【作者】 史昀青；

【导师】 王春生；

【作者基本信息】 复旦大学 ， 心脏外科学， 2010， 博士

【摘要】 心房颤动(Atrial Fibrillation,AF)是临床上最为常见的心律失常,其主要表现为≥400次/分钟的不规则电节律所引起的心房各部分快速颤动,它对人类健康的影响不容小觑。不论导致何种并发症,AF均可显著增加患者的致残率和死亡率。它常伴有左心室收缩功能减退和充血性心力衰竭,还可以引起脑血管栓塞。AF可以恶化患者的生活质量,并消耗大量的医疗资源。但由于目前对于AF的发生机制尚不十分清楚,因此目前临床上对AF的治疗尚缺乏有效手段。钙离子通道蛋白是一类由多种不同亚型构成的离子通道家族,广泛存在于各种组织中,其作用涉及生长发育的调控、自律性的调节等多方面。在心肌细胞表面,目前已证实存在的钙离子通道仅有T型及L型,参与了钙稳态的调控。研究发现,无论是临床AF患者还是快速心房起搏(Rapid Atrial Pacing)诱发心房颤动的动物的心房肌细胞中,胞浆内游离钙的水平均明显升高,提示心肌细胞胞浆内钙超负荷(Intracellular Calcium Overloading)可能与AF的发生密切相关。心肌细胞胞浆内的游离Ca2+水平([Ca2+]i)取决于两个因素：一方面是出胞的Ca2+减少,如SERCA／PMCA系统及Na+-Ca2+／H+-Na+交换系统异常时,均可导致肌质网及细胞膜回摄及外排Ca2+减少,从而导致细胞内游离钙水平增高；另一方面是入胞Ca2+增加,入胞的Ca2+有两个来源,一是肌质网表面的钙离子通道(兰利碱受体,1,4,5-三磷酸肌醇受体)释放Ca2+增加,二是细胞膜表面的钙离子通道(心肌表面为T型及L型钙离子通道)介导的细胞外Ca2+入胞增加。由于心房肌细胞膜表面的钙离子通道(T型及L型钙离子通道)是心房肌细胞胞浆内游离ca2+的重要来源之一,因此,研究其在AF时表达和功能的改变对于揭示AF时细胞内钙超负荷形成的机制及作用有着重要的意义。本研究目的是采用多种技术手段研究AF时心房肌细胞膜表面钙离子通道的表达及功能变化,以此阐述细胞膜钙离子通道在AF时心房肌细胞胞浆内钙超负荷形成中的作用。标本是采自于接受开胸心脏手术患者的少量心房肌组织,按照基础疾病类型和心律分组,借助于分子生物学及免疫组织化学方法比较了风湿性AF组患者、风湿性窦性心律患者和正常窦性心律患者之间T型和L型钙离子通道的表达水平,并用激光共聚焦显微镜钙成像技术比较了两种钙离子通道不同心律组患者中对Ca2+通量的影响。目的：研究正常窦律(NSR)、风湿性瓣膜病窦律(RSR)及风湿性瓣膜病房颤(RAF)三组患者右房组织中L型钙离子通道α1C和T型钙离子通道α1G、α1H亚基mRNA丰度及蛋白定位和表达差异。方法：术中获取各组患者(NSR=10,RSR=11,RAF=16)少量右房标本,所有纳入患者在建立体外循环后、灌注心肌停搏液前切取少量心房肌组织(约200mg-400mg),于冰生理盐水中洗净血迹,分为两部分,一部分(大小约200mg)剪切为小块组织后放入无RNA酶的冻存管中,迅速置入液氮中冷冻,随后转入-80℃深低温冰箱保存；剩下组织放入10%中性福尔马林溶液中固定,留作免疫组化染色用。提取总RNA,设计引物,实时荧光定量RT-PCR对各组α1C、α1G及α1H亚基的mRNA丰度相对定量(2-△△Ct法)；提取总蛋白,蛋白免疫印迹法比较三种α1亚基蛋白表达水平；HE染色及免疫组织化学染色比较三组标本的差异。结果：与NSR及RSR组相比,RAF组的α1C及α1H的mRNA丰度及蛋白水平显著上调(P<0.05),α1G表达水平三组间差异不显著；三种α1亚基在NSR及RSR两组间表达水平无显著差异；HE染色及免疫组化染色发现RAF组较另外两组间质增生明显,核粗大,细胞膜表面通道蛋白重染,RSR组及NSR组则差异不明显。结论：房颤时,α1C及α1H亚基表达水平上调,而α1G亚基表达水平不变；风湿性因素对L及T型钙离子通道的表达并无直接影响,但不能排除其对钙离子通道功能发生影响的可能。目的：1、在分离人单个心房肌细胞的经典方法基础上,探索适合本中心所获得的心房颤动患者心房肌组织的心肌细胞分离方法；2、在分离获得的单个心房肌细胞基础上,借助于激光共聚焦荧光倒置显微镜及钙成像技术,观察心房颤动组及窦率组不同钙离子通道激活前后胞浆内游离Ca2+水平的改变；3、探索成人心房肌活组织切片制备及培养方法,建立一种保留心肌完整结构的多细胞电生理研究模型,为下一步电生理研究作准备。方法：1、改良Bustamante法急性分离成人单个心房肌细胞：所有纳入患者在建立体外循环后、灌注心肌停搏液前切取少量心房肌组织,立即保存于37℃氧饱和的无钙液中,并在5-10min内转运至实验室；将标本切成细小的组织块(<2mm3),在36-37℃下以氧饱和的无钙液冲洗3次；将组织块在含有胶原酶Ⅰ(0.4mg/ml)和蛋白酶XXIV(0.2mg/ml)的无钙液中孵育20-30min,37℃下持续充氧,磁力搅拌器4Hz频率持续搅拌；用无钙液冲洗lmin；将组织块在含有胶原酶Ⅰ(0.4mg/ml)的无钙液中孵育,37℃下持续充氧,磁力搅拌器4Hz频率持续搅拌,每10min镜下观测一次,直至出现单个心肌细胞；将组织块放入KB液中轻轻吹打,所获得的细胞悬液以200目滤网过滤,KB液保存；取KB液保存30-60min的心肌悬液,吹匀,经200目滤网过滤,1000r/min离心5 min,弃上清,按由低到高梯度复钙至1.5mmol/L,吹匀后加入培养皿中,于倒置显微镜下观察。加入台盼蓝(4g/L台盼蓝1份+9份细胞悬液,放置3min),血球计数板分别计数活细胞及死细胞数。2、激光共聚焦荧光倒置显微镜观察T型及L型钙离子通道对Ca2+的通量在房颤组及窦律组的差异：分离获得AF组及对照组单个心房肌细胞,在细胞悬液中加入Thapsigargin(TG,毒胡萝卜内酯)以耗竭肌质网内的储备钙,终浓度1μmol/L,放置10min后,用无钙液洗净；将每例标本所获取的存活心肌细胞悬液分为两份,滴入激光扫描共聚焦荧光显微镜专用培养皿中放置半小时,让其自然贴壁。在培养皿中加入荧光染料Fluo-4/AM(终浓度20p.mol/L),孵育30min,用无钙液洗净细胞膜表面染料,然后细胞外液换用1mmol/L的含钙液,在其中一份的培养皿中加入L型钙通道阻断剂Verapamil(终浓度10μmol/L),另一份加入T型钙通道阻断剂Mibefradil(终浓度1μmol/L),分别孵育20min；在LSCFM下,用氪氩离子激光激发(激发波长494nm,发射波长516nm),观察并记录钙离子通道静息状态下人心房肌细胞内游离Ca2+的荧光强度(OD1)；在培养皿中加入40mmol/L KCl溶液,使细胞膜除极化,激活未被阻断的钙离子通道,记录钙离子通道激活后心肌细胞内游离Ca2+的荧光强度(OD2)；将所测得的激活后细胞内荧光强度除以静息状态下的细胞内荧光强度(OD2/OD1),所得即为统计量。组间t检验比较两组荧光强度比值。