠心肌细胞的原代培养及鉴定目的建立新生SD大鼠心肌细胞体外原代培养模型。方法采用胰酶分步消化法、差速贴壁法及化学抑制法分离、纯化新生SD大鼠心肌细胞后作原代培养,采用倒置显微镜、透射电镜观察细胞。结果倒置显微镜下观察原代培养的心肌细胞呈不规则三角形、多边形,多数单个或成团细胞出现自发性搏动。培养的心肌细胞在3～7天内细胞形态和细胞搏动良好。透射电镜下观察心肌细胞线粒体丰富,肌丝排列整齐。结论采用胰酶消化法、差速贴壁法及化学抑制法建立体外原代培养的新生SD大鼠心肌细胞,经倒置显微镜和透射电镜观察证实所培养的细胞为心肌细胞。第二部分TGF-β1诱导的心肌细胞肥大和凋亡及其与Smads通路和癌基因相关蛋白的关系目的1.观察TGF-β1诱导的心肌细胞肥大和凋亡及其相互关系;2.探讨Samds通路在TGF-β1诱导心肌细胞肥大时的作用;3.了解TGF-β1诱导心肌细胞肥大时癌基因相关蛋白p15、c-myc的表达及其与Samds通路的相关性。方法应用不同浓度的TGF-β1(0.1μg/L、1μg/L、3μg/L)分别刺激15min、30min、1h、2h、24h后观察TGF-β1对心肌细胞的影响,单纯DMEM培养液组为空白对照组。TGF-β1诱导孵育24h,采用3H-亮氨酸掺入法检测心肌细胞蛋白合成速率,PI标记细胞内RNA含量,实时荧光定量PCR检测心肌细胞内ANF、α-MHC及β-MHC的mRNA表达,TdT-FragEL染色及FCM检测TGF-β1诱导的心肌细胞凋亡,透射电镜观察心肌细胞形态学变化。TGF-β1(3μg/L)作诱导浓度孵育心肌细胞15min、30min、1h、2h、24h,Western-blot检测Smad2、pSmad2、Smad2/3、p15、c-myc的蛋白表达。同时应用Smad2siRNA与TGF-β1(3μg/L)共孵育2小时和24h,特异性抑制Smads表达后观察心肌细胞蛋白合成速率、心脏胚胎型基因和Smad2的mRNA表达及Smad2、p15和c-myc的蛋白表达。结果1.TGF-β1对心肌细胞的影响1.1 3H-亮氨酸掺入法检测心肌细胞蛋白合成速率TGF-β1刺激心肌细胞24h后,心肌细胞蛋白合成速率均高于空白对照组,呈浓度依赖性(P<0.01)。抑制Smad2后心肌细胞合成速率较TGF-β1组明显下降(P<0.01)。1.2 PI标记的RNA含量检测TGF-β1组PI荧光强度明显高于对照组,其中TGF-β1(3μg/L)组荧光强度最高(92.89±3.12)(P<0.01),呈浓度依赖性(P<0.05)。1.3实时荧光定量PCR检测心肌细胞胚胎型心脏基因与对照组比较,TGF-β1组中ANFmRNA、β-MHCmRNA水平明显增高(1.61±0.14)、(2.28±0.13()P<0.01),呈剂量依赖性;α-MHCmRNA表达相对减少(0.69±0.05)(P<0.01)。1.4 TdT-FragEL染色心肌细胞凋亡对照组心肌细胞凋亡率为(1.3%±0.08%),TGF-β1组心肌细胞凋亡率明显增加,TGF-β1(3μg/L)组凋亡率最高达(9.4%±1.16%)(P<0.01)。1.5 FCM检测心肌细胞凋亡TGF-β1刺激可使心肌细胞凋亡率增加,Caspase-3表达增加,且呈剂量依赖性(P<0.01)。Capase-3表达与心肌细胞凋亡率呈正相关。1.6透视电镜观察心肌肥大和凋亡正常心肌细胞线粒体丰富,细胞核很小。TGF-β1(3μg/L)组心肌细胞线粒体肿胀增大,高尔基体丰富,心肌细胞核及核仁增大变形,异染质丰富,并且部分心肌细胞出现染色质边集或凋亡小体。对照组细胞形态正常,TGF-β1组观察到细胞肥大和凋亡形态同时出现。2. Smads通路表达2.1 Smad2mRNA水平检测心肌细胞中Smad2的mRNA水平在TGF-β1(3μg/L)刺激2h后显著上升,在Smad2基因沉默后较TGF-β1组明显下降(P<0.01)。2.2 Smads蛋白表达空白对照组心肌细胞pSmad2、Smad2/3微量表达,Smad2有少量表达;TGF-β1(3μg/L)刺激后pSmad2、Smad2/3在30min内即表达增加,至1～2h后达高峰,24h开始下降,蛋白表达量均较对照组显著增高(P<0.01)。Smad2在TGF-β1(3μg/L)刺激后有所增加(P<0.05),但与同时段内pSmad2相比,pSmad2增加明显(P<0.01)。抑制Smad2mRNA表达后,心肌细胞pSmad2蛋白水平较TGF-β1对照组明显下降(P<0.01)。心肌细胞蛋白合成速率与Smad2mRNA水平及pSmad2蛋白表达呈正相关(P<0.05)。3. p15及c-myc蛋白表达p15蛋白在对照组中表达极微(0.08±0.02),TGF-β1刺激后24h后p15蛋白表达增加(0.28±0.03)(P<0.01)。TGF-β1刺激后,c-myc表达也增高,作用2小时后表达最高(0.42±0.03)(P<0.01),24h后表达开始下降。与TGF-β1组相比,抑制Smad2表达后,p15蛋白降低(P<0.01),c-myc无明显改变(P>0.05)。心肌细胞蛋白合成速率与c-myc、p15蛋白表达呈正相关(P<0.05)。结论1. TGF-β1诱导大鼠心肌细胞肥大的同时也诱导心肌细胞凋亡增加。2. TGF-β1诱导心肌细胞肥大的过程中Smad2蛋白磷酸化显著增高,Smad2/3、Smad2表达也增加。3. TGF-β1诱导心肌细胞肥大时伴随原癌基因c-myc和抑癌基因p15的蛋白表达增加;p15是Smad2通路的下游信号分子。第三部分葛根素对TGF-β1诱导心肌细胞肥大的干预研究目的1.观察葛根素对TGF-β1诱导的心肌细胞肥大和凋亡的影响;2.观察葛根素、Smad2siRNA对Smad2、p15、c-myc表达的影响;探讨葛根素对心肌细胞肥大的作用机制。方法葛根素(0.g1/L、1g/L、5g/L)或Smad2siRNA分别与TGF-β1(3μg/L)共同诱导培养心肌细胞24h后,采用3H-亮氨酸掺入法检测心肌细胞蛋白合成速率,实时荧光定量PCR检测心肌细胞内ANF、α-MHC及β-MHC的mRNA水平, FCM检测心肌细胞凋亡率。Western-blot检测分别培养2h及24h后Smad2、pSmad2、Smad2/3、p15、c-myc的表达。结果1.葛根素可明显改善TGF-β1诱导的心肌细胞肥大,葛根素干预后心肌细胞3H-掺入量为(20463.10±3052.15),比TGF-β1组(48625.18±3325.47)明显下降(P<0.01)。Smad2siRNA也可有效干预心肌细胞肥大(28675.08±2168.34),两组比较葛根素组干预更为明显(P<0.01)。2.经葛根素干预后,心肌细胞ANFmRNA、β-MHCmRNA水平较TGF-β1对照组下降,并且葛根素组(1.41±0.15、2.36±0.14)较Smad2siRNA组下降更明显(1.83±0.12、3.85±0.23)(P<0.01)。