急慢性心肌缺血心肌细胞凋亡的机制研究
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摘要
研究背景：传统观念认为，心肌缺血与其导致的心肌坏死是一种全
或无的关系。近年认为缺血对心肌的影响是多方面的：缺血时间在5min
内时心肌处于可逆性损伤，恢复再灌注后缺血心肌迅速恢复正常；经短
暂缺血的心肌对以后的缺血发作具保护作用，即心肌缺血预适应
（IPC）；冠状动脉阻塞5～20min所致心肌短暂缺血后引起的左室收缩功
能不全，可在数小时或数天后恢复，无不可逆损伤，称为心肌顿抑（MS）；
冠状动脉病变所致低灌注引起持续性左心功能不全变化，当冠脉血流恢
复正常后，心肌功能可部分或全部恢复正常，称为冬眠心肌（HM）；冠
状动脉阻塞时间超过30min，可引起心肌不可逆的损伤、坏死。
既往认为细胞坏死是心肌缺血所致细胞死亡的唯一形式。近年来普
遍承认细胞死亡有两种方式，即坏死和凋亡。缺血致心肌细胞凋亡是近
年研究的热点，有关心肌缺血后细胞凋亡的演变规律及调控机制的研究
尚处于起步阶段。MS本质上是缺血再灌注的后果，尚不清楚MS是否
存在细胞凋亡。急性心肌梗塞（AMI）、HM以及缺血再灌注损伤，IPC
时凋亡相关蛋白的表达缺乏系统研究。Losartan对心肌缺血再灌注损伤
的预防作用是否也涉及到细胞凋亡尚不清楚。
研究目的：本课题在AMI、HM、MS、IPC及缺血再灌注模型基础
上，探讨急慢性心肌缺血所致心肌细胞凋亡及其机制，以及ATII拮抗
剂Losartan对心肌缺血再灌注细胞凋亡的影响。
研究方法：
1.采用结扎大鼠冠脉LAD15min，再灌注2h及6h方法建立MS
模型。观察MS心功能和MDA的改变，以及心肌细胞凋亡和
Bcl-2、Bax、FAS的表达。
2.采用闭塞大鼠冠脉LAD致缺血5min，随后再灌注5min，重复
3次的方法造成IPC。观察IPC对缺血40min、再灌注6h的缺
血再赐注的保护作用。
3、采用结扎大鼠LAD的方法建立AMI模型。观察AMI后Zh、4h、
6h、24h、48h及7d心肌细胞凋亡和Bcl．2、Bax、FAS的表达。
4、采用给幼猪冠状动脉LAD第一对角支近端植人缩窄器，术后饲
养 ZI周的方法建立 HM模型。观察 HM心肌细胞凋亡及 BcIZ、
Bax、FAS的表达。
5．上述心肌组织标本细胞凋亡的检测采用TUNEL及DNA凝胶电
泳检测DNA片断。Bcl2、Bax及FAS蛋白的表达采用免疫组
织化学方法。用图象分析法定量分析心肌细胞凋亡及BclZ、Bax
及FAS蛋白的表达。
6、观察ATll诱导培养心肌细胞凋亡及Losartan对其影响；观察
L。satan对培养心肌缺血再涯注心肌细胞凋亡的影响。用碘化
丙陡染色流式细胞仪检测及透射电镰检查来观察心肌细胞凋
亡。
％：
1．MS与心肌细胞凋亡 缺血 15min再灌 Zh及 6力可致心功能可逆
性障碍，MDA增加，但CK正常，光镜下组织结构正常；TUN’EL检测
备组未见阳性细胞，DNA Ladder各组均阴性。
2．预适应与心肌细胞凋亡：0厂PC组较缺血再蠢注组梗塞面积减
少（ 13．8土 4．互％ VS 31．3土 6．互％，尸＜0刀 1），CK水平降低（．7土 10．8 IU4
VS 247．7土67．7则几，P＜0．of）。（2 IPC组凋亡指数明显低于缺血再灌
注组（10＊土2．3％ vs ZI．6土5．6％，尸＜0．of）。＊ IPC组 DNA Ladder阳
性率口2．2％）较缺血再灌注组＊5．6％）明显要低（P＜0．OI人＊）缺血再灌
注组 BCIE 表达p9．9 i 4．7O）较正常对照组p．5上 l．lo）明显增加 呵
（P功．of人 而 IPC组 BC12的表达（45．吕士 6．l％）较缺血再蠢注组高
（尸＜0刀1）；各组间 Bax表达差别不大，但 IPC组 Bcl－hax比值较缺
血再灌注组高门．9土5．l％vs6尸土3．3％，P＜0刀5h缺血再沤注组FAS表
达（32．2土7．l％）较对照组a．6t0．8％）增加（P＜0．OI人 而 IPC组 FAS
表达门．8土4．20）低于缺血再灌注组（P＜0．01L但高于对照组（P＜0．01人
3．AMI与，C’肌细胞凋亡（l），0脏大体观察见 AMI 24h组心脏增大，
－6－
梗塞处心肌呈污秽暗红色；7d组梗塞处向外膨出、壁薄如羊皮纸、色
苍白。o）光镜下见AMI 6h以前各组无明显组织形态改变；24h组可见
梗塞区心肌细胞肿胀、空泡形成、横纹消失，出现多形核白细胞浸润。
48h心肌纤维出现玻璃样变，嗜酸性增强，核溶解，白细胞浸润明显增
多。7d时梗塞灶肌纤维消失，为炎性肉芽肿及成纤维细胞所代替。m
结扎冠脉后受累区 Zh即出现心肌细胞凋亡，6h达高峰（33，SM．2％），
至24h时梗塞区仍有较多凋亡；48h梗塞区凋亡较少，7d时梗塞区未见
凋亡细胞。48h及7d时梗塞边缘区见少量心肌细胞凋亡。（）对照组及
梗塞 Zh组 DNA Ladder检测阴性，梗塞 4 ，J’时组 l例阳性（阳性率 20o／o），
6刁时组5例阳性（83．3％），24小时组4例阳性（66．7％），48J时组1例
Background: It was believed that cardiac ischemia and ischeniia-caused
cardiomyocyte necrosis is a all or none?relationship. However, recent
reports have demonstrated that the impacts of ischemia to myocardium are in
many ways: when the ischemic period is shorter than 5mm, myocardial injury
is reversible, the ischemia myocardium will return to normal rapidly after
reperfusion; brie.f periods of acute myocardial ischemia protect against
ischemia reperfusion injury, this phenomenon is called ischemia
