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内皮-1预处理对培养乳鼠心肌细胞低氧损伤的保护作用

潘燕霞, 林丽, 袁文俊, 吴国宏, 唐朝枢

第二军医大学生理学教研室，上海200433福建医科大学生理学教研室，福州350004；北京大学医学部生和病理生理学系，北京100083理

摘要

实验观察了 01一1 nmol/ L内皮素一1 (ET-1)预处理对低氧孵育((3 % 02 -5 % C02, 12 h)的培养乳鼠心肌细胞乳酸脱氢酶(LDH)释放量、培养液上清超氧化物歧化酶(SOD)活性以及丙二醛( MDA)含量的影响。用Fluo3/ AM负载培养的心肌细胞，在激光扫描共聚焦显微镜下监测急性低氧的心肌细胞「Ca~(2+)];的变化和ET1预处理对低氧所致「Ca~(2+)];变化的影响。结果如下:(1)心肌细胞低氧孵育12 h后，培养液上清LDH活力和MDA含量较常氧对照组明显升高，分别为43. 33士1.21 U/ L二19.33士1. 03 U/ L和1.71士0. 02 nmol/ L二0.91士0. 03 nmol/ L ( PX0.01)，SOD活性为16. 93士1.11 U/ ml明显低于常氧对照组的33. 48士1.15 U/ ml (P<0.01);0.01一1 nmol/ LET-1预处理呈浓度依赖性抑制低氧培养心肌细胞LDH释放，减少培养液上清MDA含量、提高SOD活性(P<0.01) o (2)低氧灌流后29士1. 5 s ( n = 23)心肌细胞自发性钙瞬变完全终止，[ Ca~(2+) ];升高了107士3.2% ( P<0. 001) ; 0. 01一1 nmol/ L ET-1能明显加快心肌细胞钙瞬变的频率(P<0.01);ET-1预处理后低氧所致钙瞬变终止的时间较单纯低氧组明显推迟，[ Ca~(2+)+];过度升高被明显减轻(P<0.01)。上述结果表明，0.01一1 nmol/ L ET-1预处理可减轻培养乳鼠心肌细胞的低氧损伤和抑制低氧所致「Ca~(2+)];的变化，具有一定的细胞保护作用。

关键词： 内皮素; 低氧; 心肌细胞; 细胞内钙

Preventive effect of endothelia 1 pretreatment on hypoxix induced injury in cultured neonatal rat cardiomyoc尹e

PAN Yan Xia, Lin Li, YUAN Weir Jun, WU Guo-Hong, TANG CHAO-SHU

'Department of Physiology, Second Military Medical University, Shanghai 200433；Department of Physiology, Fujian Medical University, Fuzhou 350004；Department of Physiology and Pathophysiology，Health Sciences Center，Peking University，Beijing 100083

Abstract

This study was designed to observe the effects of endothelixrl(ET-1)pretreatment on hypoxia7induced injury and changes in[Ca~(2+)]，in cultured neonatal rat cardiomyocytes .The activity of lactate dehydrogenase(LDH) and superoxide dis mutase(S OD)，and the content of malondialdehyde
(MDA) in the supernatant were determined in the cultured cardiomyocytes su匀ected to a 12-h hypoxia induced by a 3q一5 % Cq atmosphere at 37℃with or without ET-1 pretreatment.[Ca~(2+)]，was measured with Ca~(2+)sensitive fluorescent probe fluo-3/ AM under a laser scanning confocal microscope .Fluo-
rescence intensity emitted from fluo-3/ AM-loaded cells reflected the concentration of[Ca~(2+)]，.The hypoxia model used in[Ca~(2+)]，measurement was established by continously perfusing cardiomyocytes for 30 min with 95%NZ 5%CO2 saturated D ME M solution containing 1 m mol/ L Na2S2 04.Pretreatment with ET-1 consisted of three cycles of ET-1 perfusion(5 min for each) followed by E T-1一free DME M solution (1 0 min for each) prior to hypoxia .The results showed that(1)after incubation in a 3%02 -5%C02 hypoxic atmosphere for 1 2 h，the activity of LDH and the content of MDA in the supernatant significant-
1y increase dfrom 19.33士1.03 U/ L to 43. 33士1.21 U/ L and from 0. 91士0. 03 nmol/ L to 1.71士0. 02nmol/ L，respectively，whereas the activity of SOD decreased from 33.48士1.15 U/ ml to 16. 93士1.11U/ ml(P<0. 01)·In hypoxic cardiomyocytes pretreated with 0. 01一1 nmol/ L ET-1，LDH release and
supernatant MDA content were decreased，while SOD activity was enhanced dose-dependently(P<0. 01).(2) The spontaneous calcium transient in cultured cardiomyocytes terminated at 29士1.5 s and [Cat十]，increased by 107士13.2 % during perfusion of hypoxic solution(P<0. 001)at the end of 30 min .ET-1(0. 01一1 nmol/ L) increased the frequency of[Ca~(2+)]，transient in cultured cardiomyocytes in a dose-dependent manner(P<0.01).The termination of[Ca~(2+)]，transient and the elevation of[Cat十]1caused by hypoxia were postponed by pretreatment with 0. 01一1 nmol/ L ET -I(P<0. 01).These results show that pretreatment with 0. 01一1 nmol/ L ET-1 attenuates hypoxia7induced injury and[Cat十]1changes in cultured neonatal cardiomyocytes，indicating a cytoprotective role of ET-1 pretreatment.

Key words: hypoxia;cardiomyocytes;intracellular calcium ;

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引用本文：

潘燕霞, 林丽, 袁文俊, 吴国宏, 唐朝枢. 内皮-1预处理对培养乳鼠心肌细胞低氧损伤的保护作用[J]. 生理学报 2003; 55 (2): .

PAN Yan Xia, Lin Li, YUAN Weir Jun, WU Guo-Hong, TANG CHAO-SHU. Preventive effect of endothelia 1 pretreatment on hypoxix induced injury in cultured neonatal rat cardiomyoc尹e. Acta Physiol Sin 2003; 55 (2): (in Chinese with English abstract).