【摘要】：背景介紹膜耦聯(lián)復(fù)合物(junctional membrane complexes,JMCs)是與心肌細(xì)胞興奮收縮耦聯(lián)相關(guān)的耦聯(lián)細(xì)胞表面膜與肌質(zhì)網(wǎng)膜的細(xì)胞結(jié)構(gòu)物質(zhì)。在心肌細(xì)胞中,JMCs是由心肌細(xì)胞肌漿膜向內(nèi)凹陷形成T管,以及肌質(zhì)網(wǎng)膜形成的“二聯(lián)體”結(jié)構(gòu)。功能上,JMCs是細(xì)胞膜表面膜與細(xì)胞肌漿網(wǎng)(sarcoplasmic reticulum/endo reticulum,SR/ER)離子通道之間實(shí)現(xiàn)有效交通的結(jié)構(gòu)基礎(chǔ)。Junctophilin(JP,JPH)是參與形成JMCs的蛋白分子,在哺乳類動物的可興奮細(xì)胞,JPH的基因JPH1、JPH2、JPH3和JPH4分別編碼其4個(gè)蛋白亞型:JPH1~JPH4[1]。由JPH2基因編碼的蛋白亞型Junctophilin2可促進(jìn)心肌“二聯(lián)體”的形成和穩(wěn)定,也是心肌細(xì)胞Ca2+信號系統(tǒng)穩(wěn)定傳導(dǎo)的關(guān)鍵分子[2]。RyR2(ryanodine receptor type 2)是心肌細(xì)胞肌質(zhì)網(wǎng)膜上ryanodine受體(ryanodine receptors,RyRs)的通道亞型。心肌肌質(zhì)網(wǎng)因?yàn)楹写罅緾a2+,有心肌細(xì)胞內(nèi)“鈣庫”之稱。RyR2作為心肌肌質(zhì)網(wǎng)主要的Ca2+釋放通道存在[3]。胞漿中Ca2+的產(chǎn)生途徑主要是細(xì)胞膜上的電壓門控Ca2+通道(voltage-gated Ca2+channels,VGCC)介導(dǎo)的細(xì)胞外Ca2+內(nèi)流和肌質(zhì)網(wǎng)RyRs介導(dǎo)的肌質(zhì)網(wǎng)內(nèi)Ca2+的釋放[4]。心肌細(xì)胞產(chǎn)生動作電位(action potential,AP)使VGCC開放,引起細(xì)胞外少量Ca2+內(nèi)流。內(nèi)流的Ca2+作為細(xì)胞內(nèi)的信號激活肌質(zhì)網(wǎng)RyR2通道,從而導(dǎo)致肌質(zhì)網(wǎng)內(nèi)貯存的Ca2+大量釋放進(jìn)入胞漿,此過程成為鈣離子誘導(dǎo)的鈣釋放(Ca2+-induced Ca2+release,CICR)。Ca2+是維持和調(diào)節(jié)心臟生理功能以及體內(nèi)多種生理過程的重要信號分子。JPH2的敲減引起鈣相關(guān)的興奮收縮耦聯(lián)的破壞,從而導(dǎo)致心肌病,例如心力衰竭。JPH2對細(xì)胞內(nèi)鈣離子調(diào)控系統(tǒng)起核心作用。目的本研究采用IonOptix光譜檢測系統(tǒng)記錄培養(yǎng)的單個(gè)新生小鼠心肌細(xì)胞的鈣瞬變;采用siRNA技術(shù)構(gòu)建針對JPH2基因的重組腺病毒載體,研究重組腺病毒介導(dǎo)的JPH2-siRNA對心肌細(xì)胞鈣瞬變的影響,以此探討JPH2在心肌細(xì)胞Ca2+調(diào)控中的作用。方法1.實(shí)驗(yàn)所用動物為新生1~3d的C57BL/6小鼠,生產(chǎn)許可證號為SCXK(京)2012-0001,雌雄不限,均購自于北京維通利華公司。2.實(shí)驗(yàn)所用帶有EGFP標(biāo)記的重組腺病毒顆粒Ad-JPH2-siRNA及Ad-NC-siRNA(siRNA無關(guān)對照腺病毒顆粒)由上海吉?jiǎng)P基因公司包裝完成,并放置于-80℃冰箱保存?zhèn)溆�。�?shí)驗(yàn)分為以下3組:(1)正常對照組(Ctrl-NC),即培養(yǎng)的細(xì)胞不加任何處理;(2)無關(guān)序列對照組(Ad-NC-siRNA);(3)重組腺病毒組(Ad-JPH2-siRNA)。3.新生1~3d的C57BL/6小鼠心肌細(xì)胞常規(guī)分離培養(yǎng)。培養(yǎng)的新生小鼠心肌細(xì)胞分別感染Ad-NC-siRNA與Ad-JPH2-siRNA重組腺病毒。熒光倒置顯微鏡觀察感染36-48h后的感染結(jié)果;4.采用IonOptix光譜檢測系統(tǒng)分別對感染腺病毒36h的培養(yǎng)的心肌細(xì)胞進(jìn)行鈣瞬變的檢測:心肌細(xì)胞加入終濃度為2μM的Fura-2/AM,置于37℃培養(yǎng)箱避光孵育30 min,有鈣臺氏液清洗,局部電場刺激(電壓8 V,1.0 Hz,脈寬4 ms)誘發(fā)鈣瞬變。IonOptix光譜檢測系統(tǒng)所用激發(fā)波長340/380 nm。5.采用IonWizard 6.6(美國IonOptix公司)軟件分析鈣瞬變的以下指標(biāo):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.鈣瞬變數(shù)據(jù)的分析采用IonWizard 6.6(美國IonOptix公司)。SPSS 21.0軟件對相關(guān)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)處理,以均數(shù)±標(biāo)準(zhǔn)誤(Mean±SEM)表示,同一指標(biāo)三組間數(shù)據(jù)的比較用單因素方差分析(One-way ANOVA),兩組間比較用雙側(cè)t檢驗(yàn),以p0.05為有顯著性差異。結(jié)果1.培養(yǎng)的新生小鼠心肌細(xì)胞在48-72h間長成單層并且形成細(xì)胞簇,倒置顯微鏡下觀察到心肌細(xì)胞出現(xiàn)自發(fā)的強(qiáng)有力的搏動,證明培養(yǎng)的心肌細(xì)胞生長狀態(tài)良好;2.心肌細(xì)胞轉(zhuǎn)染Ad-NC-siRNA與Ad-JPH2-siRNA重組腺病毒36h后,熒光顯微鏡下可觀察到心肌細(xì)胞大部分均有EGFP表達(dá);3.IonOptix光譜檢測系統(tǒng)對培養(yǎng)的單個(gè)心肌細(xì)胞(EGFP表達(dá)陽性)分別進(jìn)行鈣瞬變的測定,Ad-JPH2-siRNA重組腺病毒組與正常對照組(Ctrl-NC)細(xì)胞及感染Ad-NC-siRNA無關(guān)序列組細(xì)胞比較,鈣瞬變幅值(Peak height)明顯降低;細(xì)胞舒張期鈣水平(BL)無明顯改變。結(jié)論siRNA引起JPH2沉默,顯著降低心肌細(xì)胞鈣瞬變,提示膜耦聯(lián)復(fù)合體蛋白與心肌細(xì)胞Ca2+穩(wěn)態(tài)密切相關(guān)。
[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，

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