离体大鼠心肌内质网应激模型构建条件的优化

Optimization of the conditions in construction of a rat myocardial model of endoplasmic reticulum stress in vitro

目的探讨构建体外心肌内质网应激(endoplasmic reticulum stress,ERS)模型的方法及实验条件。方法应用Langendorff灌流装置制作大鼠心脏离体缺血/再灌注模型,采用PowerLab系统持续监测血流动力学参数,Western blot检测缺血(停止灌流)不同时间/再灌注120 min后心肌ERS标志性分子糖调节蛋白(GRP)78的表达,并检测C/EBP同源蛋白(CHOP)的表达;逆转录-聚合酶链反应(RT-PCR)检测二者mRNA的表达;体外孵育心肌组织切片,分别应用不同浓度的衣霉素(tunicamycin,Tm)和二硫苏糖醇(dithiothreitol,DTT)处理3 h和6 h,Western-blot检测心肌GRP78及CHOP的表达。结果与对照组相比,离体灌注心脏缺血40 min/再灌注120 min时,心肌GRP78表达最高(P<0.01),CHOP蛋白、GRP78 mRNA及CHOP mRNA表达均明显升高(P<0.01,P<0.05和P<0.05),同时,各项血流动力学参数受损(均P<0.01);Tm 10μg/mL和DTT 2 mmol/L孵育心肌组织切片3 h时,GRP78表达较对照组显著升高(均P<0.001),CHOP表达亦均明显升高(P<0.05和P<0.01)。结论使用离体大鼠心脏缺血/再灌注和孵育心肌组织切片的方法,均可成功构建体外心肌ERS模型。

Objective To investigate the methods and experimental conditions of constructing a rat myocardial model of endoplasmic reticulum stress（ ERS） in vitro. Methods Langendorff perfusion device was used to make rat heart ischemia / reperfusion model in vitro. The left ventricular end-systolic pressure（ LVESP）,left ventricular end-diastolic pressure（ LVEDP）,heart rate（ HR） and the maximum rate of change of left ventricular pressure（ ＋ /- LVdp / dt max）were continuously monitored by a PowerLab system. The expression of classic ERS marker glucose-regulated protein（ GRP） 78 in myocardium was detected by Western blot analysis after ischemia（ stop perfusion） for different times（ 30 min,35 min,40 min and 45 min） / reperfusion 120 min. The expression of C / EBP homologous protein（ CHOP） was detected,too,and the mRNA levels of GRP 78 and CHOP were detected by reverse transcription-polymerase chain reaction（ RT-PCR）. Myocardial tissue slices were incubated in vitro and treated by ERS stimulant tunicamycin（ Tm） or dithiothreitol（ DTT） at different concentrations for 3 h and 6 h,respectively,and the expressions of GRP78 and CHOP were detected by Western blot. Results The highest expression of GRP78 protein was detected in myocardium from the isolated perfused rat heart with ischemia 40 min / reperfusion 120 min（ P 0. 01）. The CHOP protein,GRP78 mRNA and CHOP mRNA were significantly increased（ P 0. 01,P 0. 05,and P 0. 05）,and at the same time,compared with the control group,HR,± LVdp / dt max,coronary perfusion flow（ CPF）,and left ventricular pressure（ △LVP,the difference between LVESP and LVEDP） were decreased,and LVEDP was increased（ P 0. 01 for all）. Compared with the control group,myocardial tissue slices incubated with Tm / DTT for 3 h,the GRP78 levels in Tm 10 μg / mL group and DTT 2 mmol / L group were significantly increased（ both P 0. 001）,and the expression of CHOP was also increased significantly（ P 0. 05 and P 0. 01）. Conclusions Using both isolated rat heart ischemia / reperfusion and myocardial tissue slices incubation methods,the in vitro myocardial ERS model can be successfully constructed.