枸杞糖肽对缺氧乳鼠心肌细胞内游离钙浓度的影响

Effects of lycium barbarum glycopeptide on the intracellular free calcium concentration of cardiomyocytes in neonatal hypoxia mice

目的:研究低氧心肌细胞内游离钙(犤Ca2+犦i)浓度的变化,枸杞糖肽减轻缺氧诱导的心肌细胞钙超载以及对缺氧心肌的保护作用。方法:实验选用出生两三天的SD乳大鼠80只进行心肌细胞培养,建立心肌缺血模型,实验分3组:对照组;缺氧组:细胞缺氧6h;枸杞糖肽组:先加入终浓度为50mg/L的枸杞多糖,再行缺氧6h。以Fluo/AM荧光指示剂负载,通过激光扫描共聚焦显微系统和Flou-3/AM荧光指示剂标记技术,观察枸杞糖肽对缺氧心肌细胞游离钙含量的影响。结果:心肌细胞经缺氧处理6h后,细胞之间相互连接减少,大部分细胞肿胀,折光性减弱,部分细胞甚至呈圆形,少部分细胞胞膜破裂,提示心肌细胞损伤。枸杞糖肽处理组心肌细胞经缺氧后,可见部分细胞肿胀,内有颗粒,折光性减弱,但较缺氧模型组有一定的改善作用。心肌细胞Fluo-3/AM负载后在激光共聚焦显微镜下观察,细胞的分布和外形与倒置显微镜下所见相符,呈不规则棱形、多角形、星形细胞,轮廓清晰,成群的细胞出现自发性,同步性、有节律的搏动。经Flu3-AM标记后,细胞出现规律性闪烁。对照组心肌细胞犤Ca2+犦i荧光强度和吸光度(A值)较低,缺氧6h荧光强度明显增加(P<0.05);枸杞糖肽组心肌细胞犤Ca2+犦i荧光密度(62.86±28.71)低于缺氧组(156.76±55.39)(P<0.01)。

AIM:To study the changes of concentration of intracellular free calcium i) under hypoxia,and investigate the relieving effect of lycium barbarum glycopeptide(LbGp) on myocardial calcium overload induced by hypoxia and the protective effects of LbGp on the hypoxic cardiomyocytes. METHODS:Eighty neonatal rats of 2 or 3 days old were selected for culture of cardiomyocytes,and then made into models of myocardial ischemia. The rats were divided into control group, hypoxia group (cardiomyocytes under hypoxia for 6 hours) and LbGp group(50 mg/L LbGp was added,then hypoxia for 6 hours).The(i concentration of cardiomyocytes induced by ischemia was observed with the laser confocal microscope and the calcium fluorescent probe Flou 3/AM after loaded with Fluo/AM fluorescent indicator. RESULTS:After 6 hour hypoxia, there were fewer connection between cardiomyocytes,most of the cells became swelling,refraction was weaken,part of the cells even turned round, and the cellular membrane of a few cells were broken, indicating that there was myocardial injury.After hypoxia, cardiomyocytes in the LbGp group became swelling and had particles inside,refraction was weaken,but had the role of amelioration as compared with the hypoxia model group.The results observed under laser confocal microscope after loaded with Fluo/AM showed that the distribution and outline form of cells were coincident with those observed under inverted microscope:the cells appeared irregular edges and multi angles,astrocyte, clear outline,spontaneous,synchronic and rhythmic beats were observed in the clustering cardiomyocytes.After Flu3 AM labeling,the cells appeared regular sparkling.The myocardial i fluorescent intensity and absorbance(A) in the control group were lower, and the fluorescent intensity after hypoxia for 6 hours was obviously increased(P< 0.05); the myocardiali fluorescent density was significantly lower in the LbGp group(62.86 ± 28.71) than in the hypoxia group(156.76± 55.39) (P< 0.01). CONCLUSION: LbGp can relieve the calcium overload induced by hypoxia.