TGF-β_1/Smad3在心肌细胞缺血预适应的信号转导中的作用的实验研究
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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-β_1。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-β_1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-β_1/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-β_1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-β_1/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-β_1/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-β_1(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-β_1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(N_2 95%,CO_2 5%)中培养48小时后,10%胎牛血清DMEM培养基,37℃、5%CO_2、95%空气培养箱中培养3小时;
3、TGF-β_1干预组(C组):不含血清的低糖DMEM培养基加入TGF-β_1(5ng/ml),厌氧培养箱(N_2 95%,CO_2 5%)中培养48小时后,10%胎牛血清、TGF-β_1.(5ng/ml)DMEM培养基,37℃、5%CO_2、95%空气培养箱中培养3小时;
4、TGF-β_1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(N_295%,CO_2 5%)中培养6小时后,10%胎牛血清DMEM培养基,37℃、5%CO_2、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-β_1+缺血预适应组(F组):不含血清的低糖DMEM培养基加入TGF-β_1(5ng/ml),厌氧培养箱(N_2 95%,CO_2 5%)中培养6小时后,10%胎牛血清、TGF-β_1(5ng/ml)DMEM培养基,37℃、5%CO_2、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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1/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-β_1/Smad3信号转导通路的影响,以探讨TGF-β_1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-β_1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-β_1/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。
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-β_1.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 preparation
1.1 Neonatal rat cardiomyocytes culture
Neonatal 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 preparation
Cardiomyocytes 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-β_1 was added in the culture solution.
1.3 To identify the efficacy of the ischemia-reperfusion and ischemic preconditioning model
The 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-β_1/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%CO_2) 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%N_2 and 5%CO_2;the O_2 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%CO_2) for 3h;
Group C(Ischemia-Reperfusion Injury and TGF-β_1 group):TGF-β_1(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%N_2 and 5%CO_2;the O_2 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%CO_2) for 3h,then the same to B group;
Group F(ischemic preconditioning and TGF-β_1 group):TGF-β_1(5ng/ml) was put in DMEM,the others are the same to E group.
2.1 Detection of cardiac myocyte apoptosis
2.1.1 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) staining
The 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 detection
Keep 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-β_1 during Ischemia-Reperfusion Injury and Ischemic Preconditioning
Keep 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-β_1.The cardiomyocytes apoptosis and Smad3 were detected with several methods,by which we can study the signal transduction of TGF-β_1/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-β_1 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.
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-β_1/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-β_1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-β_1/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-β_1/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-β_1(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-β_1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(N_2 95%,CO_2 5%)中培养48小时后,10%胎牛血清DMEM培养基,37℃、5%CO_2、95%空气培养箱中培养3小时;
3、TGF-β_1干预组(C组):不含血清的低糖DMEM培养基加入TGF-β_1(5ng/ml),厌氧培养箱(N_2 95%,CO_2 5%)中培养48小时后,10%胎牛血清、TGF-β_1.(5ng/ml)DMEM培养基,37℃、5%CO_2、95%空气培养箱中培养3小时;
4、TGF-β_1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(N_295%,CO_2 5%)中培养6小时后,10%胎牛血清DMEM培养基,37℃、5%CO_2、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-β_1+缺血预适应组(F组):不含血清的低糖DMEM培养基加入TGF-β_1(5ng/ml),厌氧培养箱(N_2 95%,CO_2 5%)中培养6小时后,10%胎牛血清、TGF-β_1(5ng/ml)DMEM培养基,37℃、5%CO_2、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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1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-β_1/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-β_1/Smad3信号转导通路的影响,以探讨TGF-β_1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-β_1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-β_1/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。
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-β_1.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 preparation
1.1 Neonatal rat cardiomyocytes culture
Neonatal 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 preparation
Cardiomyocytes 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-β_1 was added in the culture solution.
1.3 To identify the efficacy of the ischemia-reperfusion and ischemic preconditioning model
The 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-β_1/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%CO_2) 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%N_2 and 5%CO_2;the O_2 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%CO_2) for 3h;
Group C(Ischemia-Reperfusion Injury and TGF-β_1 group):TGF-β_1(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%N_2 and 5%CO_2;the O_2 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%CO_2) for 3h,then the same to B group;
Group F(ischemic preconditioning and TGF-β_1 group):TGF-β_1(5ng/ml) was put in DMEM,the others are the same to E group.
2.1 Detection of cardiac myocyte apoptosis
2.1.1 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) staining
The 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 detection
Keep 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-β_1 during Ischemia-Reperfusion Injury and Ischemic Preconditioning
Keep 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-β_1.The cardiomyocytes apoptosis and Smad3 were detected with several methods,by which we can study the signal transduction of TGF-β_1/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-β_1 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.
引文
[1]鄂征,主编.组织培养和分子细胞学技术。北京出版社,1995.109-112。
[2]冯伯森,王秋雨,胡玉兰。动物细胞工程原理与实践。科学出版社,2000,152-153。
[3]郝亚荣,李庚山 李建军。乳鼠心肌细胞培养。岭南心血管病杂志。2001,7(2):137-139。
[4]沈静 谢苗荣 徐雍等。乳鼠心肌细胞培养及纯化方法的改良。中国医药导刊 2001,3(3):225-226。
[5]彭黎明,王曾礼主编.细胞凋亡的基础与临床.人民卫生出版社,2000年,167.
[6]吴立玲,主编.心血管病理生理学,北京医科大学出版社,2000年,215。
[7]Hemal H.Patel,Brian P.Head,1 Heidi N.Petersen,1 Ingrid R.Niesman,3 Diane Huang,Garrett J.Gross,5 Paul A.Insel,1 and David M.Roth2 Protection of adult rat cardiac myocytes from ischemic cell death:role of caveolar microdomains and δ-opioid receptors Am J Physiol Heart Circ Physiol 291:H344-H350,2006.
[8]奚群英,祝宝华,刘晨。乳鼠心肌细胞缺血预适应模型的建立及电镜下超微结构的改变。实用临床医药杂志 2005,3(9):18-24。
[9]程先进 曾志勇 杨胜生等。缺血预适应对缺血-再灌注心肌细胞的保护作用。临床麻醉学杂志 2007,23(5)138-140。
[10]Wang HC,Zhang HF,Guo WY,Su H,Zhang KR,Li QX,Yan W,Ma XL,Lopez BL,Christopher TA,Gao F.Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation:role of peroxynitrite formation.Apoptosis.2006 Aug;11(8):1453-60.
[11]李迎斋,张万义,陈学军等。缺血预适应对心肌细胞凋亡及血浆TNF水平影响的初步观察。中国微循环杂志 2005,9(4)264-266。
[12]Haunstetter A,Izumo S.Toward Antiapoptosis as a new treatment modality.Circ Res,2000,86:371-376
[13]He-Ying Sun,Ning-Ping Wang,Faraz Kerendi,Michael Halkos,Hajime Kin,Robert A.Guyton,Jakob Vinten-Johansen,and Zhi-Qing Zhao Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+overload Am J Physiol Heart Circ Physiol 288:H1900-H1908,2005.
[14]Maclellan WR,Schneider MD.Death by design.Programmed cell death in cardiovascular biology and disease.Circ Res,1997;81:137-144.
[15]Piot CA,Padmanban D,Ursell PC,Sievers RE,Wolfe CL.Ischemic predonditioning decreases apoptosis in rat hearts in vivo.Circulation,1997;2;96(5):1598-604.
[16]Maulik N,Yoshida T,Engelman RM,Deaton D,Flack JE 3rd,Rousu JA,Das DK.Ischemic preconditioning attenuates apoptotic cell death associated with ischemia / repeRPusion.Mol Cell Biochem,1998;186(1-2):139-45.
[17]Elizabeth N,Morgan MD,Edward M,et al.An essential role for NF-K B in the cardioadaptive response to ischemia.Ann Thorac Surg;1999,68:377-382.
[18]Simpson P,Savion S.Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cell.Circ Res.1982,50:101-116.
[19]Zhao ZQ,Nakamura M,Wang NP,et al.Reperfusion induces myocardial apoptotic cell death.Cardiovasc Res,2000;45(3):651-660.
