1a，25（OH）2D3对心肌细胞生物钟基因表达的影响  

Effect of la,25 （OH）2 D3 on circadian clock gene expression in cardiac myocytes

作　　者：陈玉梅[1] 范燚[1] 李杏[1] 李华[1] 吴丽洁[1] 张奇军[1] 程子杰[1] 钱玲梅[1] 

机构地区：[1]南京医科大学第一附属医院心脏病科,210029

出　　处：《中华实用儿科临床杂志》2016年第13期1013-1016,共4页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（81370278）

摘　　要：目的探讨1a，25（OH），D，对心肌细胞生物钟基因Bmal!mRNA、Per2mRNA和Rev—erbamRNA表达的影响。方法分离培养7H龄SD大鼠乳鼠心肌细胞，并用免疫荧光法鉴定。原代心肌细胞培养72h后，设置1a，25（OH）：D，5个终浓度梯度即0nmol／L、1nmol／L、10nmol／L、50nmol／L和100nmol／L处理心肌细胞2h，然后提取细胞RNA，实时荧光定量PCR（RT—PCR）检测生物钟基因（BmalI、Per2、Rev—erba）表达量变化，以确定1a，25（OH）：D，最佳处理浓度。再将培养72h的原代心肌细胞分为3组，对照组：无血清培养基培养；血清休克组：含体积分数50％马血清的DMEM培养2h；1a，25（OH）2D3处理组：最佳1a，25（OH）2D3浓度培养2h。分别于7个时间点（0h、4h、8h、12h、16h、20h、24h）收集细胞，提取细胞总RNA，再采用RT-PCR分析心肌细胞生物钟基因（Bmall，Per2，Rev—erba）的转录水平。结果1a，25（OH）：D，培养浓度为50nmol／L时，BmallmRNA表达水平最高，Per2和Rev—erbamRNA表达水平最低。与对照组相比，1a，25（OH）：D，处理组和血清休克组均引起心肌细胞Bmall、Per2和Rev—erba基因呈日周期的节律振荡，且Bmall和Per2的表达模式呈相反的时相表达，在12h时Bmall的表达量出现在波峰，而nr2的表达量出现在波谷。Rev—erba的表达变化趋势在8h开始上升，在12～16h出现最高表达量。结论1a，25（OH）2D3可影响心肌细胞生物钟基因Bmall、nr2和Rev—erbamRNA表达。Objective To explore the effect of la,25 （OH）2 D3 on circadian clock gene expressions in cardiac myocytes. Methods Cultured cardiac myocytes isolated from 7 - day - old Sprague - Dawley（SD） rats were identified by immunofluorescence. The medium including 1 a, 25 （ OH ） 2 D3 （ final concentrations were 0 nmol/L, 1 nmo]/L, 10 nmol/L,50 nmol/L and 100 nmol/L） were added to primary myocardial cells to culture for 2 h and then total RNA was extracted. Real - time polymerase chain reaction （ RT - PCR） was applied to analyze myocardial cells circadian clock gene （ Bmall, Per2, Rev - erba） transcript levels to determine optimum concentration of 1 a, 25 （ OH ） 2 D3. Then, the primary myocardial cells cultured for 72 h were divided into 3 groups ： the control group was of serum - free culture medium ; serum shock group was of DMEM containing 50% volume fraction of horse serum cultured 2 h ; 1 a, 25 （OH） 2 D3 treatment group receiving l a, 25 （OH）2D3 at optimal concentration cultured 2 h. The cells were collected at 7 time points （0 h ,4 h,8 h, 12 h, 16 h ,20 h ,24 h） and then total RNA was extracted. RT- PC R was applied to analyze circa- dian clock gene （Bmall ,Per2 ,Rev - erba） transcript levels in the myocardial cells. Results In the presence of 50 nmol/L 1 a,25 （OH）2D3, the Bmall mRNA expression showed the highest level, but the Per2 and Rev -erba mRNA expression levels were minimum. Compared with the control group, both l a, 25 （OH）2D3 treatment group and serum shock group caused day- cycle rhythmic oscillation in circadian clock genes （Bmall, Per2, Rev -erba） in the cardiac myocytes. And the expressions pattern of Bmall and Per2 genes were in the opposite phase. While Bmall gene expres- sion appeared at peak at 12 h,Per2 gene expression appeared in a trough. Expression of Rev -erba gene trend began to rise at 8 h, and the highest expression level appeared at 12 - 16 h. Conclusions 1 a, 25 （OH） 2 D3 can affect Bmall, Per2 and Rev - erba mRNA expressions of

关 键 词：1a 25(OH)2D3 心肌细胞 生物钟 生物钟基因 

分 类 号：R54[医药卫生—心血管疾病]