【作者】 李超彦；

【导师】 沈建新；

【作者基本信息】 汕头大学 ， 生理学， 2008， 硕士

【摘要】 前言Ca²⁺作为细胞内最广泛和最重要的第二信使之一,在心肌细胞的兴奋-收缩耦联、物质代谢、细胞周期调控与细胞间通信等活动中发挥重要作用。心肌细胞胞内[Ca²⁺]信号的研究是探索心脏疾病病理生理机制的重要课题之一。鉴于[Ca²⁺])i在生物学上的重要性,研究者建立了多种在细胞和（或）亚细胞水平上对[Ca²⁺]_i活动机制进行观测的方法和技术:如单光子和双光子共聚焦显微镜、全内反射荧光显微镜、宽视野显微镜和低噪CCD照相机的联用等。近十几年来,随着激光共聚焦显微镜技术的发展及钙离子荧光指示剂的更新,其精确性及高分辨率等特征大大提高了人们对心肌胞内Ca²⁺_i信号分子的影像观测及三维重组能力;这为功能学研究提供了方便直观的活的细胞Ca²⁺信号图像,从而人们对细胞内钙信号图像特征及调控机制的认识进一步深入。在本研究中,我们采用LSM510 META型激光共聚焦显微镜,在快速面扫描和线扫描模式下记录胞外低Ca²⁺、胞外高Ca²⁺及β受体激动剂异丙肾上腺素（Isoproterenol, ISO）、阻滞剂普萘洛尔（Propranolol, pro）对心室肌细胞静息钙水平(resting[Ca²⁺])的改变及电场刺激诱发的钙瞬变的影响效应并分析其机制,探讨了胞内钙信号与心肌细胞功能活动（产生钙瞬变和收缩的能力）的关系和变化规律。希望为临床心血管疾病的治疗或防治提供一定的理论依据。目的1.建立激光共聚焦显微镜下心肌胞内钙信号研究模型及方法。2.应用激光共聚焦显微镜成像技术研究胞外低Ca²⁺及高Ca²⁺环境对心肌胞内钙信号的影响及其机制。3.应用激光共聚焦显微镜成像技术研究β受体激动剂及阻滞剂对心肌胞内钙信号的影响。材料和方法选用体重200³00g的成年SD大鼠,使用改良的Langendorff装置,以胶原酶+蛋白酶主动脉逆向灌流法急性分离心室肌单细胞,经过三步梯度复钙法复钙,静置1²h后与荧光指示剂Fluo4-AM孵育染色;置于Zeiss LSM510-META型倒置激光共聚焦显微镜下,由NEC-7203型电子刺激器提供同步阈上电场刺激心肌细胞产生兴奋,诱发胞内钙瞬变;胞内钙信号图像由共聚焦系统实时记录。有关药物通过自制多管道给药系统灌流供给;主要观测不同浓度胞外钙(分别含0.0、0.05、0.1、0.2、0.5、1.0、2.0、5.0、10.0 mmol\L Ca²⁺)、不同浓度β受体激动剂（分别含0.1、0.2、0.5、1.0μmol\L ISO）、β受体激动剂及阻滞剂（1.0μmol\L ISO+4.0μmol\L propranolol）对心肌胞内钙信号（静息钙水平和诱发钙瞬变）的作用。实验设计为自身前后对照。钙信号图象数据的处理及分析使用自编的图像分析处理程序,编程语言为IDL6.2 （Research Systems,Inc.,Boulder,CO）。胞内静息钙水平以荧光强度F0表示。而诱发钙瞬变以标准荧光强度（normalized fluorescence）的净变化即?F/F0表示,其中F0表示静息状态的荧光强度。结果1.采用本科室改进的Langendorff法急性分离的SD大鼠心肌细胞,约有85%-95%的细胞形态呈长杆状,可得40%～60%钙稳态心肌细胞;与钙荧光染料Fluo4-AM孵育后给予阈上场刺激,在共聚焦显微镜下可记录到典型的钙瞬变图像。2.胞外低钙对心肌胞内钙信号的影响:在本研究中,胞外正常灌流液含钙1mM。当胞外钙浓度逐渐变为0.5mM时,发现有3/7诱发钙瞬变变化不明显,2/7诱发钙瞬变明显增强,另有2/7诱发钙瞬变有所减弱;多数（6/7）诱发钙瞬变的时程（从上升相的半高峰处到下降相的半高峰处所持续的时间）有所延长,钙瞬变前沿不整齐现象不明显。当胞外钙浓度逐渐变为0.2mM时,诱发钙瞬变有所增强的约有40%（2/5）,其余的变化不明显;所有诱发钙瞬变的时程均延长（ P<0.05）*（由正常灌流液的222.5+/-36.9ms变为0.2mM时的247.4+/-37.9ms）,钙瞬变前沿均变得不整齐（因为刚开始时有些部位钙内流太弱,不足以触发胞内钙释放;后因为临近部位钙信号的增强,循钙诱导钙释放的原则而引起全细胞范围的钙释放）。当胞外钙浓度逐渐变为0.1mM时,约有一半（3/6）的细胞的诱发钙瞬变明显逐渐减弱,但最终并未完全消失;另有一半（3/6）细胞在灌流开始不久时钙瞬变有所增强且时程延长。当胞外钙浓度逐渐变为0.05mM时,可见开始时诱发钙瞬变有所增强,之后逐渐减弱,但最终胞内仍可见场刺激引起的微弱钙信号存在（10/10）。当胞外钙浓度逐渐变为0mM（无钙台式液）时,心肌细胞的诱发钙瞬变均逐渐减弱,并最终消失（5/5）。总之,随胞外钙浓度从正常情况下的1mM最终降至0mM的过程中,给予心肌细胞阈上场刺激,一般的变化过程是:刚开始（灌流约1分钟时）仍可诱发钙瞬变和细胞收缩,且诱发钙瞬变强度有增强趋势,后诱发钙瞬变前沿变得不整齐;随胞外钙继续降低,诱发钙瞬变减弱且不连续（有些部位有胞内钙释放,有些部位没有）,再往后胞内钙释放逐渐不明显,再往后则只有少量胞外钙内流而无胞内钙被触发释放;当胞外钙浓度为0mM时,诱发钙瞬变消失,心肌细胞失去收缩能力。3.胞外高钙对心肌胞内钙信号的影响:当胞外钙浓度由1mM逐渐变为2mM时,胞内静息钙水平（荧光强度）处理前后没有明显差异;诱发钙瞬变峰值（?F/F0）差别也无统计学意义也。当胞外钙浓度由1mM逐渐变为5mM时,胞内静息钙水平改变处理前后有明显差异（ P<0.05）;诱发钙瞬变峰值（?F/F0）灌流后峰值明显降低（ P<0.05）。当胞外钙浓度由1mM逐渐变为10mM时,胞内静息钙水平处理前后有明显差异（ P<0.05）,且高于胞外钙浓度为5mM时的静息钙水平（ P<0.05）;诱发钙瞬变峰值（?F/F0）灌流后峰值明显降低（ P<0.05）,且低于胞外钙浓度为5mM时的峰值（ P<0.05）。4.β受体激动剂及阻滞剂对心肌胞内钙信号的效应:当ISO为0.1μM时,胞内静息钙水平下降,灌流0.1μM ISO后胞内静息钙水平降低（ P<0.05）;灌流0.1μM ISO后心肌细胞的诱发钙瞬变峰值（?F/F0）升高,前后相对比值为1.24±0.07,可见灌流0.1μM ISO后心肌细胞的诱发钙瞬变峰值显著增大（ P<0.01）。当ISO为0.2μM时,胞内静息钙水平也下降,灌流0.2μM ISO后胞内静息钙水平降低（ P<0.05）;灌流0.2μM ISO后心肌细胞的诱发钙瞬变峰值升高明显,心肌细胞的诱发钙瞬变峰值显著增大（ P<0.01）。当ISO为0.5μM时,胞内静息钙水平降低（ P<0.05）,心肌细胞的诱发钙瞬变峰值显著增大（ P<0.01）。当胞外ISO为1.0μM时,胞内静息钙水平降低（ P<0.05）,诱发钙瞬变峰值增大（ P<0.05）。