[20]Liu H,Zhang HY,Zhu X,Shao Z,Yao Z,Preconditioning blocks cardiocyte apoptosis:role of K(ATP) channels and PKC-epsilon.Am J Physiol Heart Circ Physiol,2002;282(4):1380-1386.
[21]Korge P,Honda HM,Weiss JN.Protection of cardiac mitochondria by diazoxide and protein kinase C:implications for ischemic preconditioning.Proc Natl Acad Sci U S A,2002;99(5):3312-331
[22]Nozawa Y,Miura T,Miki T,Ohnuma Y,Yano T,Shimamoto K,Mitochondrial K(ATP)infarction in the rat.Basic Res Cardiol.2003;98(1):50-58.
[23]Rohr S,Scholly DM,Kléber AG.Patterned grouth of neonatal rat heart cells in culture:morphological and dectrophysiological characterization.Circ Res,1991,68:114-130.
[24]张旭晨。心血管疾病与细胞凋亡。国外医学·生理·病理科学与临床分册。1998,18:335-337。
[25]Jing Liu,Sharon Tsang and Tak Ming Wong Testosterone Is Required for Delayed Cardioprotection and Enhanced Heat Shock Protein 70 Expression Induced by Preconditioning Endocrinology Vol.147,No.10 4569-4577.
[26]Saraste A,Pulkki K,Kallajoki M,et al.Apoptosis in human acute myocardial infarction.Circulation,1997,95(2):320-323.
[27]Terry L.Vanden Hoek,Lance B.Becker,Zuo-Hui Shao,Chang-Qing Li,Paul T.Schumacker Preconditioning in Cardiomyocytes Protects by Attenuating Oxidant Stress at Reperfusion Circ.Res.2000;86;541-548.
[28]HuiPing Liu,Bradley C.Mcpherson,and ZhenHai Yao.Preconditioning attenuates apoptosis and necrosis:role of protein kinase Ce and -d isoforms.Am J Physiol Heart Circ Physiol 281:H404-H410,2001.
[29]Michael S.Marber Ischemic Preconditioning in Isolated Cells Circ.Res.2000;86;926-931.
[30]HuiPing Liu,Bradley C.Mcpherson,and ZhenHai Yao.Preconditioning blocks cardiocyte apoptosis:role of KATP channels and PKC-ε . Am J Physiol Heart Circ Physiol 282: H1380-H1386,2002;
[31] Ferran Padilla, David Garcia-Dorado, Antonio Rodri'guez-Sinovas,Marisol Ruiz-Meana, Javier Inserte, and Jordi Soler-Soler Protection afforded by ischemic preconditioning is not mediated by effects on cell-to-cell electrical coupling during myocardial ischemia-reperfusion Am J Physiol Heart Circ Physiol 285: H1909-H1916, 2003.
[32] T. Ravingerova, D. Pancza, A. Ziegelhoffer, J. STYK. Preconditioning Modulates Susceptibility to Ischemia-Induced Arrhythmias in the Rat Heart: The Role of α -Adrenergic Stimulation and K(ATP) Channels Physiol. Res. 51: 109-119, 2002
[33] Alexander E. Kabakov, Karina R. Budagova,David S. Latchman,R and Harm H. Kampinga. Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion. Am J Physiol Cell Physiol 283: C521-C534, 2002.
[34] Taro Date,Seibu Mochizuki,Adam J. Belanger,Midori Yamakawa,Zhengyu Luo, Karen A. Vincent, Seng H. Cheng, Richard J. Gregory, and Canwen Jiang. Expression of constitutively stable hybrid hypoxia-inducible factor-1α protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury Am J Physiol Cell Physiol 288: C314-C320,2005.
[1]杨明雄,袁爱国。心肌细胞凋亡的研究进展及意义。天津体育学院学报 2006,2(4):323-328。
[2]苏卫东 李会强 姚智。缺氧导致体外培养乳鼠心肌细胞凋亡的研究。放射免疫学杂志。2005,18(2):136-138。
[3]Gentzon Hall,Jeffery D.Hasday,Terry B.Rogers.Regulating the regulator:NF-κ B signaling in heart.Journal of Molecular and Cellular Cardiology 41(2006) 580 - 591.
[4]Frank Eeftinga,Benno Rensinga,Jochem Wigmana,Willem Jan Pannekoeka,Wai Ming Liua,Maarten Jan Cramera,Daniel J.Lipsa,Pieter A.Doevendans.Role of apoptosis in reperfusion injury.Cardiovascular Research 61(2004) 414-426.
[5]Asa B.Gustafsson and Roberta A.Gottlieb.Bcl-2 family members and apoptosis,taken to heart.Am J Physiol Cell Physiol 292:45-51,2007.
[6]Nakamura M,Wang NP,Zhao ZQ,et al.Preconditioning decreases Bax expression,PMN accumulation and apoptosis in reperfused rat heart.Cardiovasc Res,2000;45:661-670.
[7]郭永红,罗金燕。TGF-β超家族与Smad信号转导研究进展。医学综述 2005,11(8):685-687
[8]谷丽君。TGF-β超家族信号通路中的Smads蛋白。国外医学·生理、病理科学与临床分册。2002,22(6):580-583.
[9]Araujo-Jorge TC,Waghabi MC,Hasslocher-Moreno AM,Xavier SS,Higuchi Mde L,Keramidas M,et al.hnplication of transforming growth factor-betal in Chagas disease myocardiopathy.J Infect Dis 2002;186:1823 - 8.
[10]Hao J,Wang B,Jones SC,Jassal DS,Dixon IM.Interaction between angiotensin Ⅱ and Smad proteins in fibroblasts in failing heart and in vitro.Am J Physiol Heart Circ Physiol 2000;279:H3020-30.
[11]Dixon IM,Hao J,Reid NL,Roth JC.Effect of chronic AT receptor blockade on cardiac Smad overexpression in hereditary cardiomyopathic hamsters.Cardiovasc Res 2000;46:286-97.
[12]Schneiders D,Heger J,Best P,Piper HM,Taimor G.Smad proteins are involved in apoptosis induction in ventricular cardiomyocytes.Cardiovasc Res 2005;67:87-96.
[13]Francis JM,Heyworth CM,Spooncer E,Pierce A,Dexter TM,Whetton AD.Transforming growth factor-β 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.J Biol Chem 2000;75:39137-45.
[14]Chipuk JE,Bhat M,Hsing AY,Ma J,Danielpour D.Bcl-xL blocks transforming growth factor-b1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing apaf-1 dependent caspase activation in prostate epithelial cells.J Biol Chem 2001;276:26614-21.
[15]Alexandre D.T.Costal,Sandrine V.Pierre2,Michael V.Cohen3,4,James M.Downey3,and Keith D.Garlid1.cGMP signalling in pre- and post-conditioning:the role of mitochondria.Cardiovascular Research(2008) 77,344 - 352.
[16]Brian O'Rourke.Myocardial KATP Channels in Preconditioning.Circ.Res.2000;87;845-855.
[17]Derek M.Yellon and James M.Downey.Preconditioning the Myocardium:From Cellular Physiology to Clinical Cardiology.Physiol Rev 83:1113-1151,2003.
[18]Marisol Ruiz-Meana 1,Antonio Rodr guez-Sinovas 1,Alberto Cabestrero 1,Kerstin Boengler2,Gerd Heusch2,and David Garcia-Dorado 1.Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury.Cardiovascular Research(2008) 77,325 - 333.
[19]Madoka Matsumoto-Ida,MD;Masaharu Akao,MD,PhD;Toshihiro Takeda,MD;Masashi Kato,MD;Toru Kita,MD,PhD.Real-Time 2-Photon Imaging of Mitochondrial Function in Perfused Rat Hearts Subjected to Ischemia/Reperfusion.Circulation.2006;114:1497-1503.
[20]Liu F,Pouponnot C,Massague J.Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes.Genes Dev 1997;11:3157-67.
[21]Miehiko lliyakia,Toshio Kuroki.Role of Smad4(DP4)inactivntion in human calleet.Biochemical and Biophysical Research Communieatiom 306(2003) 799-804.
[22]Chacko BM,Qin BY,Tiwari A,Shi G,Lain S,Hayward LJ,et al.Structural basis of heteromeric Smad protein assembly in TGF-beta signaling.Mol Cell 2004;15:813-23.
