【作者】 肖健；

【导师】 王志农；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 硕士

【摘要】 TGF-β超家族由大量结构相关的多肽生长因子组成,包括TGF-β、激活素和骨形态发生蛋白（BMP）3大类,TGF-β作为TGF-β超家族成员之一,是一种多功能的生长因子,由于其作用细胞的类型、分化程度、外环境及是否有其他生长因子作用的不同,因而它的活性具有多样性。因为它的多功能,使它在胚胎发生发展、肿瘤的形成、免疫反应、炎症反应、创伤愈合中具有重要作用。随着TGF-β的下游分子Smads蛋白研究的深入,TGF-β超家族的作用也越来越受到重视,在人类组织细胞中仅存在TGF-β1、TGF-β2、TGF-β3等三种,其中研究较多的是TGF-β₁。资料表明Smads在心脏发育、细胞增殖、细胞生长和凋亡中起重要作用,基于Smads的不同亚型以及在特定情形下Smads与其它转录因子之间的相互作用和不同激酶对Smads的活性调节,Smads激活后具有不同作用。已有研究表明骨形态发生蛋白（BMP）通过Smad1的信号转导机制能够减轻心肌细胞缺血再灌注损伤。但TGF-β₁及其下游信号分子Smad3在心肌细胞缺血再灌注损伤及缺血预适应中的信号转导研究较少。缺血再灌注损伤机制及心肌保护的研究是当今倍受重视的课题。为了干预心肌缺血梗死及保护心肌,文献报道研究了许多药物如β-受体阻滞剂、自由基清除剂、钙拮抗剂等的心肌保护作用,其中对缺血预适应（ischemic preconditioning,IPC）保护心肌的研究一直是一个热点。缺血预适应的心肌保护作用表现为缩小梗死面积、促进心肌功能恢复、保护心肌超微结构,减少再灌注后心律失常等,但缺血预适应心肌保护作用的机理迄今仍未完全明了。目前对心肌细胞缺血预适应减轻心肌缺血再灌注损伤作用的机制主要集中在线粒体ATP敏感性钾离子通道、蛋白激酶C、NF-κB等分子信号转导机制。既然TGF-β₁/Smad3可能与心肌细胞凋亡有关,而缺血预适应已经被证明能够减少心肌细胞凋亡率,我们推测缺血预适应的机制可能与TGF-β/Smad3信号转导通路有关。本实验以体外培养乳鼠心肌细胞为实验研究和观察对象,建立缺血再灌注损伤的细胞模型,并以TGF-β₁及其阻断剂进行干预。采用细胞TUNEL染色、DAPI荧光染色、LDH漏出量测定及流式细胞仪检测,多角度、多时程检测心肌细胞凋亡比率并通过ELISA方法检测细胞Smad3蛋白表达,从细胞和分子水平探讨TGF-β₁/Smad3对缺血再灌注损伤中心肌细胞凋亡的作用以及缺血预适应对TGF-β₁/Smad3信号转导通路的调控。进一步探讨缺血再灌注损伤中心肌保护机制,为临床围手术期的心肌保护提供新的理论基础和可能的治疗方法。本实验主要分为两个部分:一、乳鼠心肌细胞的分离培养及缺血再灌注损伤和缺血预适应模型的制备（一）乳鼠心肌细胞分离培养及缺血再灌注和缺血预适应模型的建立1、乳鼠心肌细胞分离培养、鉴定及活力测定按改良的Simpson等的方法进行消化分离,采用差速贴壁分离纯化方法培养心肌细胞,培养4天后,细胞呈同步搏动。利用α-肌动蛋白单克隆抗体免疫组化鉴定,心肌细胞的纯度超过90%,台盼蓝染色计算细胞活力超过94%。2、缺血再灌注和缺血预适应模型的建立细胞培养成功后,对心肌细胞以缺氧缺糖模拟缺血,恢复氧和糖的供应模拟再灌注,利用细胞培养液的变化（缺氧、缺糖、乳酸堆积、酸中毒,再灌注后恢复正常）模拟缺血再灌注损伤,同时给予TGF-β₁（5ng/ml）进行干预。利用短暂的缺氧、复氧培养模拟缺血预适应。3、缺血再灌注和缺血预适应模型有效性验证有效性验证主要研究缺血再灌注和缺血预适应模型心肌细胞凋亡率,实验分为3组,正常对照组心肌细胞凋亡率为5.92±1.88%,缺血再灌注组凋亡率为28.99±6.96%,缺血预处理组凋亡率为13.85±1.40%;三者之间存在显著差异（P＜0.01）。说明采用本实验方法建立的缺血再灌注及缺血预适应模型是有效可行的。二、TGF-β₁在缺血再灌注损伤和缺血预适应信号转导中的作用及机制实验分组:培养4天的乳鼠心肌细胞随机分为6组1、正常对照组（A组）:含10%胎牛血清DMEM培养基（37℃,5%CO2、95%空气培养箱）培养48小时,更换培养基后同样条件下继续培养3小时:2、缺血再灌注诱导凋亡模型组（B组）:不含血清的低糖DMEM培养基,厌氧培养箱（N₂ 95%,CO₂ 5%）中培养48小时后,10%胎牛血清DMEM培养基,37℃、5%CO₂、95%空气培养箱中培养3小时;3、TGF-β₁干预组（C组）:不含血清的低糖DMEM培养基加入TGF-β₁（5ng/ml）,厌氧培养箱（N₂ 95%,CO₂ 5%）中培养48小时后,10%胎牛血清、TGF-β₁.（5ng/ml）DMEM培养基,37℃、5%CO₂、95%空气培养箱中培养3小时;4、TGF-β₁受体抑制剂干预组（D组）:培养基中加入LY364947（59nmol/L）,余同B组;5、缺血预适应组（E组）:不含血清的低糖DMEM培养基,厌氧培养箱（N₂95%,CO₂ 5%）中培养6小时后,10%胎牛血清DMEM培养基,37℃、5%CO₂、95%空气培养箱中培养3小时;再模拟进行缺血再灌注,同B组;6、TGF-β₁+缺血预适应组（F组）:不含血清的低糖DMEM培养基加入TGF-β₁（5ng/ml）,厌氧培养箱（N₂ 95%,CO₂ 5%）中培养6小时后,10%胎牛血清、TGF-β₁（5ng/ml）DMEM培养基,37℃、5%CO₂、95%空气培养箱中培养3小时;再模拟进行缺血再灌注,同C组。（一）针对细胞凋亡不同时程的生化特征应用多种方法检测心肌细胞凋亡。心肌细胞凋亡是心肌缺血再灌注损伤的重要表现,并在后期的心功能损伤中起主要作用1、TUNEL法原位检测细胞凋亡使用TUNEL试剂盒检测凋亡心肌细胞。结果显示:C组见较多阳性染色细胞（54.60±8.49%）,明显高于其他各组（P＜0.01）,说明TGF-β₁加重缺血再灌注损伤后心肌细胞损害;D组心肌细胞凋亡率（20.48±1.94%）低于B组（31.10±4.21%）凋亡率（P＜0.01）,说明TGF-β₁受体抑制剂可以有效减少缺血再灌注损伤后心肌凋亡;E组心肌细胞凋亡率（12.21±0.92%）与F组（14.16±1.66%）无明显差异（P＞0.05）,说明缺血预适应可以有效抑制TGF-β₁对心肌细胞的损害;A组心肌细胞阳性率（5.24±0.99%）明显较其他各组低（P＜0.01）。2、AnnexinV-FITC/PI双标记检测细胞凋亡以AnnexinV和PI标记心肌细胞,以流式细胞仪进行凋亡及坏死细胞定量检测,灵敏度高。