冷刺激对乳鼠心肌细胞缝隙连接蛋白43的影响及药物干预的研究
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摘要
目的:在急性冷刺激乳鼠心肌细胞模型下,评价缝隙连接蛋白43(Connexin43,Cx43)的表达及研究急性冷刺激时乳鼠心肌细胞间传导的变化及其机制。方法 :乳鼠心肌细胞原代培养,随机分为正常对照组,实验4℃组;实验0℃组;并在实验4℃组和实验0℃组分别加入100 nmol/L抗心律失常肽(APP)10,每组乳鼠8只。采用流式细胞术(FCM)分析各组细胞的凋亡率,应用聚合酶链反应(PCR)和蛋白免疫印迹法(Western blot)检测Cx43及其磷酸化水平在基因和蛋白上的表达。结果 :FCM结果显示:实验4℃组和实验0℃组随着冷刺激程度加强和低温时间的延长,心肌细胞的凋亡率增加;同时PCR结果显示:实验4℃组和实验0℃组Cx43 m RNA呈现不同程度的下降;Western blot结果显示:实验4℃组和实验0℃组随着刺激强度和低温时间的延长,Cx43蛋白表达亦出现不同程度的下降;磷酸化的Cx43(P-Cx43)蛋白表达亦下降,而AAP10干预后可提高两组Cx43和P-Cx43蛋白的表达。结论 :心肌细胞在急性冷刺激时Cx43在基因和蛋白水平上表达均有不同程度的下降,P-Cx43表达亦下降,而AAP10能提高Cx43和P-Cx43蛋白的表达从而改善细胞间的传导。
Objective: To study the effects of acute cold stress on connexin43(Cx43) protein expression with drug intervention, and cell to cell conduction with its mechanism in neonatal rats' myocardial cells.Methods: The primary neonatal rats' myocardial cell culture was conducted in 4 groups. Group 1, the cells were normally cultured, Group2, the cells were cultured at 4℃, Group 3, the cells were cultured at 0℃ and Group 4, the antiarrhythmia peptide(AAP 10) was added in Group2 and Group 3. The apoptosis rate of myocardial cells was evaluated by flow cytometry assay, m RNA and protein expressions of CX43 were examined by RT-PCR and Western blot analysis, and CX43 phosphorylation product(P-CX43) was detected.Results: Compared with normally cultured cells, the myocardial cell apoptosis rate was obviously increased by acute cold stress at 4℃ and 0℃ with time extension. The m RNA expression of Cx43 was decreased at varying degrees at 4℃ and 0℃ stimulation, the protein expression of Cx43 was decreased at varying degrees at 4℃ and 0℃ stimulation with time extension, and P-Cx43 level was decreased. While the APP 10 intervention may obviously elevate the protein levels of Cx43 and P-Cx43.Conclusion: Acute cold stress could reduce the protein expression of CX43 and P-CX43, while APP 10 intervention may elevate such expression and improve the cell to cell conduction in neonatal rats' myocardial cells.
Objective: To study the effects of acute cold stress on connexin43(Cx43) protein expression with drug intervention, and cell to cell conduction with its mechanism in neonatal rats' myocardial cells.Methods: The primary neonatal rats' myocardial cell culture was conducted in 4 groups. Group 1, the cells were normally cultured, Group2, the cells were cultured at 4℃, Group 3, the cells were cultured at 0℃ and Group 4, the antiarrhythmia peptide(AAP 10) was added in Group2 and Group 3. The apoptosis rate of myocardial cells was evaluated by flow cytometry assay, m RNA and protein expressions of CX43 were examined by RT-PCR and Western blot analysis, and CX43 phosphorylation product(P-CX43) was detected.Results: Compared with normally cultured cells, the myocardial cell apoptosis rate was obviously increased by acute cold stress at 4℃ and 0℃ with time extension. The m RNA expression of Cx43 was decreased at varying degrees at 4℃ and 0℃ stimulation, the protein expression of Cx43 was decreased at varying degrees at 4℃ and 0℃ stimulation with time extension, and P-Cx43 level was decreased. While the APP 10 intervention may obviously elevate the protein levels of Cx43 and P-Cx43.Conclusion: Acute cold stress could reduce the protein expression of CX43 and P-CX43, while APP 10 intervention may elevate such expression and improve the cell to cell conduction in neonatal rats' myocardial cells.
引文
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[2]Israel A,Zavala LE,Cierco M,et al,Effect of AT1 angiotensinⅡreceptor antatonists on the sympathetic response to a cold pressor test in healthy volunteers.Am J Ther,2007,14:183-188.
[3]Peters NS,Coromilas J,Severs NJ,et al.Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia.Circulation,1997,95:988-996.
[4]Gros DB,Jongsma HJ.Connexins in mammalian heart function.Bioessays,1996,18:719-730.
[5]Lampe PD,Lau AP.The effects of connexin phosphorylation on gap junctional communication.Int J Biochem Cell Biol,2004,36,1171-1186.
[6]李凤芝,颜培华,刘友梅,等,低温对培养的血管内皮细胞损伤作用的研究,解放军医学杂志,1994,19:98.
[7]Esch T,Stefano GB,Fricchione GL,et al.Stress-related diseases a potential role for nitric oxide.Med Sci Monit,2002,8:RA103-118.
[8]Dipak K Daz.Heart in stress.Annals of the New York Academy of Science.1999,874:370-378.
[9]Tillinger A,Myslivecek J,NovákováM.Gene expression of adrenoceptors in the hearts of cold-acclimated rats exposed to a novel stressor.Ann NY Acad Sci,2008,48:393-399.
[10]杨俊杰,刘志强,王海滨,等,寒冷刺激诱导棕色脂肪组织中的心脏干细胞增多,中国循环杂志,2011,10:382-385.
[11]Rover R,Dhein S.Structure-activity relationships of novel peptides related to the antiarrhythmic peptide AAP10 which reduce the dispersion of epicardial action potential duration.Peptides,2001,22:1011-1021.
[12]Weng S,Lauven M,Schaefer T,et al.Pharmacological modification of gap junction coupling by an antiarhythmic peptide via protein kinase C activation.FASEB J,2002,16:1114-1116.
[13]孙冰,齐向强,孙宝贵,等,缝隙连接阻滞剂预防缺血性室性心律失常及其机制的研究,中国循环杂志,2005,8:260-263.