[23]李春义。Smad蛋白与TGF-β信号转导。国外医学.生理、病理科学与临床分册21(2):89-92。
[24]明佳。Smads蛋白家族与TGF-β的细胞内信号转导。国外医学分子生物学分册 2003,25(2):89-92。
[25]何文喜,陈健。Smads基因家族的研究进展。体牙髓牙周病学杂志 2000,10(5):297-299。
[26]Wang G,Long J,Matsuura I,He D,Liu F.The Smad3 linker region contains a transcriptional activation domain.Biochem J 2005;386:29-34.
[27]Zhang Y,Chang C,Gehling DJ,Hemmati-Brivanlou A,Derynck R.Regulation of Smad degradation and activity by Smurf2,an E3 ubiquitin ligase.Proc Natl Acad Sci U S A 2001;98:974-9.
[28]Gerhild Euler-Taimor,Jacqueline Heger.The complex pattern of Smad signaling in the cardiovascular system. Cardiovascular Research 69 (2006) 15-25.
[29] Arimura T, Helbling-Leclerc A, Massart C, Varnous S, Niel F, Lacene E, et al. Mouse model carrying H2P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies. Hum Mol Genet 2005; 14: 155-69.
[30] Li RK, Li G, Mickle DA, Weisel RD, Merante F, Luss H, et al. Overexpression of transforming growth factor-betal and insulin-like growth factor-I in patients with idiopathic hypertrophic cardiomyopathy. Circulation 1997; 96:874-81.
[31] De Boer RF, Pokharel S, Flesch M, van Kampen DA, Suurmeijer AJH, Boomsa F, et al. Extracellular signal regulated kinase and Smad signaling both mediate the angiotensin II driven progression towards overt heart failure in homozygous TGR (mRen2)27. J Mol Med 2004; 82:678-87.
[32] Shi Y, Wang YF, Jayaraman L, Yang H, Massague J, Pavletich NP. Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell 1998; 94:585-94.
[33] Shimizu N, Yoshiyama M, Omura T, Hanatani A, Kim S, Takeuchi K,et al. Activation of mitogen-activated protein kinases and activator protein-1 in myocardial infarction in rats. Cardiovasc Res 1998; 38: 116-24.
[34] Frantz S, Fraccarollo D, Wagner H, Behr TM, Jung P, Angermann CE, et al. Sustained activation of nuclear factor kappa B and activator protein 1 in chronic heart failure. Cardiovasc Res 2003; 57:749-56.
[35] Arthur HM, Ure J, Smith AJ, Renforth G, Wilson DI, Torsney E, et al. Endoglin, an ancillary TGF beta receptor, is required for extraembryonic angiogenesis and plays a key role in heart development. Dev Biol 2000;217:42-53.
[36] Nakamura M, Wang NP, Zhao ZQ, et al. Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res, 2000; 45: 661-670.
[37] Piot CA, Padmanaban D, Ursell PC, et al. Ischemic preconditioning decreases apoptosis in rat hearts in vivo. Circulation, 1997;96(5): 1598-1604.
[38] 史永军。Smads蛋白家族的研究现状。中华腹部疾病杂志2002,2(5)：497—500。
[39] Liu F, Pouponnot C, Massague J. Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes. Genes Dev 1997; 11:3157-67.
[40] Chacko BM, Qin BY, Tiwari A, Shi G, Lam S, Hayward LJ, et al. Structural basis of heteromeric Smad protein assembly in TGF-beta signaling. Mol Cell 2004; 15:813-23.
[41] Wang G, Long J, Matsuura I, He D, Liu F. The Smad3 linker region contains a transcriptional activation domain.Biochem J 2005;386:29-34.
[42]Zhang Y,Chang C,Gehling DJ,Hemmati-Brivanlou A,Derynck R.Regulation of Smad degradation and activity by Smurf2,an E3 ubiquitin ligase.Proc Natl Acad Sci U S A 2001;98:974-9.
[43]Thomas E.Callis,Dongsun Cao,Da-Zhi Wang.Bone Morphogenetic Protein Signaling Modulates Myocardin Transactivation of Cardiac Genes.Circulation Research.2005;97:992.
[44]Hao J,Ju H,Zhao S,Junaid A,Scammell-La Fleur T,Dixon IM.Elevation of expression of Smads 2,3,and 4,decorin and TGF-beta in the chronic phase of myocardial infarct scar healing.J Mol Cell Cardiol 1999;31:667-78.
[45]赵俊芳,刘成,刘成海。转化生长因子细胞内信号转导与Smads蛋白。中国病理生理杂志 2002,18(3):321-325。
[46]原野,张明伟,于晓等。FGF2对BMP/Smads信号转导通路的负调节机制初步研究。临床口腔医学杂志 2005,21(3):134-136。
[47]Aoki H.Fuji M,Imamura T,et al.Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction[J].J Cell Sci,2001,14(Pt 8):1483-1 489.
[48]Hale A J,Smith C A,Sutherland L C,et al.Apeptosis:molecular regulation of cell death m.Eur J Biochem,1996,236(1):1-26.
[49]Bar P R.Apeptosis-The cell's silent exit.Life Science,1996,59(5/6):369-387.
[50]Carraro U,France schi C.Apeptosis of skeletal and cardiac muscles and physical exercise[J].Aging,1997,9(1-2):19-34.
[51]Schneiders D,Heger J,Best P,Piper HM,Taimor G.Smad proteins are involved in apoptosis induction in ventricular cardiomyocytes.Cardiovasc Res 2005;67:87-96.
[52]Francis JM,Heyworth CM,Spooncer E,Pierce A,Dexter TM,Whetton AD.Transforming growth factor- β1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.J Biol Chem 2000;75:39137-45.
[53]Chipuk JE,Bhat M,Hsing AY,Ma J,Danielpour D.Bcl-xL blocks transforming growth factor-b1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing apaf-1 dependent caspase activation in prostate epithelial cells.J Biol Chem 2001;276:26614- 21.
[54]Jang CW,Chen CH,Chen CC,Chen JY,Su YH,Chen RH.TGF-β induces apoptosis through Smad-mediated expression of DAP.Nat Cell Biol 2002;4:51-8.
[55]Mitsuru Masaki,MD;Masahiro Izumi,MD,PhD;Yuichi Oshima,MD;Yoshikazu Nakaoka, MD, PhD; Tadashi Kuroda, MD; Ryusuke Kimura, MD; Shoko Sugiyama, MD;Kazuo Terai, MD; Masafumi Kitakaze, MD, PhD; Keiko Yamauchi-Takihara, MD, PhD;Ichiro Kawase, MD, PhD; Hisao Hirota, MD, PhD. Smadl Protects Cardiomyocytes From Ischemia-Reperfusion Injury. Circulation. 2005; 111:2752-2759.
[56] S Jack Lin, Thomas F Lerch, Robert W Cook, Theodore S Jardetzky and Teresa K Woodruff. The structural basis of TGF-b, bone morphogenetic protein, and activin ligand binding. Reproduction (2006) 132 179-190.
[57] Ye-Guang Chen, Qiang Wang, Shi-Lung Lin,C. Donald Chang, Jody Chung,and Shao-Yao Ying. Activin Signaling and Its Role in Regulation of Cell Proliferation, Apoptosis, and Carcinogenesis. Experimental biology and medicine. 231(5):534.(2006)
[58] Alex Bobik. Transforming Growth Factor- β and Vascular Disorders. Arterioscler. Thromb. Vasc. Biol. 2006;26; 1712-1720.
[59] Victor W.T. Liu and Paul L. Huang. Cardiovascular roles of nitric oxide: A review of insights from nitric oxide synthase gene disrupted mice. Cardiovascular Research (2008) 77, 19-29.
[60] Tibor Kempf, Matthias Eden, Jens Strelau, Marian Naguib, Christian Willenbockel, Jorn Tongers, Jorg Heineke, Daniel Kotlarz, Jian Xu, Jeffery D. Molkentin, Hans W. Niessen, Helmut Drexler, Kai C. Wollert. The Transforming Growth Factor-β Superfamily Member Growth-Differentiation Factor-15 Protects the Heart From Ischemia/Reperfusion Injury. Circ. Res. 2006;98:351-360.