在另一组实验中,胞内静息钙水平由正常时的12.18±1.04降为1.0μM ISO时的10.53±0.90,再加入4.0μM Propranolol（β-受体阻断剂）后静息钙水平升高至11.62±1.11,洗去胞外ISO及Propranolol后静息钙水平为12.94±1.47（n=7）;可见ISO对胞内静息钙水平的降低作用可被其阻断剂Propranolol所阻断（ P<0.05）;灌流1.0μM ISO后心肌细胞的诱发钙瞬变峰值由正常时的8.40±0.69增强为10.82±0.77,加入4.0μM Propranolol后诱发钙瞬变峰值为7.70±0.43;洗去胞外ISO及Propranolol后诱发钙瞬变峰值为7.17±0.79;,可见ISO对诱发钙瞬变峰值的增强作用也可被其阻断剂Propranolol所阻断（ P<0.05）。结论1.成功建立了激光共聚焦显微镜下心肌胞内钙信号研究模型与方法,获得了一系列胞内钙信号图像,为完整心肌细胞钙信号数字化图谱的有机组成部分。2.胞外低钙环境下诱发钙瞬变幅度下降,但时程延长,并有兴奋-收缩脱耦联发生。3.胞外高钙环境下心肌细胞内静息钙离子荧光信号强度增强提示细胞钙稳定性下降及钙超载;随胞外钙浓度增大诱发钙瞬变相对峰值减小,兴奋-收缩耦联效率降低;并且,前者显然是后者的重要原因。4.较低浓度（0.1μM）的β受体激动剂异丙肾上腺素（ISO）即可使胞内静息钙信号水平显著下降,而诱发钙瞬变峰值明显增大;且ISO的这种效应可被其阻断剂普萘洛尔完全阻断。更多还原

【Abstract】 BackgroundAs the most extensive and important second messenger, intracellular Ca²⁺ plays an important role in cardiac excitation-contraction coupling, substance metabolism, cell cycle regulation, cell communication and other activities. Cardiac myocyte calcium signalling is an in important research field for pathophysiological mechanism of heart diseases. Because of the biological importance of intracellular calcium ([Ca²⁺]i) signaling, lots of scientists tried to establish many different techniques to investigate the cellular or subcellular calcium signalling, such as single-photon and two-photon confocal microscopy, total internal reflection fluorescence microscopy, wide vision microscope and low noise CCD camera and etc. Over the last decade, with the development of the laser scanning confocal microscopy and calcium fluorescent indicators,cardiac calcium imaging and and its three dimension reconstruction were improved greatly with good accuracy and high resolution. Calcium imaging makes cellular function study more conveniently and directly, and enhances our understanding of intracellular signaling greatly.In the present study, we used the laser scanning confocal microscope, by fast-scanning mode and line-scanning mode, to measure the effects of high extracellular Ca²⁺, low extracellular Ca²⁺, isoproterenol （ISO, aβreceptor agonist）, and propranolol （aβreceptor antagonist） on the cardiac myocytes’resting calcium and evoked calcium transients by electrical field stimulation. The mechanisms of the effects （relationship between calcium signaling and cardiac myocytes’function） were discussed. These may be theoretically helpful for the treatment and prevention of cardiovascular diseases.Objectives1. To establish a research model and method for cardiac myocytes’intracellular calcium signaling, using the confocal laser scanning microscopy system.2. To explore the effects of extracellular low Ca²⁺ and high Ca²⁺ on cardiac myocytes’intracellular calcium signals. 