体外培养高糖、高脂喂养大鼠主动脉平滑肌细胞钙化的研究

目的 检测高糖、高脂喂养Goto-Kakizaki（GK）糖尿病大鼠主动脉平滑肌细胞（SMCs）的血管钙化指标,探讨糖尿病血管钙化的相关机制.方法 高糖、高脂喂养GK及Wistar大鼠2周,同时分离培养两组大鼠的主动脉SMCs,Wistar大鼠SMCs作为对照.通过细胞计数法观察细胞生长状况,以甲基百里香酚蓝比色法测定两组大鼠细胞层及培养上清中钙的含量,实时定量PCR检测两组细胞碱性磷酸酶（ALP）、骨桥蛋白（OPN）、核心结合因子α-1（Cbfα-1）、α-平滑肌肌动蛋白（α-SMA）的基因表达.结果 与Wistar大鼠SMCs相比,GK大鼠SMCs生长速度明显缓慢（F =363.392,P＜0.05）;细胞层钙含量（0.56±0.22） vs.（0.39±0.09）,t=2.47,P＜0.05]明显增加,培养上清中钙含量（0.82±0.22）vs.（1.20±0.17）,t=-22.573,P＜0.05]明显减少.GK大鼠SMCs中ALP （t=12.963,P＜0.05）、OPN（t=8.305,P＜0.05）及Cbfα-1（t=10.109,P＜0.05）的基因表达增加,同时α-SMA（t=-8.219,P＜ 0.05）的基因表达减少.结论 高糖、高脂喂养的GK糖尿病大鼠的主动脉平滑肌细胞易发生钙化.

Calcification of aortic smooth muscle cells from high-sugar and high-fat diet rat cultured in vitro

Objective To investigate the mechanisms of diabetic vascular calcification by examining the markers related to vascular calcification of aortic smooth muscle cells （SMCs） isolated from the thoracic aortas of Goto-Kakizaki（GK） rats fed with high-sugar and high-fat diet.Methods GK and Wistar rats were fed with high-sugar and high-fat diet for two weeks.SMCs were isolated from GK rats and Wistar rats simultaneously,and SMCs from Wistar rats were used as control.The growth of SMCs was observed by cell counting.The calcium contents in SMCs layer and supernatant were measured by methyl thymol blue method.Expression of alkaline phosphatase （ALP）,osteopontin （OPN）,core binding factor α-1 （Cbfα-1）,α-smooth muscle actin （α-SMA） were detected by real-time quantitative PCR.Results Compared with SMCs from Wistar rats,SMCs from GK rats growed slowly significantly （F =363.392,P 〈0.05）,the calcium contents was significantly increased in the cells＇ layer （0.56 ± 0.22） vs.（0.39 ± 0.09）,t =2.47,P 〈0.05],and was significantly reduced in the supernatant （0.82 ± 0.22） vs.（1.20 ± 0.17）,t =-22.573,P 〈0.05] in GK rats group.The expression of ALP （t=12.963,P 〈0.05）,OPN （t=8.305,P 〈0.05）,Cbfα-1 （t =10.109,P 〈0.05） in SMCs from GK rats increased,while expression of α-SMA decreased （t =-8.219,P 〈0.05）.Conclusions SMCs from high-sugar and high-fat fed GK diabetic rats tend to appear calcification.