[61] Frank Eeftinga, Benno Rensinga, Jochem Wigmana, Willem Jan Pannekoeka, Wai Ming Liua, Maarten Jan Cramera, Daniel J. Lipsa, Pieter A. Doevendans. Role of apoptosis in reperfusion injury. Cardiovascular Research 61 (2004) 414- 426.
[62] Hideki Niimi, Katerina Pardali, Michael Vanlandewijck, Carl-Henrik Heldin, and Aristidis Moustakas. Notch signaling is necessary for epithelial growth arrest by TGF-β . The Journal of Cell Biology, Vol. 176, No. 5, February 26, 2007 695 - 707.
[63] Easwar Natarajan, John D. Omobono, II, Zongyou Guo, Susan Hopkinson, Alexander J.F. Lazar, Thomas Brenn, Jonathan C. Jones and James G. Rheinwald. A Keratinocyte Hypermotility/Growth-Arrest Response Involving Laminin 5 and p16INK4A Activated in Wound Healing and Senescence. American Journal of Pathology. 2006; 168:1821-1837.
[64] Sunil K Haider, R Daniel Beauchamp, and Pran K Datta. A Specific Inhibitor of TGF-β Receptor Kinase, SB-431542, as a Potent Antitumor Agent for Human Cancers. Neoplasia. 2005 May; 7(5): 509-521.
[65] Mitsunobu R. Kano , Younsoo Bae , Caname Iwata, Yasuyuki Morishita, Masakazu Yashiro, Masako Oka, Tomoko Fujii, Akiyoshi Komuro, Kunihiko Kiyono, Michio Kaminishi, Kosei Hirakawa, Yasuyoshi Ouchi, Nobuhiro Nishiyama, Kazunori Kataoka, and Kohei Miyazono. Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by inhibition of TGF-β signaling. Preceedings of the national academy of sciences of the United States of America. February 27, 2007 vol. 104 no. 9 3460-3465.
[66] Sheng-Ru Shiou, Pran K. Datta, Punita Dhawa, Brian K. Law, Jonathan M. Yingling, Dan A. Dixon, and R. Daniel Beauchamp. Smad4-dependent Regulation of Urokinase Plasminogen Activator Secretion and RNA Stability Associated with Invasiveness by Autocrine and Paracrine Transforming Growth Factor-β . J. Biol. Chem., Vol. 281, Issue 45, 33971-33981, November 10, 2006.
[67] Joan Massague,Joan Seoane,and David Wotton. Smad transcription factors. Genes & Dev. 2005 19:2783-2810.
[68] Aristidis Moustakas and Carl-Henrik Heldin. Non-Smad TGF-β signals. Journal of Cell Science 118 (16).
[69] Juan Rodriguez-Vita, BSc; Elsa Sanchez-Lopez, BSc; Vanesa Esteban, BSc; Monica Ruperez, BSc; Jesus Egido, MD; Marta Ruiz-Ortega, PhD. Angiotensin II Activates the Smad Pathway in Vascular Smooth Muscle Cells by a Transfomiing Growth Factor-β Independent Mechanism. Circulation May 17, 2005. 7(5): 509-521.
[70] El-Sayed Akool, Anke Doller, Roswitha Muller, Paul Gutwein, Cuiyan Xin, Andrea Huwiler, Josef Pfeilschifter, and Wolfgang Eberhardt3. Nitric Oxide Induces TIMP-1 Expression by Activating the Transforming Growth Factor β-Smad Signaling Pathway. The Journal Of Biological Chemistry VOL. 280, NO. 47, pp. 39403-39416, November 25, 2005.
[71] Shigeki Tsukada, John K. Westwick, Kenichi Ikejima, Nobuhiro Sato, and Richard A. Rippe. Smad and p38 MAPK Signaling Pathways Independently Regulate α1(I) Collagen Gene Expression in Unstimulated and Transforming. Circulation 2005. 7(5): 509-521.
[72] El-Sayed Akool, Anke Doller, Roswitha Muller, Paul Gutwein, Cuiyan Xin, Andrea Huwiler, Josef Pfeilschifter, and Wolfgang Eberhardt.Growth Factor-β -stimulated Hepatic Stellate Cells. The Journal Of Biological Chemistry Vol. 280, No. 11, Issue of March 18, pp. 10055-10064,2005.
[73] Yan Sun, Lihua Ding, Hao Zhang, Juqiang Han, Xiao Yang, Jinghua Yan, Yunfeng Zhul, Jiezhi Li, Haifeng Song2 and Qinong Ye. Potentiation of Smad-mediated transcriptional activation by the RNA-binding protein RBPMS. Nucleic Acids Research, 2006, Vol. 34, No. 21 .
[74] Nandini Ghosh-Choudhury, Prajjal K. Singha, Kathleen Woodruff, Patricia St Clair, Sameer Bsoul, Sherry L. Werner, and Goutam Ghosh Choudhury. Concerted Action of Smad and CREB-binding Protein Regulates Bone Morphogenetic Protein-2-stimulated Osteoblastic Colony-stimulating Factor-1 Expression. The Journal Of Biological Chemistry VOL. 281, NO. 29, pp. 20160-20170, July 21, 2006.
[75] Lei-Lei Chen, Hang Yin, Jun Huang. Inhibition of TGF-h1 signaling by eNOS gene transfer improves ventricular remodeling after myocardial infarction through angiogenesis and reduction of apoptosis. Cardiovascular Pathology 16 (2007) 221- 230
[1]史永军。Smads蛋白家族的研究现状。中华腹部疾病杂志 2002,2(5):497-500。
[2]Gerhild Euler-Taimor,Jacqueline Heger.The complex pattern of Smad signaling in the cardiovascular system.Cardiovascular Research 69(2006) 15 - 25
[3]Huang Jun,Qin Guo-hui,HU Chang-xing,Gong Li-ya Cheng Fang-zhou,Ma Ye-xin,LU Zai-ying.Effects of transforming growth factor-β1 and signal protein Smad3 on rat cardiomyocyte hypertrophy Chinese Medical Journal,2004,Vol.117 No.10:1471-1475.
[4]Gerald C.Ch,N.Ray Dunnl,Dorian C.Anderson,Leif Oxburgh and Elizabeth J.Robertsonl Differential requirements for Smad4 in TGFβ-dependent patterning of the early mouse embryo Development 131,3501-3512(2004)
[5]Chacko BM,Qin BY,Tiwari A,Shi G,Lam S,Hayward LJ,et al.Structural basis of heteromeric Smad protein assembly in TGF-beta signaling.Mol Cell 2004;15:813-23.
[6]李春义。Smad蛋白与TGF-β信号转导。国外医学.生理、病理科学与临床分册 21(2):89-92。
[7]Lanying Song,Wensheng Yan,Xinbin Chen,Chu-xia Deng,Qin Wang,Kai Jiao Myocardial Smad4 Is Essential for Cardiogenesis in Mouse Embryos Circulation Research.2007;101:277
[8]Erika A.Bosman 1,Kirstie A.Lawson2,Joke Debruyn1,Lisette Beek 1,Annick Francis 1,Luc Schoonjans3,Danny Huylebroeck1 and An Zwijsen1 Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels Development 133,3399-3409(2006)
[9]Wang G,Long J,Matsuura I,He D,Liu F.The Smad3 linker region contains a transcriptional activation domain.Biochem J 2005;386:29- 34.
[10]Zhang Y,Chang C,Gehling DJ,Hemmati-Brivanlou A,Derynck R.Regulation of Smad degradation and activity by Smurf2,an E3 ubiquitin ligase.Proc Natl Acad Sci U S A 2001;98:974- 9.
[11]Gerhild Euler-Taimor*,Jacqueline Heger.The complex pattern of Smad signaling in the cardiovascular system.Cardiovascular Research 69(2006) 15 - 25
[12]Lieve Umans,Luk Cox,Marc Tjwa,Virginie Bito,Liesbeth Vermeire,Kjell Laperre,Karin Sipido,Lieve Moons,Danny Huylebroeck and An Zwijsen Inactivation of Smad5 in Endothelial Cells and Smooth Muscle Cells Demonstrates that Smad5 Is Required for Cardiac Homeostasis American Joumal of Pathology.2007;170:1460-1472
[13]赵俊芳 刘成 刘成海。转化生长因子细胞内信号转导与Smads蛋白.中国病理生理杂志 2002,18(3):321-325。
[14]Thomas E.Callis,Dongsun Cao,Da-Zhi Wang Bone Morphogenetic Protein Signaling Modulates Myocardin Transactivation of Cardiac Genes Circulation Research.2005;97:992.
