脂联素通过减轻内质网应激介导的凋亡抑制心肌细胞钙化  

Adiponectin Inhibited Calcification in Cultured Rat Neonatal Cardiomyocytes via Reducing Endoplasmic Reticulum Stress-mediated Apoptosis

作　　者：赵旭静[1] 边云飞[1] 白瑞[1] 王敏[1] 肖传实[2] 

机构地区：[1]山西医科大学第二临床医学院心内科,山西省太原市030001 [2]山西医科大学第一临床医学院心内科,山西省太原市030001

出　　处：《中国动脉硬化杂志》2012年第4期321-326,共6页Chinese Journal of Arteriosclerosis

摘　　要：目的通过培养SD大鼠乳鼠心室肌细胞并建立细胞钙化模型,观察脂联素对心肌细胞钙化的影响及其机制。方法采用Ⅱ型胶原酶消化法原代培养SD大鼠乳鼠心室肌细胞,α肌动蛋白免疫荧光法进行鉴定。选用培养72 h的单层心肌细胞,实验分为3组:对照组、钙化组、脂联素+钙化组。β-甘油磷酸钠和丙酮酸钠联合诱导制备心肌细胞钙化模型,茜素红和Von Kossa染色法对钙化心肌细胞进行鉴定,通过细胞钙含量、碱性磷酸酶活性和骨钙素的测定判断钙化程度。通过乳酸脱氢酶活性测定和流式细胞术检测心肌细胞凋亡。用定量实时聚合酶链反应测定骨保护素及内质网应激指标葡萄糖调节蛋白78、半胱氨酸天冬氨酸蛋白酶12的表达。结果与对照组相比,单纯心肌细胞钙化后,钙含量、碱性磷酸酶活性和骨钙素分泌量显著增加(P<0.01),骨保护素及内质网应激指标葡萄糖调节蛋白78和半胱氨酸天冬氨酸蛋白酶12在mRNA水平表达明显增高,乳酸脱氢酶活性和细胞凋亡率显著增加(P<0.01)。用脂联素进行干预后,可较大程度地逆转上述指标变化,与心肌细胞钙化组相比均具有显著差异(P<0.05),并且呈现一定的浓度依赖性。结论钙化心肌细胞可以诱导心肌细胞内质网应激介导的凋亡;脂联素可促进血管保护因子骨保护素的表达,并通过减轻内质网应激来减轻心肌细胞钙化及凋亡,对心肌细胞有保护作用。Aim To investigate the effects of adiponectin on calcification in cultured cardiomyocytes via reducing endoplasmie retieulum stress-mediated apoptosis in vitro. Methods Primary cultured cardiomyoeytes were obtained from neonatal rats by collagenase II digestion method. The α-actin expression as molecular marker of the cardiomyocytes was observed by immunocytochemistry. The cells cultured for 72 h were used in the experiment and divided into groups randomly： control group, calcified myocardial cells （CMC） group, adiponectin ＋ CMC （3, 10, 20, 30 mg/L） groups. The morphological changes of the cardiomyocytes were observed under phase contracted microscope. CMC was prepared by incubation with β-glycerophosphate （BGP）. Calcification was confirmed by Von Kossa staining and Alizarin Red S. Calcium content and alkaline phosphatases （ALP）. Alkaline phosphatase was measured by spectrophotometric measure- ment of p-nitorphenol release. Osteocalcin was detected by radioimmunoassay. The content of lactate dehydrogenase （LDH） was measured. The cardiomyoeyte apoptosis was detected by flow cytometry. The expression levels of glucose regulated protein 78 （ GRP78 ）/eysteine-containing aspartate-specific proteases-12 （ Caspase-12 ）/osteoprotegerin （OPG） mRNA were detected by quantitative real-time polymerase chain reaction （qRT-PCR）. Results The immunocyto- chemical stain for smooth muscle α-actin confirmed cardiomyocytes phenotype and the multieellular nodules spontaneously appeared in cardiomyocytes culture. ALP activity and the content of calcium and osteocalcin in calcified myocardial cells was increased by 138% and 136. 9% （ all P 〈0. 05）, respectively, compared with control. The apoptotic rate and the levels of LDH was also significantly increased. Compared to untreated cells, the mRNA levels of GRP78 and Caspase-12 increased in CMC, but the mRNA levels of OPG decreased. Adiponectin elevated the level of OPG mRNA, but suppressed ALP activity, the content of calcium, osteocalcin

关 键 词：脂联素 钙化 内质网应激 心肌细胞 凋亡 

分 类 号：R363[医药卫生—病理学]