preconditioning (IPC); 5mm to 20 mm myocardial ischemia can cause a fully
reversible yet prolonged mechanical dysfunction despite restoration of
normal coronary flow, this is called myocardial stunning (MS); chronic
reduced coronary blood flow can cause persistently impaired myocardial and
LV function at rest , but the impaired heart function could be partially or
completely restored to normal by improving blood flow or reducing oxygen
demand, this is hibernating myocardium (I-fM); prolonged periods of
myocardial ischemia (longer than 30 mm ) can cause tissue injury and cell
death.
For a long time it is believed that necrosis is the only way to death caused
by myocardia ischemia and reperfion. Recently, considerable attention has
been directed to another form of cell death , referred to as apoptosis. Recent
reports have demonstrated that apoptosis does occur in cardiomyocyte by
agents that have traditionally been thought to produce necrosis such as
ischemia and hypoxia. The reseach relating to the developing rule and
regulatory mechanism of cardiomyocyte apoptosis caused by ischeniia are on
the early stage. It is lack systemic research on the expression of apoptotic
ralated protein after acute myocardial infarction, hibernating rnyocardium,
ischemia-reperfusion injury and ischemia preconditioning. Angiotensin II can
induce cardiomycyte apoptosis, and .Angiotensin II type 1 antagonist
Losartan can pretect against ischemic reperfusion injury, but whether the
protect effect of Losartan to is relatd to cardiomyocyte apoptosis is unclear.
Objectives: On the basis of replicated AIvfl,HM, MS. IPC and ischemic
reperfusion(IR) model, we investigating cardiomyocyte apoptosis and the
expression of Bcl-2,Bax and FAS in acute and chronic myocardium
ischemic, the effect of IPC and Losartan to cardiiomyocyte apoptosis of
ischemic reperfusion are also observed.
Materials and methods:
I .The rat in vivo myocardial stunning model was made by ligating the LAD
1 5mm followed by 2h and 6h reperfusion. Inspecting cardiomyocyte
apoptosis and BcI-2,Bax and FAS protein expression in MS.
2.The IPC model of rat was made with the protocol consisting of 3 cycles of
5mm of myocardial ischemia and 5mm of reperfusion. Inspecting the effect
of IPC to ischemia reperfusion with 40mm ischemia and 6h reperfusion.
Collecting blood sample for biochemistry measurement, measuring the
infarct area, Inspecting cardiomyocyte apoptosis and Bcl-2,Bax and FAS
protein.
3.Making AMI model with the method of ligating the left anterior descending
coronary artery of rat. Investigating cadiomyocyte apoptosis and Bcl-
2,Bax and FAS proteins in the group of 2h,4h, 6h, 24h, 48h and 7d after
AMI.