[15]Ping Dai,Takuo Nakagami,Hideo Tanaka,Toshiaki Hitomi,and Tetsuro Takamatsu Cx43Mediates TGF-β Signaling through Competitive Smads Binding to Microtubules Molecular biology of the CELL Vol.18,Issue 6,2264-2273,June 2007.
[16]原野,张明伟,于晓 等。FGF2对BMP/Smads信号转导通路的负调节机制初步研究。临床口腔医学杂志 2005,21(3):134-136。
[17]Aoki H.Fuji M。Imamura T,et al.Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction[J].J Cell Sci,2001,14(Pt 8):1483-1 489.
[18]Schneiders D,Heger J,Best P,Piper HM,Taimor G.Smad proteins are involved in apoptosis induction in ventricular cardiomyocytes.Cardiovasc Res 2005;67:87-96.
[19]Francis JM,Heyworth CM,Spooncer E,Pierce A,Dexter TM,Whetton AD.Transforming growth factor-h1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.J Biol Chem 2000;75:39137 -45.
[20]Chipuk JE,Bhat M,Hsing AY,Ma J,Danielpour D.Bcl-xL blocks transforming growth factor-b1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing apaf-1 dependent caspase activation in prostate epithelial cells.J Biol Chem 2001;276:26614- 21.
[21]Jang CW,Chen CH,Chen CC,Chen JY,Su YH,Chen RH.TGF-β induces apoptosis through Smad-mediated expression of DAP.Nat Cell Biol 2002;4:51-8.
[22]Mitsuru Masaki,MD*;Masahiro Izumi,MD,PhD*;Yuichi Oshima,MD;Yoshikazu Nakaoka,MD,PhD;Tadashi Kuroda,MD;Ryusuke Kimura,MD;Shoko Sugiyama,MD;Kazuo Terai,MD;Masafumi Kitakaze,MD,PhD;Keiko Yamauchi-Takihara,MD,PhD;Ichiro Kawase,MD,PhD;Hisao Hirota,MD,PhD.Smad1 Protects Cardiomyocytes From Ischemia-Reperfusion Injury.Circulation.2005;111:2752-2759.
[2]冯伯森,王秋雨,胡玉兰。动物细胞工程原理与实践。科学出版社,2000,152-153。
[3]郝亚荣,李庚山 李建军。乳鼠心肌细胞培养。岭南心血管病杂志。2001,7(2):137-139。
[4]沈静 谢苗荣 徐雍等。乳鼠心肌细胞培养及纯化方法的改良。中国医药导刊 2001,3(3):225-226。
[5]彭黎明,王曾礼主编.细胞凋亡的基础与临床.人民卫生出版社,2000年,167.
[6]吴立玲,主编.心血管病理生理学,北京医科大学出版社,2000年,215。
[7]Hemal H.Patel,Brian P.Head,1 Heidi N.Petersen,1 Ingrid R.Niesman,3 Diane Huang,Garrett J.Gross,5 Paul A.Insel,1 and David M.Roth2 Protection of adult rat cardiac myocytes from ischemic cell death:role of caveolar microdomains and δ-opioid receptors Am J Physiol Heart Circ Physiol 291:H344-H350,2006.
[8]奚群英,祝宝华,刘晨。乳鼠心肌细胞缺血预适应模型的建立及电镜下超微结构的改变。实用临床医药杂志 2005,3(9):18-24。
[9]程先进 曾志勇 杨胜生等。缺血预适应对缺血-再灌注心肌细胞的保护作用。临床麻醉学杂志 2007,23(5)138-140。
[10]Wang HC,Zhang HF,Guo WY,Su H,Zhang KR,Li QX,Yan W,Ma XL,Lopez BL,Christopher TA,Gao F.Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation:role of peroxynitrite formation.Apoptosis.2006 Aug;11(8):1453-60.
[11]李迎斋,张万义,陈学军等。缺血预适应对心肌细胞凋亡及血浆TNF水平影响的初步观察。中国微循环杂志 2005,9(4)264-266。
[12]Haunstetter A,Izumo S.Toward Antiapoptosis as a new treatment modality.Circ Res,2000,86:371-376
[13]He-Ying Sun,Ning-Ping Wang,Faraz Kerendi,Michael Halkos,Hajime Kin,Robert A.Guyton,Jakob Vinten-Johansen,and Zhi-Qing Zhao Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+overload Am J Physiol Heart Circ Physiol 288:H1900-H1908,2005.
[14]Maclellan WR,Schneider MD.Death by design.Programmed cell death in cardiovascular biology and disease.Circ Res,1997;81:137-144.
[15]Piot CA,Padmanban D,Ursell PC,Sievers RE,Wolfe CL.Ischemic predonditioning decreases apoptosis in rat hearts in vivo.Circulation,1997;2;96(5):1598-604.
[16]Maulik N,Yoshida T,Engelman RM,Deaton D,Flack JE 3rd,Rousu JA,Das DK.Ischemic preconditioning attenuates apoptotic cell death associated with ischemia / repeRPusion.Mol Cell Biochem,1998;186(1-2):139-45.
[17]Elizabeth N,Morgan MD,Edward M,et al.An essential role for NF-K B in the cardioadaptive response to ischemia.Ann Thorac Surg;1999,68:377-382.
[18]Simpson P,Savion S.Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cell.Circ Res.1982,50:101-116.
[19]Zhao ZQ,Nakamura M,Wang NP,et al.Reperfusion induces myocardial apoptotic cell death.Cardiovasc Res,2000;45(3):651-660.
[20]Liu H,Zhang HY,Zhu X,Shao Z,Yao Z,Preconditioning blocks cardiocyte apoptosis:role of K(ATP) channels and PKC-epsilon.Am J Physiol Heart Circ Physiol,2002;282(4):1380-1386.
[21]Korge P,Honda HM,Weiss JN.Protection of cardiac mitochondria by diazoxide and protein kinase C:implications for ischemic preconditioning.Proc Natl Acad Sci U S A,2002;99(5):3312-331
[22]Nozawa Y,Miura T,Miki T,Ohnuma Y,Yano T,Shimamoto K,Mitochondrial K(ATP)infarction in the rat.Basic Res Cardiol.2003;98(1):50-58.
[23]Rohr S,Scholly DM,Kléber AG.Patterned grouth of neonatal rat heart cells in culture:morphological and dectrophysiological characterization.Circ Res,1991,68:114-130.
[24]张旭晨。心血管疾病与细胞凋亡。国外医学·生理·病理科学与临床分册。1998,18:335-337。
[25]Jing Liu,Sharon Tsang and Tak Ming Wong Testosterone Is Required for Delayed Cardioprotection and Enhanced Heat Shock Protein 70 Expression Induced by Preconditioning Endocrinology Vol.147,No.10 4569-4577.
[26]Saraste A,Pulkki K,Kallajoki M,et al.Apoptosis in human acute myocardial infarction.Circulation,1997,95(2):320-323.
[27]Terry L.Vanden Hoek,Lance B.Becker,Zuo-Hui Shao,Chang-Qing Li,Paul T.Schumacker Preconditioning in Cardiomyocytes Protects by Attenuating Oxidant Stress at Reperfusion Circ.Res.2000;86;541-548.
[28]HuiPing Liu,Bradley C.Mcpherson,and ZhenHai Yao.Preconditioning attenuates apoptosis and necrosis:role of protein kinase Ce and -d isoforms.Am J Physiol Heart Circ Physiol 281:H404-H410,2001.
[29]Michael S.Marber Ischemic Preconditioning in Isolated Cells Circ.Res.2000;86;926-931.
[30]HuiPing Liu,Bradley C.Mcpherson,and ZhenHai Yao.Preconditioning blocks cardiocyte apoptosis:role of KATP channels and PKC-ε . Am J Physiol Heart Circ Physiol 282: H1380-H1386,2002;
[31] Ferran Padilla, David Garcia-Dorado, Antonio Rodri'guez-Sinovas,Marisol Ruiz-Meana, Javier Inserte, and Jordi Soler-Soler Protection afforded by ischemic preconditioning is not mediated by effects on cell-to-cell electrical coupling during myocardial ischemia-reperfusion Am J Physiol Heart Circ Physiol 285: H1909-H1916, 2003.