3. To investigate the effects ofβreceptor agonists and antagonist on cardiac myocytes’intracellular calcium signals.Materials and MethodsAdult SD rats, weighing 200-300 g, were employed to get single cardiac myocytes acutely, using an improved Langendorff device,reverse-aortic perfusion of collagenase plus protease solution. After a three-step gradient of calcium for calcium recovery, single cardiac myocytes were kept for 1 to 2 h. Then the cells were incubated with Fluo4-AM and placed on the stage of Zeiss510- META inverted laser scanning confocal microscope. A NEC-7203 electronic stimulator was used to provide electrical field stimulation onto cardiac myocytes, working with the confocal system synchronously by a trigger signal. Intracellular calcium transients were activated and recoded by the confocal system simultaneously. Chemical reagents were delivered to the cells by a self-made multi-channel system. Effects of extracellular calcium in different concentration (including 0.0, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mmol \ L Ca²⁺ respectively),βreceptor agonist （including 0.1, 0.2, 0.5, 1.0μmol \ L ISO respectively）、βreceptor agonist and antagonist（1.0μmol\L ISO+4.0μmol\L propranolol） on cardiac myocytes’intracellular calcium signals (including resting [Ca²⁺]i and evoked calcium transients) were observed. Experimental data were collected before, during and after the delivery of chemical reagents from the same single cells. Computer programs for Calcium signal image analysis was coded with IDL6.2 （Research Systems, Inc, Boulder, CO） software. Resting intracellular calcium level was measured by the background fluorescence intensity （F0）. And the evoked calcium transients were measured by normalized fluorescence （F/F0）, Where F0 refers to the present fluorescence signal intensity, and F0 the background fluorescence signal intensity.Results1. By using an improved Langendorff device, about 85% -95% of the single isolated heart cells were long-rod-shaped, among which 40% to 60% were calcium homeostasis cardiac cells. After incubation with fluo4-AM, a calcium fluorescent dye,single cardiac myocytes were triggered by electrical field stimulation and typical calcium transient images were successfully recorded by the confocal microscope system.2. Effects of extracellular low calcium on Myocardial intracellular calcium signalling: In this study, the calcium concentration in normal perfusion of extracellular is 1mM. When extracellular calcium concentration lowered to 0.5 mM, about 3/7 induced calcium transient of the cells did not change significantly, 2/7 induced calcium transient significantly enhanced, and 2/7 induced calcium transient has weakened; majority （6/7） induced calcium transient time-range （up from the peak of the half fell to half of the premises sustained peak time） has extended,the forefront of calcium transient phenomenon didn’tvary obviously.When extracellular calcium concentration has gradually lowered to 0.2 mM, induced calcium transient have increased about 40% （2/5）, and the rest did not change significantly; calcium transients time-range extension （ P<0.05） * （by normal perfusion fluid 222.5 + / -36.9 ms 0.2 mM into the 247.4 + / -37.9 ms）, the calcium