Junctophilin2对小鼠心肌细胞钙瞬变的影响
发布时间：2018-10-11 07:39
【摘要】：背景介绍膜耦联复合物(junctional membrane complexes,JMCs)是与心肌细胞兴奋收缩耦联相关的耦联细胞表面膜与肌质网膜的细胞结构物质。在心肌细胞中,JMCs是由心肌细胞肌浆膜向内凹陷形成T管,以及肌质网膜形成的“二联体”结构。功能上,JMCs是细胞膜表面膜与细胞肌浆网(sarcoplasmic reticulum/endo reticulum,SR/ER)离子通道之间实现有效交通的结构基础。Junctophilin(JP,JPH)是参与形成JMCs的蛋白分子,在哺乳类动物的可兴奋细胞,JPH的基因JPH1、JPH2、JPH3和JPH4分别编码其4个蛋白亚型:JPH1~JPH4[1]。由JPH2基因编码的蛋白亚型Junctophilin2可促进心肌“二联体”的形成和稳定,也是心肌细胞Ca2+信号系统稳定传导的关键分子[2]。RyR2(ryanodine receptor type 2)是心肌细胞肌质网膜上ryanodine受体(ryanodine receptors,RyRs)的通道亚型。心肌肌质网因为含有大量Ca2+,有心肌细胞内“钙库”之称。RyR2作为心肌肌质网主要的Ca2+释放通道存在[3]。胞浆中Ca2+的产生途径主要是细胞膜上的电压门控Ca2+通道(voltage-gated Ca2+channels,VGCC)介导的细胞外Ca2+内流和肌质网RyRs介导的肌质网内Ca2+的释放[4]。心肌细胞产生动作电位(action potential,AP)使VGCC开放,引起细胞外少量Ca2+内流。内流的Ca2+作为细胞内的信号激活肌质网RyR2通道,从而导致肌质网内贮存的Ca2+大量释放进入胞浆,此过程成为钙离子诱导的钙释放(Ca2+-induced Ca2+release,CICR)。Ca2+是维持和调节心脏生理功能以及体内多种生理过程的重要信号分子。JPH2的敲减引起钙相关的兴奋收缩耦联的破坏,从而导致心肌病,例如心力衰竭。JPH2对细胞内钙离子调控系统起核心作用。目的本研究采用IonOptix光谱检测系统记录培养的单个新生小鼠心肌细胞的钙瞬变;采用siRNA技术构建针对JPH2基因的重组腺病毒载体,研究重组腺病毒介导的JPH2-siRNA对心肌细胞钙瞬变的影响,以此探讨JPH2在心肌细胞Ca2+调控中的作用。方法1.实验所用动物为新生1~3d的C57BL/6小鼠,生产许可证号为SCXK(京)2012-0001,雌雄不限,均购自于北京维通利华公司。2.实验所用带有EGFP标记的重组腺病毒颗粒Ad-JPH2-siRNA及Ad-NC-siRNA(siRNA无关对照腺病毒颗粒)由上海吉凯基因公司包装完成,并放置于-80℃冰箱保存备用。实验分为以下3组:(1)正常对照组(Ctrl-NC),即培养的细胞不加任何处理;(2)无关序列对照组(Ad-NC-siRNA);(3)重组腺病毒组(Ad-JPH2-siRNA)。3.新生1~3d的C57BL/6小鼠心肌细胞常规分离培养。培养的新生小鼠心肌细胞分别感染Ad-NC-siRNA与Ad-JPH2-siRNA重组腺病毒。荧光倒置显微镜观察感染36-48h后的感染结果;4.采用IonOptix光谱检测系统分别对感染腺病毒36h的培养的心肌细胞进行钙瞬变的检测:心肌细胞加入终浓度为2μM的Fura-2/AM,置于37℃培养箱避光孵育30 min,有钙台氏液清洗,局部电场刺激(电压8 V,1.0 Hz,脉宽4 ms)诱发钙瞬变。IonOptix光谱检测系统所用激发波长340/380 nm。5.采用IonWizard 6.6(美国IonOptix公司)软件分析钙瞬变的以下指标:BL:Baseline(RU);Peak h:Peak height(RU);Peak t:Time to peak(s);Dep V:Departure velocity(RU/s);Ret V:Return velocity(RU/s);TP 90%:Time to 90%peak(s);TB 90%:Time to 90%baseline(s)6.钙瞬变数据的分析采用IonWizard 6.6(美国IonOptix公司)。SPSS 21.0软件对相关实验数据进行统计学处理,以均数±标准误(Mean±SEM)表示,同一指标三组间数据的比较用单因素方差分析(One-way ANOVA),两组间比较用双侧t检验,以p0.05为有显著性差异。结果1.培养的新生小鼠心肌细胞在48-72h间长成单层并且形成细胞簇,倒置显微镜下观察到心肌细胞出现自发的强有力的搏动,证明培养的心肌细胞生长状态良好;2.心肌细胞转染Ad-NC-siRNA与Ad-JPH2-siRNA重组腺病毒36h后,荧光显微镜下可观察到心肌细胞大部分均有EGFP表达;3.IonOptix光谱检测系统对培养的单个心肌细胞(EGFP表达阳性)分别进行钙瞬变的测定,Ad-JPH2-siRNA重组腺病毒组与正常对照组(Ctrl-NC)细胞及感染Ad-NC-siRNA无关序列组细胞比较,钙瞬变幅值(Peak height)明显降低;细胞舒张期钙水平(BL)无明显改变。结论siRNA引起JPH2沉默,显著降低心肌细胞钙瞬变,提示膜耦联复合体蛋白与心肌细胞Ca2+稳态密切相关。
[Abstract]:BACKGROUND OF THE INVENTION Membrane coupled complexes (JS1) are cellular structures that are associated with myocyte excitation and contraction coupling and are associated with myenteric membrane. In cardiomyocytes, JS1 was formed by the formation of T-tube and myenteric membrane from the serosa of myocyte myocyte. "diad" Structure. Functionally, JS1 is a structural basis for effective transportation between membrane vesicles and sarcoplaser reticulum/ endo reticulum (SR/ ER) ion channels. JPH1, JPH1, JPH2, JPH3 and JPH4 encode four protein subtypes: JPH1-JPH4[1]. Junctophilin2, a protein subtype encoded by JPH2 gene, can promote myocardial ischemia "diad" The formation and stabilization of myocardial cells is also the key molecule of the stable conduction of Ca2 + signaling system in cardiomyocytes[2]. RyR2 (ryanodine receptor type 2) is a channel subtype of ryanodine receptor (RyRs) on myocyte myocyte membrane. The myocardial sarcoplasmic reticulum contains a large amount of Ca 2 +, and there