[32] T. Ravingerova, D. Pancza, A. Ziegelhoffer, J. STYK. Preconditioning Modulates Susceptibility to Ischemia-Induced Arrhythmias in the Rat Heart: The Role of α -Adrenergic Stimulation and K(ATP) Channels Physiol. Res. 51: 109-119, 2002
[33] Alexander E. Kabakov, Karina R. Budagova,David S. Latchman,R and Harm H. Kampinga. Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion. Am J Physiol Cell Physiol 283: C521-C534, 2002.
[34] Taro Date,Seibu Mochizuki,Adam J. Belanger,Midori Yamakawa,Zhengyu Luo, Karen A. Vincent, Seng H. Cheng, Richard J. Gregory, and Canwen Jiang. Expression of constitutively stable hybrid hypoxia-inducible factor-1α protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury Am J Physiol Cell Physiol 288: C314-C320,2005.
[1]杨明雄,袁爱国。心肌细胞凋亡的研究进展及意义。天津体育学院学报 2006,2(4):323-328。
[2]苏卫东 李会强 姚智。缺氧导致体外培养乳鼠心肌细胞凋亡的研究。放射免疫学杂志。2005,18(2):136-138。
[3]Gentzon Hall,Jeffery D.Hasday,Terry B.Rogers.Regulating the regulator:NF-κ B signaling in heart.Journal of Molecular and Cellular Cardiology 41(2006) 580 - 591.
[4]Frank Eeftinga,Benno Rensinga,Jochem Wigmana,Willem Jan Pannekoeka,Wai Ming Liua,Maarten Jan Cramera,Daniel J.Lipsa,Pieter A.Doevendans.Role of apoptosis in reperfusion injury.Cardiovascular Research 61(2004) 414-426.
[5]Asa B.Gustafsson and Roberta A.Gottlieb.Bcl-2 family members and apoptosis,taken to heart.Am J Physiol Cell Physiol 292:45-51,2007.
[6]Nakamura M,Wang NP,Zhao ZQ,et al.Preconditioning decreases Bax expression,PMN accumulation and apoptosis in reperfused rat heart.Cardiovasc Res,2000;45:661-670.
[7]郭永红,罗金燕。TGF-β超家族与Smad信号转导研究进展。医学综述 2005,11(8):685-687
[8]谷丽君。TGF-β超家族信号通路中的Smads蛋白。国外医学·生理、病理科学与临床分册。2002,22(6):580-583.
[9]Araujo-Jorge TC,Waghabi MC,Hasslocher-Moreno AM,Xavier SS,Higuchi Mde L,Keramidas M,et al.hnplication of transforming growth factor-betal in Chagas disease myocardiopathy.J Infect Dis 2002;186:1823 - 8.
[10]Hao J,Wang B,Jones SC,Jassal DS,Dixon IM.Interaction between angiotensin Ⅱ and Smad proteins in fibroblasts in failing heart and in vitro.Am J Physiol Heart Circ Physiol 2000;279:H3020-30.
[11]Dixon IM,Hao J,Reid NL,Roth JC.Effect of chronic AT receptor blockade on cardiac Smad overexpression in hereditary cardiomyopathic hamsters.Cardiovasc Res 2000;46:286-97.
[12]Schneiders D,Heger J,Best P,Piper HM,Taimor G.Smad proteins are involved in apoptosis induction in ventricular cardiomyocytes.Cardiovasc Res 2005;67:87-96.
[13]Francis JM,Heyworth CM,Spooncer E,Pierce A,Dexter TM,Whetton AD.Transforming growth factor-β 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.J Biol Chem 2000;75:39137-45.
[14]Chipuk JE,Bhat M,Hsing AY,Ma J,Danielpour D.Bcl-xL blocks transforming growth factor-b1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing apaf-1 dependent caspase activation in prostate epithelial cells.J Biol Chem 2001;276:26614-21.
[15]Alexandre D.T.Costal,Sandrine V.Pierre2,Michael V.Cohen3,4,James M.Downey3,and Keith D.Garlid1.cGMP signalling in pre- and post-conditioning:the role of mitochondria.Cardiovascular Research(2008) 77,344 - 352.
[16]Brian O'Rourke.Myocardial KATP Channels in Preconditioning.Circ.Res.2000;87;845-855.
[17]Derek M.Yellon and James M.Downey.Preconditioning the Myocardium:From Cellular Physiology to Clinical Cardiology.Physiol Rev 83:1113-1151,2003.
[18]Marisol Ruiz-Meana 1,Antonio Rodr guez-Sinovas 1,Alberto Cabestrero 1,Kerstin Boengler2,Gerd Heusch2,and David Garcia-Dorado 1.Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury.Cardiovascular Research(2008) 77,325 - 333.
[19]Madoka Matsumoto-Ida,MD;Masaharu Akao,MD,PhD;Toshihiro Takeda,MD;Masashi Kato,MD;Toru Kita,MD,PhD.Real-Time 2-Photon Imaging of Mitochondrial Function in Perfused Rat Hearts Subjected to Ischemia/Reperfusion.Circulation.2006;114:1497-1503.
[20]Liu F,Pouponnot C,Massague J.Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes.Genes Dev 1997;11:3157-67.
[21]Miehiko lliyakia,Toshio Kuroki.Role of Smad4(DP4)inactivntion in human calleet.Biochemical and Biophysical Research Communieatiom 306(2003) 799-804.
[22]Chacko BM,Qin BY,Tiwari A,Shi G,Lain S,Hayward LJ,et al.Structural basis of heteromeric Smad protein assembly in TGF-beta signaling.Mol Cell 2004;15:813-23.
[23]李春义。Smad蛋白与TGF-β信号转导。国外医学.生理、病理科学与临床分册21(2):89-92。
[24]明佳。Smads蛋白家族与TGF-β的细胞内信号转导。国外医学分子生物学分册 2003,25(2):89-92。
[25]何文喜,陈健。Smads基因家族的研究进展。体牙髓牙周病学杂志 2000,10(5):297-299。
[26]Wang G,Long J,Matsuura I,He D,Liu F.The Smad3 linker region contains a transcriptional activation domain.Biochem J 2005;386:29-34.
[27]Zhang Y,Chang C,Gehling DJ,Hemmati-Brivanlou A,Derynck R.Regulation of Smad degradation and activity by Smurf2,an E3 ubiquitin ligase.Proc Natl Acad Sci U S A 2001;98:974-9.
[28]Gerhild Euler-Taimor,Jacqueline Heger.The complex pattern of Smad signaling in the cardiovascular system. Cardiovascular Research 69 (2006) 15-25.
[29] Arimura T, Helbling-Leclerc A, Massart C, Varnous S, Niel F, Lacene E, et al. Mouse model carrying H2P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies. Hum Mol Genet 2005; 14: 155-69.
[30] Li RK, Li G, Mickle DA, Weisel RD, Merante F, Luss H, et al. Overexpression of transforming growth factor-betal and insulin-like growth factor-I in patients with idiopathic hypertrophic cardiomyopathy. Circulation 1997; 96:874-81.
[31] De Boer RF, Pokharel S, Flesch M, van Kampen DA, Suurmeijer AJH, Boomsa F, et al. Extracellular signal regulated kinase and Smad signaling both mediate the angiotensin II driven progression towards overt heart failure in homozygous TGR (mRen2)27. J Mol Med 2004; 82:678-87.
[32] Shi Y, Wang YF, Jayaraman L, Yang H, Massague J, Pavletich NP. Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell 1998; 94:585-94.
[33] Shimizu N, Yoshiyama M, Omura T, Hanatani A, Kim S, Takeuchi K,et al. Activation of mitogen-activated protein kinases and activator protein-1 in myocardial infarction in rats. Cardiovasc Res 1998; 38: 116-24.
[34] Frantz S, Fraccarollo D, Wagner H, Behr TM, Jung P, Angermann CE, et al. Sustained activation of nuclear factor kappa B and activator protein 1 in chronic heart failure. Cardiovasc Res 2003; 57:749-56.