3.葛根素与TGF-β1共孵育24h后,可明显降低TGF-β1诱导的心肌细胞凋亡率(12.54%±1.12%),其中葛根素(5g/L)+TGF-β1(3μg/L)组凋亡率最低(2.83%±0.40%),呈剂量依赖性(P<0.01)。葛根素干预后Caspase-3水平明显下降,呈剂量依赖性(P<0.01)。4.与TGF-β1对照组相比,葛根素的干预使Smad2mRNA、pSmad2蛋白表达均明显下降(P<0.01),p15蛋白表达下降(P<0.01),c-myc表达无明显改变(P>0.05)。Smad2siRNA干预后,Smad2mRNA、pSmad2蛋白表达较葛根素组下降更明显(P<0.01),但对c-myc、p15的作用与葛根素组相比无明显差别(P>0.05)。结论1.葛根素可有效改善TGF-β1诱导的心肌细胞肥大和细胞凋亡;葛根素可通过明显降低Smad2磷酸化而改善心肌细胞肥大。2.葛根素干预可下调TGF-β1诱导下心肌细胞中p15的表达,而c-myc表达则无明显改变;3. Smad2siRNA也可有效干预心肌细胞肥大,两者比较葛根素干预效果更为明显。更多还原

【Abstract】 Left ventricular hypertrophy (LVH)was one of the most important functional lesion of target organ(TO)about hypertension. 30% hypertension patients were complicated by LVH and 17% patients died of coronary heart disease(CHD)or heart failure (HF) coherented with hypertension. LVH was the independent risk factor for cardiovascular events, and for this reason retroconversion of LVH may became one of the end-all targets for hypertension therapy. The main pathological change of LVH was cardiomyocytes hypertrophy. Although the characters and mechanisms were thinked highly of people there was still lack of deeply learning about cardiomyocytes hypertrophy development. The study of the mechanisms about signal transduction intra-cellular may be helpful to investigate pathogenesy of cardiomyocytes hypertrophy to move forward a signle step.Transtorming growth factor beta 1(TGF-β1) was one of the significant cell factors who regulated growth with various functions. TGF-β1 has been predicted to contribute to the pathologies of LVH、CHD and HF after myocardial infarction(MI). Angiotensin II receptor (ATR II) was reported to stimulate paracrine and autocrine about TGF-β1 that a wide array of regulater for proliferation and hypertrophy of cardiac fibroblasts( CFs )、hyperplasy of extracellular matrix ( ECM ) and cardiomyocytes hypertrophy.The specific mechanisms of the regulation about TGF-β1 and TGF/Smad (Drosophlia mothers against dpp and Celegans Sma) for cardiomyocytes were still unknown. In our study cardiomyocytes hypertrophy were induced by TGF-β1 in rat to research mechanisms about cardiomyocytes hypertrophy though detecting cardiomyocytes hypertrophy and apoptosis, expressions of Smads, changes in p15 and c-myc. Intervention of puerarin was also observated to supply exprement data for drug prevention and cure. The experiments contained there parts as below.Part One: Primary culture and identify of cardiomyocytes in rat ObjectiveTo construct the cardiomyocytes primary culture model in rat.MethodsCardiomyocytes were isolated by trypsin digestion and differential attachment methods from neonatal Sprague Dawley (SD) rats, identified by inverted microscope and transmission electron microscope (TEM) observation.ResultsObserved with inverted microscope, primary cardiomyocytes appeared irregularity triangle and polygonm, and the most part of cardiomyocytes spontaneous beated rhythmically. Observed with