are myocardial cells. "Calcium Library" It's called. RyR2 was used as the main Ca 2 + releasing channel in myocardial sarcoplasmic reticulum[3]. The pathway of Ca 2 + in cytoplasm is mainly the intracellular Ca 2 + channel mediated by voltage gated Ca 2 + channel (VGCC) on the cell membrane and the release of Ca2 + in muscle cells mediated by muscle-like RyRs[4]. The action potential (AP) of cardiac myocytes opened the VGCC, causing a small amount of Ca 2 + internal flow outside the cells. Ca2 + in the inner stream activates the muscle-like RyR2 pathway as a signal within the cell, resulting in a substantial release of Ca2 + stored in the sarcoplasmic reticulum into the cytoplasm, which is a calcium ion-induced calcium release (Ca2 +-induced Ca2 + release, CICR). Ca2 + is an important signal molecule that maintains and regulates the physiological functions of the heart and various physiological processes in the body. The knock-down of JPH2 results in the destruction of calcium-associated excitation and contraction coupling, leading to cardiomyopathy, such as heart failure. JPH2 plays a central role in intracellular calcium ion regulation system. Objective To investigate the effect of recombinant adenovirus-mediated JPH2-siRNA on myocardial calcium transient in cultured neonatal rat cardiac myocytes using an IonOptix spectrum detection system, and to construct a recombinant adenovirus vector targeting the JPH2 gene by using siRNA technology. In order to investigate the role of JPH2 in myocardial cell Ca 2 + regulation. Method 1. The animals used in the experiment were C57BL/ 6 mice from 1 to 3 days, and the production license number was SCXK (Beijing) 2012-0001, and both sexes were not limited. They were purchased from Beijing WeiTongli Company. The recombinant adenovirus particle Ad-JPH2-siRNA with EGFP tag and Ad-NC-siRNA (siRNA-independent control adenovirus particle) used in the experiment are packaged by Shanghai Jikai gene company and placed at -80 DEG C for standby. The experiment was divided into three groups: (1) Normal control group (Ctrl-NC), i.e. cultured cells were not treated; (2) unrelated sequence control group (Ad-NC-siRNA); (3) recombinant adenovirus group (Ad-JPH2-siRNA). 3. Normal isolation and culture of neonatal C57BL/ 6 mouse cardiac myocytes from newborn 1 to 3d. The cultured neonatal rat cardiomyocytes respectively infect Ad-NC-siRNA and Ad-JPH2-siRNA recombinant adenovirus. The results of infection after 36-48h were observed by fluorescence inversion microscope; 4. Myocardial cells cultured with adenovirus 36h were detected by IonOptix spectrum detection system. The myocardial cells were cultured with Fura-2/ AM at a final concentration of 2. m u.M, incubated at 37 鈩，
本文编号：2263390
[Abstract]:BACKGROUND OF THE INVENTION Membrane coupled complexes (JS1) are cellular structures that are associated with myocyte excitation and contraction coupling and are associated with myenteric membrane. In cardiomyocytes, JS1 was formed by the formation of T-tube and myenteric membrane from the serosa of myocyte myocyte. "diad" Structure. Functionally, JS1 is a structural basis for effective transportation between membrane vesicles and sarcoplaser reticulum/ endo reticulum (SR/ ER) ion channels. JPH1, JPH1, JPH2, JPH3 and JPH4 