3、探索成人心房肌活组织切片制备及培养方法：所有纳入患者在建立体外循环后、灌注心肌停搏液前切取少量心房肌组织,立即保存于37℃氧饱和的无钙液中,并在5-10min内转运至实验室；在标本到达之前,将低熔点琼脂糖粉在70℃水浴下用超纯水溶解成液体(浓度4%),然后保存于45℃水浴中；标本运到后用冷无钙液冲洗干净血迹；将组织块放入标本固定槽中,将液态的琼脂糖溶液倒入槽中,使其没过组织块,并立即在标本固定槽外周放置冰屑,让装置快速冷却,使琼脂糖溶液在极短的时间内凝固并将心房肌组织块包埋其中；小心切除多余琼脂,保留含有心房肌组织的部分；安装好自动组织振动切片机的工作槽及刀片,在槽外填入冰屑,槽内加入无钙液的冰水混合物；用快速黏合剂将包埋有心房肌组织的琼脂糖块固定在物台上,放入切片机内切片,切片厚度为150μm-300μm；将获得的心房肌组织切片4℃下放入含钙液中梯度复钙共计30min,液体持续用100%O2饱和；将复钙后的心房肌组织切片移入高糖DMEM细胞培养液中,37℃水浴下孵育30min,持续用5%CO2+95%O2混合气体饱和液体；孵育后的心肌组织切片放入红外倒置相差显微镜载物台的灌流槽中观察,灌流槽中为100%O2饱和的含钙液(1mmol/L)结果：1、采用改良的Bustamante两步酶消化法,可以分离获得形态呈杆状、横纹清晰、细胞膜完整的具有典型特征的单个人心房肌细胞。复钙后,倒置显微镜下观察可见部分细胞恢复自主收缩活动,完全贴壁后收缩消失,形态无明显变化。经台盼蓝染色后,死细胞被染成淡蓝色,活细胞拒染,细胞存活率约在20%-50%；2、阻滞L型钙通道后,NSR组细胞激动前后OD比值(OD2/OD1)低于RAF组(1.38±0.20 vs 1.88±0.32),阻滞T型钙通道后,NSR组细胞激动前后细胞内钙水平的升高亦低于RAF组(1.43±0.24 vs 2.48±0.40)。通过比较各组在阻断不同钙离子通道后所得的OD比值发现,在NSR组中,阻断两种钙离子通道对细胞内钙离子水平的影响相当,而在RAF组中,阻断T型钙通道对细胞内钙离子水平的影响要大于阻断L型钙通道；3、采用本方法制备所得成人心肌活组织切片心态良好,横纹清晰,间质及闰盘结构清楚,复钙后可见自发性搏动。结论：1、利用改良Bustamante两步酶消化法能够成功分离获得形态学良好单个人心房肌细胞；2、RAF组对比NSR组,其T型钙通道和L型钙通道的钙通量均明显增加,提示其可能参与了心肌细胞内钙超负荷的形成；AF发生后,L型钙通道钙通量的增加明显高于T型钙通道钙通量的增加,提示L型钙通道在AF时心肌细胞内钙超负荷形成中所起的作用较T型钙通道更大；3、采用低熔点琼脂糖包埋及自动组织振动切片机切片培养的方法,能够获得形态良好、具有正常机械活动的心肌活组织切片。创新点：1.首次研究了T型钙离子通道的两个亚型(α1G、α1H)在风湿房颤、风湿窦律及正常窦律患者心房组织中的mRNA丰度及蛋白表达水平,并发现L型钙离子通道(α1C)及T型钙离子通道(α1H)亚型在AF组表达上调。2.首次探讨了T型钙离子通道在AF时钙通量(Ca2+ Influx)的变化,发现T型钙离子通道及L型钙离子通道在AF组钙通量均明显上调。3.首次建立成人心房肌活组织切片的制备和培养方法,该方法能够保留心肌组织正常结构及细胞间联系,是一种良好的多细胞体外研究模型,可用于后续的电生理研究。更多还原

【Abstract】 As the most popular arrhythmia in clinic, atrial fibrillation (AF) manifests over 400 beat-per-minute’s irregular rhythm which causes the fibrillating contraction of atrial myocardium. The effect of AF to public health can not be underestimated. It can increase the disability and mortality, no matter which complications take place. As it is often accompanied by the contractile dysfunction of left ventricle, congestive heart failure and cerebral infarction, AF can deteriorate patients’life quality and expend medical resources. However, there is no efficient treatment to AF, because the mechanism of it is still unclear.Calcium channels, as a big family constituted by different subtype channel proteins, exsit wildly in almost all kinds of tissues and play a part in the control of growing up and autorhythmicity. By now, only T and L-type calcium channels has been found in the sufface of myocardium. It is reported that the intracellular free calcium ion concentration in rapid atrial pacing animal’s atrial myocytes increases, which indicates intracellular calcium overloading may play an important role in AF, and this result has been improved not only in clinic experiments of AF patients atrial myocytes but also in atrial myocytes of rapid atrial pacing animal models.The level of intracellular Ca2+([Ca2+]i) is determined by two pathways:the first is the reduction of Ca2+ efflux when the SERCA/PMCA system and Na+-Ca2+/H+-Na+ exchanger system are abnormal which will reduce the ability of Ca2+ uptaking and excretion; the second is the increasing of the Ca2+ influx which also includes two ways-the calcium channel on the surface of sarcoplasmic reticulum (Ryanodine Receptor and 1,4,5-risphosphate rinositol receptor) and the calcium channel on the surface of cardiomyocytes (T-type and L-type Voltage Dependent Calcium Channels).As an important resource of intracellular calcium, calcium channels on the surface of cardiomyocytes became a hot spot in revealing the mechanism of AF.The aim of this study is to survey and evaluate the expressional and