4.To establishing HM animal model, implanting the proximal LAD of
juvenile pigs with a 5mm-long and 2.0mm fixed internal diameter Delran
occluder, then housing the animals for 21 weeks. Confirming the MM with
selective left coronary angiography, low dose dobutamine
echocar
或无的关系。近年认为缺血对心肌的影响是多方面的：缺血时间在5min
内时心肌处于可逆性损伤，恢复再灌注后缺血心肌迅速恢复正常；经短
暂缺血的心肌对以后的缺血发作具保护作用，即心肌缺血预适应
（IPC）；冠状动脉阻塞5～20min所致心肌短暂缺血后引起的左室收缩功
能不全，可在数小时或数天后恢复，无不可逆损伤，称为心肌顿抑（MS）；
冠状动脉病变所致低灌注引起持续性左心功能不全变化，当冠脉血流恢
复正常后，心肌功能可部分或全部恢复正常，称为冬眠心肌（HM）；冠
状动脉阻塞时间超过30min，可引起心肌不可逆的损伤、坏死。
既往认为细胞坏死是心肌缺血所致细胞死亡的唯一形式。近年来普
遍承认细胞死亡有两种方式，即坏死和凋亡。缺血致心肌细胞凋亡是近
年研究的热点，有关心肌缺血后细胞凋亡的演变规律及调控机制的研究
尚处于起步阶段。MS本质上是缺血再灌注的后果，尚不清楚MS是否
存在细胞凋亡。急性心肌梗塞（AMI）、HM以及缺血再灌注损伤，IPC
时凋亡相关蛋白的表达缺乏系统研究。Losartan对心肌缺血再灌注损伤
的预防作用是否也涉及到细胞凋亡尚不清楚。
研究目的：本课题在AMI、HM、MS、IPC及缺血再灌注模型基础
上，探讨急慢性心肌缺血所致心肌细胞凋亡及其机制，以及ATII拮抗
剂Losartan对心肌缺血再灌注细胞凋亡的影响。
研究方法：
1.采用结扎大鼠冠脉LAD15min，再灌注2h及6h方法建立MS
模型。观察MS心功能和MDA的改变，以及心肌细胞凋亡和
Bcl-2、Bax、FAS的表达。
2.采用闭塞大鼠冠脉LAD致缺血5min，随后再灌注5min，重复
3次的方法造成IPC。观察IPC对缺血40min、再灌注6h的缺
血再赐注的保护作用。
3、采用结扎大鼠LAD的方法建立AMI模型。观察AMI后Zh、4h、
6h、24h、48h及7d心肌细胞凋亡和Bcl．2、Bax、FAS的表达。
4、采用给幼猪冠状动脉LAD第一对角支近端植人缩窄器，术后饲
养 ZI周的方法建立 HM模型。观察 HM心肌细胞凋亡及 BcIZ、
Bax、FAS的表达。
5．上述心肌组织标本细胞凋亡的检测采用TUNEL及DNA凝胶电
泳检测DNA片断。Bcl2、Bax及FAS蛋白的表达采用免疫组
织化学方法。用图象分析法定量分析心肌细胞凋亡及BclZ、Bax
及FAS蛋白的表达。
6、观察ATll诱导培养心肌细胞凋亡及Losartan对其影响；观察
L。satan对培养心肌缺血再涯注心肌细胞凋亡的影响。用碘化
丙陡染色流式细胞仪检测及透射电镰检查来观察心肌细胞凋
亡。
％：
1．MS与心肌细胞凋亡 缺血 15min再灌 Zh及 6力可致心功能可逆
性障碍，MDA增加，但CK正常，光镜下组织结构正常；TUN’EL检测
备组未见阳性细胞，DNA Ladder各组均阴性。
2．预适应与心肌细胞凋亡：0厂PC组较缺血再蠢注组梗塞面积减
少（ 13．8土 4．互％ VS 31．3土 6．互％，尸＜0刀 1），CK水平降低（．7土 10．8 IU4
VS 247．7土67．7则几，P＜0．of）。（2 IPC组凋亡指数明显低于缺血再灌
注组（10＊土2．3％ vs ZI．6土5．6％，尸＜0．of）。＊ IPC组 DNA Ladder阳
性率口2．2％）较缺血再灌注组＊5．6％）明显要低（P＜0．OI人＊）缺血再灌
注组 BCIE 表达p9．9 i 4．7O）较正常对照组p．5上 l．lo）明显增加 呵
（P功．of人 而 IPC组 BC12的表达（45．吕士 6．l％）较缺血再蠢注组高
（尸＜0刀1）；各组间 Bax表达差别不大，但 IPC组 Bcl－hax比值较缺
血再灌注组高门．9土5．l％vs6尸土3．3％，P＜0刀5h缺血再沤注组FAS表
达（32．2土7．l％）较对照组a．6t0．8％）增加（P＜0．OI人 而 IPC组 FAS
表达门．8土4．20）低于缺血再灌注组（P＜0．01L但高于对照组（P＜0．01人
3．AMI与，C’肌细胞凋亡（l），0脏大体观察见 AMI 24h组心脏增大，
－6－
梗塞处心肌呈污秽暗红色；7d组梗塞处向外膨出、壁薄如羊皮纸、色
苍白。o）光镜下见AMI 6h以前各组无明显组织形态改变；24h组可见
梗塞区心肌细胞肿胀、空泡形成、横纹消失，出现多形核白细胞浸润。
48h心肌纤维出现玻璃样变，嗜酸性增强，核溶解，白细胞浸润明显增
多。7d时梗塞灶肌纤维消失，为炎性肉芽肿及成纤维细胞所代替。m
结扎冠脉后受累区 Zh即出现心肌细胞凋亡，6h达高峰（33，SM．2％），
至24h时梗塞区仍有较多凋亡；48h梗塞区凋亡较少，7d时梗塞区未见
凋亡细胞。48h及7d时梗塞边缘区见少量心肌细胞凋亡。（）对照组及
梗塞 Zh组 DNA Ladder检测阴性，梗塞 4 ，J’时组 l例阳性（阳性率 20o／o），
6刁时组5例阳性（83．3％），24小时组4例阳性（66．7％），48J时组1例
Background: It was believed that cardiac ischemia and ischeniia-caused
cardiomyocyte necrosis is a all or none?relationship. However, recent
reports have demonstrated that the impacts of ischemia to myocardium are in
many ways: when the ischemic period is shorter than 5mm, myocardial injury
is reversible, the ischemia myocardium will return to normal rapidly after
reperfusion; brie.f periods of acute myocardial ischemia protect against
ischemia reperfusion injury, this phenomenon is called ischemia
preconditioning (IPC); 5mm to 20 mm myocardial ischemia can cause a fully
