钙敏感受体参与乳鼠心肌细胞缺氧／复氧损伤的机制研究  

Calcium-sensing receptors induce calcium overload in cultured neonatal rat ventricular cardiomyocytes during anoxia/reoxygenation

作　　者：姜春明[1] 韩钢[1] 米延[1] 徐长庆[2] 韩丽萍[2] 李鸿珠[2] 

机构地区：[1]哈尔滨医科大学第一临床医学院新生儿科,150001 [2]哈尔滨医科大学病理生理学教研室,150086

出　　处：《中国小儿急救医学》2008年第5期445-447,共3页Chinese Pediatric Emergency Medicine

摘　　要：目的观察钙敏感受体（CaSR）对缺氧／复氧（A／R）乳鼠心肌细胞内钙的影响，探讨CaSR参与缺氧／复氧乳鼠心肌细胞损伤的机制及信号传导途径。方法原代培养乳鼠的心肌细胞，建立A／R乳鼠心肌细胞模型（采用95％N2＋5％CO2饱和2h的低糖、无血清DMEM液孵育60min，再更换成正常氧合细胞培养液孵育30min的方法）。心肌细胞随机分为6组：（1）正常对照组；（2）A／R组；（3）A／R＋GdCl3组：在复氧开始时给予GdCl3（CaSR激动剂）300umol／L；（4）A／R＋NiCl2＋CdCl2＋GdCl3组：在复氧时细胞培养液内加入10mmol／L NiCl2（Na^＋、Ca^2＋交换阻断剂）和200umol／L CdCl2（L—Ca^2＋阻断剂）孵育10min，再加入300umol／L GdCl3；（5）A／R＋U73122＋GdCl3组：在复氧时细胞培养液内加入10umol／L磷脂酶C（PLC）阻断剂U73122孵育10min，再加入300umol／L GdCl3；（6）A／R＋thapsigargin＋GdCl3组：在复氧时细胞培养液内加入10umol／L肌浆网三磷酸肌醇（IP3）敏感Ca^2＋泵阻断剂thapsigargin孵育10min，再加入300umol／L GdCl3。应用Fluo-3／AM荧光指示剂负载心肌细胞，激光共聚焦显微镜连续观察细胞内钙荧光强度的动态变化。结果A／R使心肌细胞内钙离子浓度（[Ca^2＋]i）增加，GdCl3使A／R乳鼠心肌细胞[Ca^2＋]进一步增加；NiCl2和CdCl2对GdCl3引起的心肌细胞[Ca^2＋]i增加无影响；而U73122和thapsigargin则抑制了GdCl3引起的心肌细胞[Ca^2＋]i增加。结论CaSR通过磷脂酶C-三磷酸肌醇途径参与了A／R所致心肌细胞钙超载。Objective To observe the influence of calcium-sensing receptor （CaSR） on intracellular calcium and to investigate the mechanism of CaSR induced injury of cultured neonatal rat cardiomyocytes under anoxia/reoxygenation（A/R）. Methods We incubated primarily neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 60 min, and re-incubated cells in normal culture medium for 30 min to establish a model of A/R. The cultured cardiomyocytes were randomly divided into six groups： （ 1 ） control group ; （2） A/ R group; （3） A/R ＋ GdCl3 group： 300 umol/L GdCl3 （a specific CaSR activator） was added to the culture medium at the beginning of reoxygenation; （4） A/R ＋ NiCl2 ＋ CdCl2 ＋ GdCl3 group：at the beginning of reoxygenation, NiCl2 （Na^＋/Ca^2＋ exchanger inhibitor） 10 mmol/L and CdCl2 （L-typecalcium channel blocker） 200umol/L were added to the culture medium prior to GdCl3 （300umol/L） ; （5） A/R ＋ U73122 ＋ GdCl3 group：at the beginning of reoxygenation, cardiomyocytes were preincubated with U73122 10umol/L（ phospholipase C inhibitor） l0 min prior to GdCl3（300umol/L） ; （6） A/R ＋ thapsigargin（TG） ＋ GdCl3 group： at the beginning of reoxygenation, cardiomyocytes were preincubated with thapsigargin （inositolt riphosphate inhibitor） 10umol/L 10 min prior to GdCl3 （300umol/L）. The changes of fluorescent intensities in [ Ca^2＋ ]i were recorded continuously with laser scanning confocal microscopy in different treatments. Results The results showed that A/R increased the intracellular calcium of cardiomyocytes. GdCl3, a specific activator of CaSR, further enhanced cardiomyocytes [Ca^2＋ ]i during A/R. NiCl2 and CdCl2 did not affect the increase in [Ca^2＋ ]i induced by GdCl3. However, U73122 and thapsigargin inhibited the increase in [Ca^2＋ ]i. Conclusion CaSR is involved in A/R-induced calcium overload of neonatal rat cardiomyocytes through PLC-IP3 signaling pathway.

关 键 词：钙敏感受体 缺氧/复氧 心肌细胞 钙 大鼠 

分 类 号：R285.5[医药卫生—中药学]