CAJ | 학술논문

目的观察去甲肾上腺素转运蛋白(NET)和蛋白激酶C结合蛋白1(PICK1)的相互作用,并检测慢性心力衰竭小鼠心脏NET和PICK1 mRNA和蛋白的表达变化.方法 (1)细胞实验:293T细胞分为4组,分别转染质粒NET(NET组)、GFP-PICK1(GFP-PICK1组)、NET+ GFP-PICK1(NET+ GFP-PICK1组)以及NET+ GFP-PICK1 (KD-AA)[NET+ GFP-PICK1 (KD-AA)组],48 h后行免疫荧光化学染色进行亚细胞定位检测.(2)动物实验:雄性C57BL/6J小鼠分为阿霉素组和正常对照组(对照组),每组各20只小鼠,分别于腹腔注射阿霉素(2 mg·kg-1·次-1,累计用量22 mg/kg)和生理盐水.实验第10周末采用实时荧光定量聚合酶链反应和蛋白质免疫印迹法检测心脏NET、PICK1和肾上腺素能受体(β1-AR)的mRNA和蛋白表达水平.结果 (1)PICK1与NET结合并介导NET的亚细胞定位.(2)阿霉素组和对照组小鼠颈交感神经节中NET mRNA的表达量分别为0.59±0.15和0.64±0.18,差异无统计学意义(P＞0.05).但阿霉素组PICK1 mRNA表达量明显低于对照组(0.73 ±0.13比1.18 ±0.15,P＜0.05),且β1-AR的mRNA表达量亦显著低于对照组(0.62 ±0.12比1.00 ±0.11,P＜0.05).(3)阿霉素组小鼠心脏NET蛋白表达量明显低于对照组(0.72±0.04比0.75 ±0.03,P＜0.05),而酪氨酸羟化酶蛋白表达量则显著高于对照组(0.95±0.06比0.84 ±0.07,P＜0.01).(4)阿霉素组和对照组小鼠的心脏交感神经纤维密度无明显差异.结论慢性心力衰竭小鼠心脏NET、PICK1的mRNA和蛋白表达均下调,PICK1介导的NET亚细胞定位障碍可能是心力衰竭时NET功能障碍的机制之一.
목적 관찰거갑신상선소전운단백(NET)화단백격매C결합단백1(PICK1)적상호작용,병검측만성심력쇠갈소서심장NET화PICK1 mRNA화단백적표체변화.방법 (1)세포실험:293T세포분위4조,분별전염질립NET(NET조)、GFP-PICK1(GFP-PICK1조)、NET+ GFP-PICK1(NET+ GFP-PICK1조)이급NET+ GFP-PICK1 (KD-AA)[NET+ GFP-PICK1 (KD-AA)조],48 h후행면역형광화학염색진행아세포정위검측.(2)동물실험:웅성C57BL/6J소서분위아매소조화정상대조조(대조조),매조각20지소서,분별우복강주사아매소(2 mg·kg-1·차-1,루계용량22 mg/kg)화생리염수.실험제10주말채용실시형광정량취합매련반응화단백질면역인적법검측심장NET、PICK1화신상선소능수체(β1-AR)적mRNA화단백표체수평.결과 (1)PICK1여NET결합병개도NET적아세포정위.(2)아매소조화대조조소서경교감신경절중NET mRNA적표체량분별위0.59±0.15화0.64±0.18,차이무통계학의의(P＞0.05).단아매소조PICK1 mRNA표체량명현저우대조조(0.73 ±0.13비1.18 ±0.15,P＜0.05),차β1-AR적mRNA표체량역현저저우대조조(0.62 ±0.12비1.00 ±0.11,P＜0.05).(3)아매소조소서심장NET단백표체량명현저우대조조(0.72±0.04비0.75 ±0.03,P＜0.05),이락안산간화매단백표체량칙현저고우대조조(0.95±0.06비0.84 ±0.07,P＜0.01).(4)아매소조화대조조소서적심장교감신경섬유밀도무명현차이.결론 만성심력쇠갈소서심장NET、PICK1적mRNA화단백표체균하조,PICK1개도적NET아세포정위장애가능시심력쇠갈시NET공능장애적궤제지일.
Objective To investigate the interaction between myocardial norepinephrine (NET) and protein interacting with kinase Cα (PICK1),and examine the myocardial expression pattern of NET and PICK1 in mice with adriamycin-induced congestive heart failure.Methods (1) Cellular experiments: 293T cells were transfected with NET,GFP-PICK1,NET + GFP-PICK1 or NET + GFP-PICK1 (KD-AA),respectively.Immunofluorescence staining was performed 48 h after the transfection.(2) Animal experiments: 40 male C57BL/6J mice were divided into control group and adriamycin group (intraperitoneal injection of 2 mg/kg adriamycin with a cumulative amount of 22 mg/kg).The myocardial mRNA and protein expression level of NET,PICK1 and adrenergic receptor (β1-AR) were detected by real-time PCR and Western blot after 10 weeks.Results (1) PICK1 mediates the intracellular trafficking of NET.(2) Compared to controls,cardiac mRNA expression of NET remained unchanged,but PICK1 and β1-AR mRNA level were significantly reduced in the heart failure mice.(3) Myocardial NET protein expression level was significantly reduced,whereas tyrosine hydroxylase (TH) protein expression was significantly upregulated in heart failure mice.(4) The myocardial density of sympathetic nerve fibers remained unchanged in heart failure mice.Conclusions Cardiac expression of NET and PICK1 are down-regulated in heart failure mice.Reduced PICK1-mediated intracellular trafficking of NET may be involved in the impairment of NET function in this congestive heart failure mice model.