reversible yet prolonged mechanical dysfunction despite restoration of
normal coronary flow, this is called myocardial stunning (MS); chronic
reduced coronary blood flow can cause persistently impaired myocardial and
LV function at rest , but the impaired heart function could be partially or
completely restored to normal by improving blood flow or reducing oxygen
demand, this is hibernating myocardium (I-fM); prolonged periods of
myocardial ischemia (longer than 30 mm ) can cause tissue injury and cell
death.
For a long time it is believed that necrosis is the only way to death caused
by myocardia ischemia and reperfion. Recently, considerable attention has
been directed to another form of cell death , referred to as apoptosis. Recent
reports have demonstrated that apoptosis does occur in cardiomyocyte by
agents that have traditionally been thought to produce necrosis such as
ischemia and hypoxia. The reseach relating to the developing rule and
regulatory mechanism of cardiomyocyte apoptosis caused by ischeniia are on
the early stage. It is lack systemic research on the expression of apoptotic
ralated protein after acute myocardial infarction, hibernating rnyocardium,
ischemia-reperfusion injury and ischemia preconditioning. Angiotensin II can
induce cardiomycyte apoptosis, and .Angiotensin II type 1 antagonist
Losartan can pretect against ischemic reperfusion injury, but whether the
protect effect of Losartan to is relatd to cardiomyocyte apoptosis is unclear.
Objectives: On the basis of replicated AIvfl,HM, MS. IPC and ischemic
reperfusion(IR) model, we investigating cardiomyocyte apoptosis and the
expression of Bcl-2,Bax and FAS in acute and chronic myocardium
ischemic, the effect of IPC and Losartan to cardiiomyocyte apoptosis of
ischemic reperfusion are also observed.
Materials and methods:
I .The rat in vivo myocardial stunning model was made by ligating the LAD
1 5mm followed by 2h and 6h reperfusion. Inspecting cardiomyocyte
apoptosis and BcI-2,Bax and FAS protein expression in MS.
2.The IPC model of rat was made with the protocol consisting of 3 cycles of
5mm of myocardial ischemia and 5mm of reperfusion. Inspecting the effect
of IPC to ischemia reperfusion with 40mm ischemia and 6h reperfusion.
Collecting blood sample for biochemistry measurement, measuring the
infarct area, Inspecting cardiomyocyte apoptosis and Bcl-2,Bax and FAS
protein.
3.Making AMI model with the method of ligating the left anterior descending
coronary artery of rat. Investigating cadiomyocyte apoptosis and Bcl-
2,Bax and FAS proteins in the group of 2h,4h, 6h, 24h, 48h and 7d after
AMI.
4.To establishing HM animal model, implanting the proximal LAD of
juvenile pigs with a 5mm-long and 2.0mm fixed internal diameter Delran
occluder, then housing the animals for 21 weeks. Confirming the MM with
selective left coronary angiography, low dose dobutamine
echocar
引文
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