encode four protein subtypes: JPH1-JPH4[1]. Junctophilin2, a protein subtype encoded by JPH2 gene, can promote myocardial ischemia "diad" The formation and stabilization of myocardial cells is also the key molecule of the stable conduction of Ca2 + signaling system in cardiomyocytes[2]. RyR2 (ryanodine receptor type 2) is a channel subtype of ryanodine receptor (RyRs) on myocyte myocyte membrane. The myocardial sarcoplasmic reticulum contains a large amount of Ca 2 +, and there are myocardial cells. "Calcium Library" It's called. RyR2 was used as the main Ca 2 + releasing channel in myocardial sarcoplasmic reticulum[3]. The pathway of Ca 2 + in cytoplasm is mainly the intracellular Ca 2 + channel mediated by voltage gated Ca 2 + channel (VGCC) on the cell membrane and the release of Ca2 + in muscle cells mediated by muscle-like RyRs[4]. The action potential (AP) of cardiac myocytes opened the VGCC, causing a small amount of Ca 2 + internal flow outside the cells. Ca2 + in the inner stream activates the muscle-like RyR2 pathway as a signal within the cell, resulting in a substantial release of Ca2 + stored in the sarcoplasmic reticulum into the cytoplasm, which is a calcium ion-induced calcium release (Ca2 +-induced Ca2 + release, CICR). Ca2 + is an important signal molecule that maintains and regulates the physiological functions of the heart and various physiological processes in the body. The knock-down of JPH2 results in the destruction of calcium-associated excitation and contraction coupling, leading to cardiomyopathy, such as heart failure. JPH2 plays a central role in intracellular calcium ion regulation system. Objective To investigate the effect of recombinant adenovirus-mediated JPH2-siRNA on myocardial calcium transient in cultured neonatal rat cardiac myocytes using an IonOptix spectrum detection system, and to construct a recombinant adenovirus vector targeting the JPH2 gene by using siRNA technology. In order to investigate the role of JPH2 in myocardial cell Ca 2 + regulation. Method 1. The animals used in the experiment were C57BL/ 6 mice from 1 to 3 days, and the production license number was SCXK (Beijing) 2012-0001, and both sexes were not limited. They were purchased from Beijing WeiTongli Company. The recombinant adenovirus particle Ad-JPH2-siRNA with EGFP tag and Ad-NC-siRNA (siRNA-independent control adenovirus particle) used in the experiment are packaged by Shanghai Jikai gene company and placed at -80 DEG C for standby. The experiment was divided into three groups: (1) Normal control group (Ctrl-NC), i.e. cultured cells were not treated; (2) unrelated sequence control group (Ad-NC-siRNA); (3) recombinant adenovirus group (Ad-JPH2-siRNA). 3. Normal isolation and culture of neonatal C57BL/ 6 mouse cardiac myocytes from newborn 1 to 3d. The cultured neonatal rat cardiomyocytes respectively infect Ad-NC-siRNA and Ad-JPH2-siRNA recombinant adenovirus. The results of infection after 36-48h were observed by fluorescence inversion microscope; 4. Myocardial cells cultured with adenovirus 36h were detected by IonOptix spectrum detection system. The myocardial cells were cultured with Fura-2/ AM at a final concentration of 2. m u.M, incubated at 37 鈩，
本文编号：2263390
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