transient forefront are becoming vary （because some sites at the beginning of calcium flow is too weak to trigger the release of intracellular calcium; later, near the site of calcium signals enhanced through calcium-induced calcium release from the principle of the whole-cell calcium release）.When extracellular calcium concentration has gradually changed to 0.1 mM, about half （3/6）of the cells induced calcium transient significantly weakened, but in the end did not completely disappear; the other half （3/6） in the beginning of reperfusion the calcium transient was soon enhanced and time-extended.When extracellular calcium concentration has gradually changed to 0.05 mM, at the beginning of that induced calcium transient has been enhanced, then gradually weaken, but in the end still visible intracellular calcium signaling weak presence （10/10）.When extracellularcalcium concentration has gradually changed 0 mM （without calcium Tyrode’s solution）, the myocardial cells induced calcium transient gradually weakened, and eventually disappeared （5/5）.In short, with the extracellular calcium concentration from the normal circumstances of 1 mM to 0 mM final in the process , myocardial cells were gaven over threshold stimulate, the general process of change was: at the beginning （about 1 minute perfusion） calcium transient and cell contraction cound be induced, and induced calcium transient strength showed increasing trend, the forefront of induced calcium transient became varied; along with the decline of extracellular calcium, induced calcium transient weakened without continuous（Some are intracellular calcium release, some not）, then on non-induced calcium transient gradually obviously, until only a few extracellular Ca²⁺without intracellular calcium release was triggered when the extracellular calcium concentration became to 0 mM, induced calcium transient disappeared, myocardial contractility lost.3. Effect of extracellular high calcium on myocardial intracellular calcium signalling:When extracellular calcium concentration has gradually ranged from 1 mM to 2 mM,Resting intracellular calcium levels （fluorescence intensity） has no significant change; calcium transient peak（F/F0）has no significant change also.When extracellular calcium concentration has gradually ranged from 1 mM to 5 mM,Resting intracellular calcium levels change has obvious differences （ P<0.05）; calcium transient peak decline obviously （ P<0.05）. When extracellular calcium concentration has gradually changed from 1 mM to 10 mM,Resting intracellular calcium levelschange has obvious differences（ P<0.05）, and above the resting calcium levels of extracellular calcium concentration of 5 mM （ P<0.05）; calcium transient peak change has obvious decline（ P<0.05）, and below the transient peak of extracellular calcium concentration of 5 mM（ P<0.05）.4. Effect ofβreceptor agonists and antagonist on myocardial intracellular calcium