检测结果与TUNEL结果相似:C组检出较多凋亡细胞（54.19±11.86%）,明显高于其他各组（P＜0.01）;D组（21.31±3.78%）心肌细胞凋亡率与B组（30.75±4.88%）有显著差异（P＜0.01）,说明TGF-β₁受体抑制剂可以有效减少缺血再灌注损伤后心肌凋亡;E组心肌细胞凋亡率（12.45±1.52%）与F组（14.10±1.78%）明显低于B组心肌细胞凋亡率（P＜0.01）,但二者之间无明显差异（P＞0.05）,说明缺血预适应可以抑制TGF-β₁诱导的缺血再灌注损伤后心肌细胞凋亡,A组心肌细胞阳性率（5.50±1.38%）明显低于其他各组（P＜0.01）。3、乳酸脱氢酶漏出量测定留取各组细胞处理后培养上清,4℃保存待测。HITACH全自动生化分析仪检测。乳酸脱氢酶漏出量可以间接反映心肌细胞凋亡情况,结果与以上两种方法检测结果相似:C组中LDH漏出量（121.94±12.98U/L）明显高于其他组（P＜0.01）;D组（39.73±3.23 U/L）低于B组（52.98±4.94 U/L）的LDH漏出量（P＜0.01）;E组（14.80±2.49 U/L）与F组（15.70±1.29%）无明显差异（P＞0.05）;A组LDH漏出量（6.59±1.45 U/L）阳性率明显低（P＜0.01）。（二）心肌细胞缺血再灌注损伤与Smad3表达量的关系收集各组处理后细胞上清液,4℃保存。机械法收集细胞加入已收集的细胞上清液,加入等体积裂解液,超声裂解细胞,ELISA方法进行检测。正常心肌细胞（A组）Smad3表达量为21.77±2.32ng/ml,C组Smad3表达量（47.51±4.60ng/ml）较其他各组明显高（P＜0.01）,说明TGF-β₁可以诱导缺血再灌注损伤中Smad3表达增加;D组（25.66±1.51ng/ml）与B组（35.7±2.43ng/ml）相比Smad3表达量明显低,有显著差异（P＜0.01）,但与A组之间无明显差异（P＞0.05）,说明TGF-β₁受体抑制剂可以有效抑制Smad3表达;E组（29.47±1.36 ng/ml）与D组、F组（34.12±1.90 ng/ml）无明显差异（P＞0.05）。说明缺血预适应与TGF-β₁受体抑制剂作用相似,同样可以减少心肌细胞缺血再灌注损伤中Smad3表达。主要结论:本研究应用体外培养乳鼠心肌细胞,建立缺血再灌注损伤的细胞模型,以TGF-β₁进行干预。针对细胞凋亡的各种特征和时程特点,应用多种方法检测心肌细胞凋亡以及缺血再灌注损伤后心肌细胞Smad3表达的变化,以阐明TGF-β₁/Smad3对心肌细胞缺血再灌注损伤的作用,并进一步评价IPC对TGF-β₁/Smad3信号转导通路的影响,以探讨TGF-β₁在缺血预适应心肌保护中的作用。主要结论如下:1、按改良的Simpson等的方法进行消化分离,采用差速贴壁分离纯化方法培养的心肌细胞纯度和活力可达到满意效果。2、TGF-β₁可以促进Smad3表达增加,同时加重缺血再灌注损伤后心肌细胞损害,即Smad3可以促进缺血再灌注损伤后心肌细胞凋亡。3、缺血预适应可以抑制TGF-β₁/Smad3的信号转导,从而减少缺血再灌注损伤引起的心肌细胞凋亡。更多还原

【Abstract】 Members of the TGF-βsuperfamily,comprising the TGF-β,activin and BMP family, are the classical activators of Smads proteins.They participate in a wide range of processes, from tissue differentiation during development through to regulation of mesenchymal and immune cell functions.In recent years,elevated expression of proteins of the transcription factor family Smads was found under several pathophysiological situations in the heart,i.e. after myocardial infarction or in diverse forms of cardiomyopathy.Smads proteins are described to have different effects on heart development,cell proliferation,cell growth, and apoptosis.These different consequences of Smads activation are dependent on different Smad isoforms,interaction of Smads with other transcription factors in the particular situation,and modulation of Smads activity by various kinases.Some studies showed that BMP/Smad1 Protects Cardiomyocytes from Ischemia-Reperfusion Injury.But the study on TGF-β/Smads in the process of Ischemic preconditioning and Ischemia-Reperfusion Injury was very few.Studies on mechanisms of ischemia-reperfusion injury and myocardial protection are the spotlight in present day.In order to fight for cardiomyocytes ischemic necrosis and protect myocardium,lots of medicine was studied for myocardial protection（for example,β-antagonist,free-radical scavenger,calcium antagonist）.The studies on cardioprotection of ischemic preconditioning were always the most fascinating.Ischemic preconditioning may lead to a reduction in infarct size,enhance the heart function,protect the ultrastructure of myocardium and reduce the arrhythmia