[35] Arthur HM, Ure J, Smith AJ, Renforth G, Wilson DI, Torsney E, et al. Endoglin, an ancillary TGF beta receptor, is required for extraembryonic angiogenesis and plays a key role in heart development. Dev Biol 2000;217:42-53.
[36] Nakamura M, Wang NP, Zhao ZQ, et al. Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res, 2000; 45: 661-670.
[37] Piot CA, Padmanaban D, Ursell PC, et al. Ischemic preconditioning decreases apoptosis in rat hearts in vivo. Circulation, 1997;96(5): 1598-1604.
[38] 史永军。Smads蛋白家族的研究现状。中华腹部疾病杂志2002,2(5)：497—500。
[39] Liu F, Pouponnot C, Massague J. Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes. Genes Dev 1997; 11:3157-67.
[40] Chacko BM, Qin BY, Tiwari A, Shi G, Lam S, Hayward LJ, et al. Structural basis of heteromeric Smad protein assembly in TGF-beta signaling. Mol Cell 2004; 15:813-23.
[41] Wang G, Long J, Matsuura I, He D, Liu F. The Smad3 linker region contains a transcriptional activation domain.Biochem J 2005;386:29-34.
[42]Zhang Y,Chang C,Gehling DJ,Hemmati-Brivanlou A,Derynck R.Regulation of Smad degradation and activity by Smurf2,an E3 ubiquitin ligase.Proc Natl Acad Sci U S A 2001;98:974-9.
[43]Thomas E.Callis,Dongsun Cao,Da-Zhi Wang.Bone Morphogenetic Protein Signaling Modulates Myocardin Transactivation of Cardiac Genes.Circulation Research.2005;97:992.
[44]Hao J,Ju H,Zhao S,Junaid A,Scammell-La Fleur T,Dixon IM.Elevation of expression of Smads 2,3,and 4,decorin and TGF-beta in the chronic phase of myocardial infarct scar healing.J Mol Cell Cardiol 1999;31:667-78.
[45]赵俊芳,刘成,刘成海。转化生长因子细胞内信号转导与Smads蛋白。中国病理生理杂志 2002,18(3):321-325。
[46]原野,张明伟,于晓等。FGF2对BMP/Smads信号转导通路的负调节机制初步研究。临床口腔医学杂志 2005,21(3):134-136。
[47]Aoki H.Fuji M,Imamura T,et al.Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction[J].J Cell Sci,2001,14(Pt 8):1483-1 489.
[48]Hale A J,Smith C A,Sutherland L C,et al.Apeptosis:molecular regulation of cell death m.Eur J Biochem,1996,236(1):1-26.
[49]Bar P R.Apeptosis-The cell's silent exit.Life Science,1996,59(5/6):369-387.
[50]Carraro U,France schi C.Apeptosis of skeletal and cardiac muscles and physical exercise[J].Aging,1997,9(1-2):19-34.
[51]Schneiders D,Heger J,Best P,Piper HM,Taimor G.Smad proteins are involved in apoptosis induction in ventricular cardiomyocytes.Cardiovasc Res 2005;67:87-96.
[52]Francis JM,Heyworth CM,Spooncer E,Pierce A,Dexter TM,Whetton AD.Transforming growth factor- β1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.J Biol Chem 2000;75:39137-45.
[53]Chipuk JE,Bhat M,Hsing AY,Ma J,Danielpour D.Bcl-xL blocks transforming growth factor-b1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing apaf-1 dependent caspase activation in prostate epithelial cells.J Biol Chem 2001;276:26614- 21.
[54]Jang CW,Chen CH,Chen CC,Chen JY,Su YH,Chen RH.TGF-β induces apoptosis through Smad-mediated expression of DAP.Nat Cell Biol 2002;4:51-8.
[55]Mitsuru Masaki,MD;Masahiro Izumi,MD,PhD;Yuichi Oshima,MD;Yoshikazu Nakaoka, MD, PhD; Tadashi Kuroda, MD; Ryusuke Kimura, MD; Shoko Sugiyama, MD;Kazuo Terai, MD; Masafumi Kitakaze, MD, PhD; Keiko Yamauchi-Takihara, MD, PhD;Ichiro Kawase, MD, PhD; Hisao Hirota, MD, PhD. Smadl Protects Cardiomyocytes From Ischemia-Reperfusion Injury. Circulation. 2005; 111:2752-2759.
[56] S Jack Lin, Thomas F Lerch, Robert W Cook, Theodore S Jardetzky and Teresa K Woodruff. The structural basis of TGF-b, bone morphogenetic protein, and activin ligand binding. Reproduction (2006) 132 179-190.
[57] Ye-Guang Chen, Qiang Wang, Shi-Lung Lin,C. Donald Chang, Jody Chung,and Shao-Yao Ying. Activin Signaling and Its Role in Regulation of Cell Proliferation, Apoptosis, and Carcinogenesis. Experimental biology and medicine. 231(5):534.(2006)
[58] Alex Bobik. Transforming Growth Factor- β and Vascular Disorders. Arterioscler. Thromb. Vasc. Biol. 2006;26; 1712-1720.
[59] Victor W.T. Liu and Paul L. Huang. Cardiovascular roles of nitric oxide: A review of insights from nitric oxide synthase gene disrupted mice. Cardiovascular Research (2008) 77, 19-29.
[60] Tibor Kempf, Matthias Eden, Jens Strelau, Marian Naguib, Christian Willenbockel, Jorn Tongers, Jorg Heineke, Daniel Kotlarz, Jian Xu, Jeffery D. Molkentin, Hans W. Niessen, Helmut Drexler, Kai C. Wollert. The Transforming Growth Factor-β Superfamily Member Growth-Differentiation Factor-15 Protects the Heart From Ischemia/Reperfusion Injury. Circ. Res. 2006;98:351-360.
[61] Frank Eeftinga, Benno Rensinga, Jochem Wigmana, Willem Jan Pannekoeka, Wai Ming Liua, Maarten Jan Cramera, Daniel J. Lipsa, Pieter A. Doevendans. Role of apoptosis in reperfusion injury. Cardiovascular Research 61 (2004) 414- 426.
[62] Hideki Niimi, Katerina Pardali, Michael Vanlandewijck, Carl-Henrik Heldin, and Aristidis Moustakas. Notch signaling is necessary for epithelial growth arrest by TGF-β . The Journal of Cell Biology, Vol. 176, No. 5, February 26, 2007 695 - 707.
[63] Easwar Natarajan, John D. Omobono, II, Zongyou Guo, Susan Hopkinson, Alexander J.F. Lazar, Thomas Brenn, Jonathan C. Jones and James G. Rheinwald. A Keratinocyte Hypermotility/Growth-Arrest Response Involving Laminin 5 and p16INK4A Activated in Wound Healing and Senescence. American Journal of Pathology. 2006; 168:1821-1837.
[64] Sunil K Haider, R Daniel Beauchamp, and Pran K Datta. A Specific Inhibitor of TGF-β Receptor Kinase, SB-431542, as a Potent Antitumor Agent for Human Cancers. Neoplasia. 2005 May; 7(5): 509-521.
[65] Mitsunobu R. Kano , Younsoo Bae , Caname Iwata, Yasuyuki Morishita, Masakazu Yashiro, Masako Oka, Tomoko Fujii, Akiyoshi Komuro, Kunihiko Kiyono, Michio Kaminishi, Kosei Hirakawa, Yasuyoshi Ouchi, Nobuhiro Nishiyama, Kazunori Kataoka, and Kohei Miyazono. Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by inhibition of TGF-β signaling. Preceedings of the national academy of sciences of the United States of America. February 27, 2007 vol. 104 no. 9 3460-3465.
[66] Sheng-Ru Shiou, Pran K. Datta, Punita Dhawa, Brian K. Law, Jonathan M. Yingling, Dan A. Dixon, and R. Daniel Beauchamp. Smad4-dependent Regulation of Urokinase Plasminogen Activator Secretion and RNA Stability Associated with Invasiveness by Autocrine and Paracrine Transforming Growth Factor-β . J. Biol. Chem., Vol. 281, Issue 45, 33971-33981, November 10, 2006.
[67] Joan Massague,Joan Seoane,and David Wotton. Smad transcription factors. Genes & Dev. 2005 19:2783-2810.