TEM, cardiomyocytes were plentiful with mitochondria, and actin filaments were lined up in order.ConclusionIt was succeed to primary culture neonatal SD rat cardiomyocytes by trypsin digestion and differential attachment methods.Part Two: Cardiomyocytes hypertrophy and apoptosis induced by TGF-β1 to observe Smads pathway and oncogene protein expressionsObjectivesTo observe the hypertrophy and apoptosis of cardiomyocytes induced by TGF-β1 and determine the expression of Smads pathway in neonatal SD rat catdiomyocytes, and the expressions of oncogene protein p15 and c-myc were also detected. MethodsCardiomyocytes were stimulated by TGF-β1 with different concentrations (0.1μg/L、1μg/L、3μg/L) , and TGF-β1(3μg/L) stimulated cardiomyocytes for different periods of 15min、30min、1h、2h and 24h. Velocity of cardiomyocytes protein were observed by 3H-Leu incorporation, and Propidium iodide (PI)staining was used to assayed RNA contents. Apoptosis were assayed by flow cytometry (FCM) and FragEL staining. The hypertrophic response was assayed by measuring the expressions of ANF、α-MHC andβ-MHCmRNA detected by real-time PCR. Expressions of hypertrophy and apoptosis were detected by TEM. The Western-blot was performed to detected protein expressions of Smad2、pSmad2、Smad2/3、p15 and c-myc. RNAi was used to be a specific inhibitor to Smad2 as a control. Cardiomyocytes in Smad2siRNA control group were essayed by 3H-Leu incorporation、real-time PCR and western-blot.Results1. The effects of TGF-β1 on cardiomyocytes3H-Leu incorporation in cardiomyocytes Stimulated with TGF-β1 for 24h, to compare with the black control, the rate of protein synthesis in cardiomyocytes was elevated significantly in concentration-dependent manner (P﹤0.01). While in the Smad2siRNA control group the rate of protein synthesis was decreased than TGF-β1group(P﹤0.01).RNA contents detected by PI staining Fluorescence intensity of PI in TGF-β1 group was obviously higher than control group (P﹤0.01).Embryon gene detected by real-time PCR Expression Levels of ANFmRNA andβ-MHCmRNA were higher in TGF-β1 group and elevated significantly in concentration-dependent manner (P﹤0.01).Apoptosis detected by TdT-FragEL staining Rates of apoptosis in cardiomyocytes induced by TGF-β1were improved(P<0.01).FCM assaied apoptosis Changes of cardiomyocytes apoptosis induced by TGF-β1 was associated with upregulation of Caspase-3(P<0.01).TEM observed cardiomyocytes hypertrophy and apoptosis Cardiomyocytes in control group were plenty of cytomicrosome. Cardiomyocytes in TGF-β1 group showed hypertrophy with increase of actin filament and tumefaction of cytomicrosome, and a part of cardiomyocytes in TGF-β1 group also expressed apoptosis by concentrate of caryon and vacuoled of cytomicrosome. Hypertrophy and apoptosis apperanced in TGF-β1 group cardiomyocytes at the same time.2. TGF-β1 activates Smads signalSmad2 mRNA levels Smad2 mRNA level was higher than black control group in