after Ischemia-Reperfusion Injury.But the mechanisms of these protective effects remain unclear.During recent years,the mechanisms of ischemic preconditioning have focused on mito-KATP channels,PKC and NF-κB,ect.In our experiments cultured neonatal rat cardiomyocytes were performed to prepared ischemia-reperfusion injury and ischemic preconditioning model in vitro,and were interfered with TGF-β₁.TUNEL,LDH detection,fluorescence staining of DAPI and flow cytometry were performed to identify the apoptosis of cardiaomyocytes during ischemia-reperfusion injury,and ELISA was performed to detect the Smad3.The main methods and results are described as follows: 1.Neonatal rat cardiomyocytes culture and ischcmia-reperfusion and ischemic preconditioning model preparation1.1 Neonatal rat cardiomyocytes cultureNeonatal rat cardiomyocytes were digested according the Simpson’s method and cultured by a selective attached technique.The purity of cardiomyocytes was over 90%. The synchronous pulsation was seen after 4 days.1.2 ischemia-reperfusion and ischemic preconditioning model preparationCardiomyocytes subjected to hypoxic substrate-free solution containing elevated potassium,acidosis,lactate accumulation and reinstated with normal culture solutions to mimic Ischemia-Reperfusion process.At the same time,TGF-β₁ was added in the culture solution.1.3 To identify the efficacy of the ischemia-reperfusion and ischemic preconditioning modelThe method to identify the efficacy of the ischemia-reperfusion and ischemic preconditioning model is to study the ratio of apoptosis.The ratio of Control group is 5.92±1.88%,the ratio of IR group is 28.99±6.96%,the ratio of IPC group is 13.85±1.40%, and there is significient deference among these groups.2.The signal transduction of TGF-β₁/Smad3 during ischemia-reperfusion injury and ischemic preconditioning.The myocardiocytes of neonatal SD rats were randomized into 6 groups:Group A（Control group）:The cells were cultured with modified DMEM with 10% FBS in a modified chamber and mixture gas（95%air.5%CO₂） for 48h,then change the culture solution and continue culturing for 3h;Group B（Ischemia-Reperfusion Injury group）:The cells were cultured with low carbohydrates DMEM under the condition of hypoxia（95%N₂ and 5%CO₂;the O₂ partial pressure was lower than 5 mmHg） for 48h,then cultured with modified DMEM with 10% FBS in a modified chamber and mixture gas（95%air,5%CO₂） for 3h;Group C（Ischemia-Reperfusion Injury and TGF-β₁ group）:TGF-β₁（5ng/ml） was put in DMEM,the others are the same to B group;Group D（Ischemia-Reperfusion Injury and Smad3 inhibitor group）:LY364947 （60nmol/L） were put in DMEM,the others are the same to B group; Group E（Ischemic Preconditioning group）:The cells were cultured with low carbohydrates DMEM under the condition of hypoxia（95%N₂ and 5%CO₂;the O₂ partial pressure was lower than 5 mmHg） for 6h,then cultured with modified DMEM with 