signalling: When the ISO at 0.1μM,Calcium background fluorescence intensity was decreased （ P<0.05）, Before and after perfuse 0.1μM ISO, calcium transient peak ratio was 1.24±0.07, peak value significantly increased（ P<0.01）. When the ISO at 0.2μM,Calcium background fluorescence intensity values were significantly decreased （ P<0.01）, after perfuse 0.1μM ISO calcium transient peak significantly increased （ P<0.01）. When the ISO at 0.5μM,Calcium background fluorescence intensity values were decreased （ P<0.05）, after perfuse 0.5μM ISO calcium transient peak significantly increased also（ P<0.01）.When the ISO at 1.0μM,Calcium background fluorescence intensity values were decreased （ P<0.05）; After perfuse 1.0μM ISO calcium transient peak also increased （ P<0.05）.In another experiment, when the ISO at 1.0μM resting intracellular calcium level reduce from the normal 12.18±1.04 （absolute value of the fluorescence intensity） to 10.53±0.90, after added 4.0μM Propranolol （β-receptor antagonist） resting calcium level increased to 11.62±1.11, then, wash away the extracellular ISO and Propranolol resting level of calcium turn to 12.94±1.47 （n=7）; This shows that the effects of ISO on the resting levels of intracellular calcium can be block by its antagonist （Propranolol） （P <0.05）. After 1.0μM ISO reperfusion, induced calcium transient peak （F/F0） of myocardial cells enhanced from the normal 8.40±0.69 to 10.82±0.77; then adding 4.0μM Propranolol, induced calcium transient peak reduce to 7.70±0.43; and next wash away the extracellular ISO and Propranolol, induced calcium transient peak turn to 7.17±0.79, This shows that the role of ISO induced calcium transient peak enhance may also be blocked by its antagonist Propranolol （ P<0.05）.Conclusions1. A research model and method for confocal imaging of cardiac intracellular calcium signalling was established successfully. A series of intracellular calcium signal images were collected, which will be an important part for the digital atlas of intracellular calcium signals.2. Under the condition of lower extracellular calcium, peak of evoked calcium transients decreased, but the duration of calcium transients prolonged, and some excitation-contraction coupling failure could occur in cardiac myocytes.3. Under the condition of higher extracellular calcium, resting intracellular calcium increased, which implied the declined stability of calcium and calcium overload; with the increase in the concentration of extracellular calcium, peak of evoked calcium transients and efficiency of excitation-contraction coupling decreased. It is obviously that the former is the main cause of the latter.4. Low concentration（0.1μM）ofβreceptor agonist （ISO） could make the resting intracellular calcium signal level decrease significantly, and the evoked calcium transient peak increase significantly; this effect of ISO could be completely blocked by its antagonist propranolol.更多还原