functional changes of calcium channels on the surface of cardiomyocytes in order to explain its mechanism in inducing of intracellular calcium overloading. Atrial myocardial tissues are obtained from different patients who accepted open-chest cardiac operation. Specimens are divided into different groups according to the donor’s heart rhythm and underlaying diseases. Using molecular biological technique, we investigate the abundance of mRNA and the expression level of proteins of T and L-type calcium channel, and we also observe the function of these two channels in Ca2+ influx changing with the help of Laser Scanning Confocal Fluorescence Microscopy and calcium imaging technique. Moreover, we try to establish a method in preparing living atrial myocardium slices of adult human hearts.Objective To investigate whether there are changes in the expression, at mRNA and protein level, ofα1 subunits of L-type (α1C) and T-type (α1G andα1H) calcium channels in the human atrial myocardium among normal sinal rhythm (NSR), rheumatic sinal rhythm (RSR) and rheumatic atrial fibrillation (RAF) patients.Methods The right atrial tissue was obtained from three distinct groups of patients during the cardiac surgeries, and the numbers of the patient were 10 for NSR,11 for RSR, and 16 for RAF, respectively. All specimens (about 200mg-400mg) were obtained before the perfusing of cardioplegic solution when extracorporeal circulation has been built. Tissues were divided into two parts, one was kept in liquid nitrogen and the rest was kept in the solution of formaldehyde (concentration:10%) for immunohistochemistry research. Extract the total RNA, design the pimers and Real-time quantative RT-PCR was used to measure the abundance of mRNA of three different calcium channelα1 subunits (CACNA1C, CACNA1G, CACNA1H) by the 2-ΔΔCt method. Get out the total protein and Western Blotting was also used to examine the expression of these three channel proteins by a semi-quantative method. Immunohistochemistry technique was used to compared the location of these three channel proteins among these groups.Results In comparison with the NSR and RSR groups, the mRNA abundance and protein expression level ofα1C andα1H subunits was significantly up-regulated in the RAF group (P<0.05). However, the level ofα1G subunit was not significantly different among the three groups. Between the NSR and RSR groups, there were no significant differences of the expression of all three subunits. Compared with NSR and RSR groups, Stained preparation of RAF shows mesenchymal hyperplasia, enlargement of nucleus and heavy steined of the surface channel proteins.Conclusions The present study demonstrated that the expression ofα1C andα1H subunits, but notα1G subunit, was up-regulated in atrial fibrillation, suggesting rheumatics may not affect the expression level of L-type and T-type calcium channel. Whether the rheumatics might still change calcium channel functions remains elusive.Objective 1. To explore and modify a proper method in isolating human single atrial myocardial cell depending on the traditional isolating methods.2. To evaluate the intracellular changes of calcium level induced by T and L-type calcium channels between normal sinal