10% FBS in a modified chamber and mixture gas（95%air,5%CO₂） for 3h,then the same to B group;Group F（ischemic preconditioning and TGF-β₁ group）:TGF-β₁（5ng/ml） was put in DMEM,the others are the same to E group.2.1 Detection of cardiac myocyte apoptosis2.1.1 In situ terminal deoxynucleotidy transferase（TdT） labeling（TUNEL）TUNEL positive apoptotic cells could be detected in all groups,and the radio of positive cells in group C（54.60±8.49%） was significantly higher compared to the other groups,group D（20.48±1.94%） is less than group B（31.10±4.21%）;group E（12.21±0.92%） and group F（14.16±1.66%） have no significant difference;group A（5.24±0.99%） is least.2.1.2 Cardiac myocyte apoptosis index were determined by flow cytometry with Annexin-V and propidinm iodiode（PI） stainingThe radio of positive cells in group C（54.19±11.86%） was significantly higher compared to the other groups,group D（21.31±3.78%） is less than group B（30.75±4.88%）, group E（12.45±1.52%） and group F（14.10±1.78%） have no significant difference; group A（5.50±1.38%） is least.2.1.3 LDH detectionKeep the culture solution in refrigerator of 4℃for detection,and the LDH will identify the ratios of cardiomyocyte apoptosis.Group C（121.94±12.98U/L） was significantly higher compared to the other groups,group D（39.73±3.23 U/L） is less than group B（52.98±4.94 U/L）;group E（14.80±2.49 U/L） and group F（15.70±1.29%） have no significant difference;group A（6.59±1.45 U/L） is least.2.2 detection of the Smad3 that induced by TGF-β₁ during Ischemia-Reperfusion Injury and Ischemic PreconditioningKeep the culture solution in refrigerator of 4℃and collect the cell by mechanical method.Add the cell to the collected culture solution and split the cell with ultrasound and detect the Smad3 with ELISA method.Group A:21.77±2.32 ng/ml,Group B:35.7±2.43 ng/ml,Group C:47.51±4.60 ng/ml,Group D:25.66±1.51ng/ml,Group E:29.47±1.36 ng/ml,Group F:34.12±1.90 ng/ml.There are singnal deference between group C and the other groups,but there is no significant deference between group B and group D.There is no significant deference between group E and group F.There is no significant deference between group E and group D.Conclusion:In our experiments cultured neonatal rat cardiomyocytes were performed to prepared ischemia-reperfusion injury and ischemic preconditioning model in vitro,and were interfered with TGF-β₁.The cardiomyocytes apoptosis and Smad3 were detected with several methods,by which we can study the signal transduction of TGF-β₁/Smad3 during ischemic preconditioning.1.The purity of cardiomyocytes that were digested according the Simpson’s method and cultured by a selective attached technique is over 90%.2.TGF-β₁ may induce the Smad3 and increase the cardiomyocytes apoptosis during Ischemia-Reperfusion injury.3.Ischemic Preconditioning may prevent the cardiomyocytes apoptosis during Ischemia-Reperfusion injury by inhibit the Smad3.更多还原