cardiomyocytes induced by TGF-β1 and the Smad2 mRNA level was also efficiently downregulated by RNAi to Smad2(P<0.01).Smads proteins TGF-β1 induced early activation of pSmad2 and Smad2/3 at 30 minutes and max expression at 2h, and TGF-β1 induced significant pSmad2 expressions until 24 hours(P<0.01). Expressions of Smad2 were not as obviously as pSmad2 ( P<0.05 ) . Smad2 phosphorylation was dowenregulated efficiently in Smad2siRNA control group(P<0.01)3. Oncogene p15 and c-myc TGF-β1 induced the early activation of c-myc and late activation of p15 in cardiomyocytes compared with control group (P<0.01). In Smad2siRNA control group p15 was inhibited alone with pSmad2(P<0.01).Conclusions1. TGF-β1 induced cardiomyocytes hypertrophy and apoptosis at the same time.2. TGF-β1 activatied early and late Smad2 activation and upregulation to induced cardiomyocytes hypertrophy.3. TGF-β1 also activated ealy upregulation of c-myc and late upregulation of p15. C-myc and p15 expressions maybe assiocatied with cardiomyocytes hypertrophy. p15 could be the downstream signaling molecule of Smads pathway.Part Three: Study of puerarin intervetion on hypertrophic cardiomyocytes induced by TGF-β1ObjectiveTo investigate the effects of puerain intervention on hypertrophic cardiomyocytes induced by TGF-β1 and obveried the inhibition of puerarin to phosphorylation of Smad2 that was compared with Smad2siRNA intervention. MethodsPuerarin(0.1g/L、1g/L、5g/L) with TGF-β1(3μg/L ) co-stimulated or Smad2siRNA with TGF-β1(3μg/L) co-stimulated for 2h and 24h for assessing protein synthesis rate, ANF,β-MHC andα-MHCmRNA expression, pSmad2, Smad2, p15 and c-myc proteins changes in cardiomyocyte.The methods of 3H-Leu incorporation, real time PCR, FCM and Western-blot were used.Results1. Hypertrophy induced by TGF-β1 were obviousily changed by intervention of puerain and Smad2siRNA. Compared with TGF-β1 group, the protein synthesis rate in cardiomyocytes was downregulated significantly in puerain group than in siRNA group (P<0.01).2. It was shown that levels of ANFmRNA andβ-MHCmRNA were downregulated by puerain and Smad2siRNA especialiy by puerain (P<0.01).3. Puerarin(1g/L) with TGF-β1(3μg/L ) co-stimulated cardiomyocytes were able to decrease apoptosis rate compared with TGF-β1(3μg/L) group (P<0.01). Level of Caspase-3 was also downregulated (P<0.01).4. PSmad2 protein was significantly downregulated by puerarin. p15 protein was inhibited by puerarin intervention. And Smad2siRNA intervention could also inhibited pSmad2 and p15 protein expression(P<0.01).Conclusions1. Puerarin was able to amendment cardiomyocytes hypertrophy and apoptosis induced by TGF-β1 in rat.2. Hypertrophy of cardiomyocytes was depressed by puerain with marked downregulation of Smad2 phosphorylation. Intervention of puerarin could drcreased p15 protein expressions in cardiomyocytes.3. Compared with Smad2siRNA intervention, puerarin intervention had preferable effect.更多还原