rhythm (NSR) group and rheumatic atrial fibrillation (RAF) group with calcium imaging technique of Laser Scanning Confocal Fluorescence Microscope (LSCFM).3. To establish a method of preparing multicellular myocardium model in vitro which may preserve the normal structures of myocardium and the connections among myocardial cells for the next electrophysiological studies.Methods 1. To isolate human single atrial cardiomyocytes by modified Bustamante’ method:All atrial myocardial tissues were obtained before the perfusing of cardioplegic solution when extracorporeal circulation has been built. Tissues were kept in oxygen saturated calcium-free solution in 37℃and sent to laboratory with 5-10 minutes. Tissues were cut into small pieces (less than 2mm3) and washed by oxygen saturated calcium-free solution in 36℃-37℃for three times. Put these tiny tissue blocks into the oxygen saturated calcium-free solution which contains collagenase typeⅠ(0.4mg/ml) and proteinase typeⅩⅩⅣ(0.2mg/ml), and incubate for 20-30 minutes in 37℃with 4Hz stirring. Wash the tissue blocks with calcium-free solution for 1 minute. Then, incubate the tissue blocks in the oxygen saturated calcium-free solution contained collagenase typeⅠ(0.4mg/ml) with 4Hz stirring. Check the solution every 10 minutes under the invert microscope until the single myocardial cells are found. Remove the tissue blocks into oxygen saturated KB solution and keep blowing slightly with pipette. The suspensions were filtered through a 200-mesh screen and kept in KB solution for 30-60 minutes. Take the suspension in keeping and blow it well-distributed, then filter through a 200-mesh screen. Centrifuge the suspension in the speed of 1000 rpm for 5 minutes, remove the supernatant, then recalcificate gradiently to 1.5mmol/L. Blow it well-distributed and check under the invert microscope. Add the Trypan Blue into the suspension (1 part Trypan Blue solution,4g/L+9 parts suspensions) and count the number of viable cells and dead cells.2. To investigate the differences of Ca2+ influx of two types calcium channels between AF group and NSR group:Isolate the single atrial myocardial cells of both groups. Exhausting the calcium storage of sarcoplasmic reticulum (SR) by Thapsigargin (a blocker of SERCA) with the concentration of 1μmol/L for 10 minutes, then washed by calcium-free solution. Divide the suspension of each specimen into two dishes. Add fluorescence dye of Fluo-4/AM (20μmol/L) and incubate togather for 30 minutes, then wash out the dye with calcium-free solution. Replace the incubating solution with recalcification solution (1mmol/L). Add Verapamil (10mmol/L), a kind of L-type calcium channel blocker, into one dish; and add Mibefradil (1μmol/L), a kind of T-type calcium channel blocker, into the other dish for 20 minutes incubation. Put the specimen under the LSCFM. Using a Argon-Krypton ion laser (excitation wave length:494nm; emission wave length:516nm) to activate the fluorescence dye and record the optical density (OD1) within the cells when the surface calcium channels are resting. Depolarize the myocardial cells with 40mmol/L KCl solution, and record the optical density (OD2) within the cells when the surface calcium channels are activated. Statistic