[68] Aristidis Moustakas and Carl-Henrik Heldin. Non-Smad TGF-β signals. Journal of Cell Science 118 (16).
[69] Juan Rodriguez-Vita, BSc; Elsa Sanchez-Lopez, BSc; Vanesa Esteban, BSc; Monica Ruperez, BSc; Jesus Egido, MD; Marta Ruiz-Ortega, PhD. Angiotensin II Activates the Smad Pathway in Vascular Smooth Muscle Cells by a Transfomiing Growth Factor-β Independent Mechanism. Circulation May 17, 2005. 7(5): 509-521.
[70] El-Sayed Akool, Anke Doller, Roswitha Muller, Paul Gutwein, Cuiyan Xin, Andrea Huwiler, Josef Pfeilschifter, and Wolfgang Eberhardt3. Nitric Oxide Induces TIMP-1 Expression by Activating the Transforming Growth Factor β-Smad Signaling Pathway. The Journal Of Biological Chemistry VOL. 280, NO. 47, pp. 39403-39416, November 25, 2005.
[71] Shigeki Tsukada, John K. Westwick, Kenichi Ikejima, Nobuhiro Sato, and Richard A. Rippe. Smad and p38 MAPK Signaling Pathways Independently Regulate α1(I) Collagen Gene Expression in Unstimulated and Transforming. Circulation 2005. 7(5): 509-521.
[72] El-Sayed Akool, Anke Doller, Roswitha Muller, Paul Gutwein, Cuiyan Xin, Andrea Huwiler, Josef Pfeilschifter, and Wolfgang Eberhardt.Growth Factor-β -stimulated Hepatic Stellate Cells. The Journal Of Biological Chemistry Vol. 280, No. 11, Issue of March 18, pp. 10055-10064,2005.
[73] Yan Sun, Lihua Ding, Hao Zhang, Juqiang Han, Xiao Yang, Jinghua Yan, Yunfeng Zhul, Jiezhi Li, Haifeng Song2 and Qinong Ye. Potentiation of Smad-mediated transcriptional activation by the RNA-binding protein RBPMS. Nucleic Acids Research, 2006, Vol. 34, No. 21 .
[74] Nandini Ghosh-Choudhury, Prajjal K. Singha, Kathleen Woodruff, Patricia St Clair, Sameer Bsoul, Sherry L. Werner, and Goutam Ghosh Choudhury. Concerted Action of Smad and CREB-binding Protein Regulates Bone Morphogenetic Protein-2-stimulated Osteoblastic Colony-stimulating Factor-1 Expression. The Journal Of Biological Chemistry VOL. 281, NO. 29, pp. 20160-20170, July 21, 2006.
[75] Lei-Lei Chen, Hang Yin, Jun Huang. Inhibition of TGF-h1 signaling by eNOS gene transfer improves ventricular remodeling after myocardial infarction through angiogenesis and reduction of apoptosis. Cardiovascular Pathology 16 (2007) 221- 230
[1]史永军。Smads蛋白家族的研究现状。中华腹部疾病杂志 2002,2(5):497-500。
[2]Gerhild Euler-Taimor,Jacqueline Heger.The complex pattern of Smad signaling in the cardiovascular system.Cardiovascular Research 69(2006) 15 - 25
[3]Huang Jun,Qin Guo-hui,HU Chang-xing,Gong Li-ya Cheng Fang-zhou,Ma Ye-xin,LU Zai-ying.Effects of transforming growth factor-β1 and signal protein Smad3 on rat cardiomyocyte hypertrophy Chinese Medical Journal,2004,Vol.117 No.10:1471-1475.
[4]Gerald C.Ch,N.Ray Dunnl,Dorian C.Anderson,Leif Oxburgh and Elizabeth J.Robertsonl Differential requirements for Smad4 in TGFβ-dependent patterning of the early mouse embryo Development 131,3501-3512(2004)
[5]Chacko BM,Qin BY,Tiwari A,Shi G,Lam S,Hayward LJ,et al.Structural basis of heteromeric Smad protein assembly in TGF-beta signaling.Mol Cell 2004;15:813-23.
[6]李春义。Smad蛋白与TGF-β信号转导。国外医学.生理、病理科学与临床分册 21(2):89-92。
[7]Lanying Song,Wensheng Yan,Xinbin Chen,Chu-xia Deng,Qin Wang,Kai Jiao Myocardial Smad4 Is Essential for Cardiogenesis in Mouse Embryos Circulation Research.2007;101:277
[8]Erika A.Bosman 1,Kirstie A.Lawson2,Joke Debruyn1,Lisette Beek 1,Annick Francis 1,Luc Schoonjans3,Danny Huylebroeck1 and An Zwijsen1 Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels Development 133,3399-3409(2006)
[9]Wang G,Long J,Matsuura I,He D,Liu F.The Smad3 linker region contains a transcriptional activation domain.Biochem J 2005;386:29- 34.
[10]Zhang Y,Chang C,Gehling DJ,Hemmati-Brivanlou A,Derynck R.Regulation of Smad degradation and activity by Smurf2,an E3 ubiquitin ligase.Proc Natl Acad Sci U S A 2001;98:974- 9.
[11]Gerhild Euler-Taimor*,Jacqueline Heger.The complex pattern of Smad signaling in the cardiovascular system.Cardiovascular Research 69(2006) 15 - 25
[12]Lieve Umans,Luk Cox,Marc Tjwa,Virginie Bito,Liesbeth Vermeire,Kjell Laperre,Karin Sipido,Lieve Moons,Danny Huylebroeck and An Zwijsen Inactivation of Smad5 in Endothelial Cells and Smooth Muscle Cells Demonstrates that Smad5 Is Required for Cardiac Homeostasis American Joumal of Pathology.2007;170:1460-1472
[13]赵俊芳 刘成 刘成海。转化生长因子细胞内信号转导与Smads蛋白.中国病理生理杂志 2002,18(3):321-325。
[14]Thomas E.Callis,Dongsun Cao,Da-Zhi Wang Bone Morphogenetic Protein Signaling Modulates Myocardin Transactivation of Cardiac Genes Circulation Research.2005;97:992.
[15]Ping Dai,Takuo Nakagami,Hideo Tanaka,Toshiaki Hitomi,and Tetsuro Takamatsu Cx43Mediates TGF-β Signaling through Competitive Smads Binding to Microtubules Molecular biology of the CELL Vol.18,Issue 6,2264-2273,June 2007.
[16]原野,张明伟,于晓 等。FGF2对BMP/Smads信号转导通路的负调节机制初步研究。临床口腔医学杂志 2005,21(3):134-136。
[17]Aoki H.Fuji M。Imamura T,et al.Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction[J].J Cell Sci,2001,14(Pt 8):1483-1 489.
[18]Schneiders D,Heger J,Best P,Piper HM,Taimor G.Smad proteins are involved in apoptosis induction in ventricular cardiomyocytes.Cardiovasc Res 2005;67:87-96.
[19]Francis JM,Heyworth CM,Spooncer E,Pierce A,Dexter TM,Whetton AD.Transforming growth factor-h1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.J Biol Chem 2000;75:39137 -45.
[20]Chipuk JE,Bhat M,Hsing AY,Ma J,Danielpour D.Bcl-xL blocks transforming growth factor-b1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing apaf-1 dependent caspase activation in prostate epithelial cells.J Biol Chem 2001;276:26614- 21.
[21]Jang CW,Chen CH,Chen CC,Chen JY,Su YH,Chen RH.TGF-β induces apoptosis through Smad-mediated expression of DAP.Nat Cell Biol 2002;4:51-8.
[22]Mitsuru Masaki,MD*;Masahiro Izumi,MD,PhD*;Yuichi Oshima,MD;Yoshikazu Nakaoka,MD,PhD;Tadashi Kuroda,MD;Ryusuke Kimura,MD;Shoko Sugiyama,MD;Kazuo Terai,MD;Masafumi Kitakaze,MD,PhD;Keiko Yamauchi-Takihara,MD,PhD;Ichiro Kawase,MD,PhD;Hisao Hirota,MD,PhD.Smad1 Protects Cardiomyocytes From Ischemia-Reperfusion Injury.Circulation.2005;111:2752-2759.