is the value of (OD2/OD1). Analyze the differences between two groups with student t test.3. To explore the perparation and culturing of living atrial myocardium slices of adult human hearts:All atrial myocardial tissues were obtained before the perfusing of cardioplegic solution when extracorporeal circulation has been built. Tissues were kept in oxygen saturated calcium-free solution in 37℃and sent to laboratory with 5-10 minutes. Before the specimen arriving, dissolve the low-melting-point agarose (LMP agarose) in ultrapure water (concentration:4%) in 70℃, and keep it under 45℃in water bath. Wash the tissue with cold calcium-free solution to remove the blood, and put it into the chamber on the surface of ice. Pour the LMP agarose solution into the chamer to cover the tissue, and cover the chamber with flake ice to chill it down quickly. Cut off redundant agarose, and keep the agarose block which embed the cardiac tissue. Fix the vibratome correctly and put the flake ice aroud the cutting chamber. Fill the cutting chamber with ice-water mixture of calcium-free solution saturated by pure oxygen. Stick the agarose block to the object stage with adhesive and start slicing (thickness:150μm-300μm). Remove the slices of myocardial tissue into 4℃cold recalcification solution saturated by pure oxygen to recalcificate gradiently for 30 minutes. Then remove the slices carefully into the DMEM solution with high glucose which is saturated by mixed gas of 5% CO2+95%O2 to culture for 30 minutes. Put the slice into the perfusing chamber and observe under the Infrared Differential Interference Contrast Invert Microscope.Results 1. Using the modified Bustamante’s method of two-step enzyme digestion, we can successfully gather qualified adult human single atrial myocardial cells with rod shape, clear transverse striation and intact membrane. After recalcification, some of the single atrial myocardial cells regain the automatic contraction which disappears after complete adherence. Dying by Trypan Blue, the viable cells can not be dyed while the dead cells shows slight blue. Survival rate of cells is 20%-50%.2. After blocking the L-type calcium channel, OD ratio (OD2/OD1) of RAF group is significantly higer than NSR group (1.38±0.20 vs 1.88±0.32); while afer blocking the T-type calcium channel, OD ratio of RAF group is also significantly higer than NSR group (1.43±0.24 vs 2.48±0.40). Compared the OD ratio of each group of blocking L-type calcium channel and T-type calcium channel, it is found that, in NSR group, blocking these two channels makes nearly the same influence of the intracellular calcium changing (1.38±0.20 vs 1.43±0.24), while in RAF group, blocking T-type channel affect the intracellular calcium level much serious than blocking L-type channel (1.88±0.32 vs 2.48±0.40).3. The slices obtained are qualified with clear transverse striation, mesenchyme and intercalated disc structure. After recalcification, some part of the myocardium regain the ablitlity of automatic contraction.Conclusions 1. It is available to obtain qualified adult human single atrial myocardial cells by the modified Bustamante’s method of two-step enzyme digestion.2. Compared to NSR group, the calcium influx of T and L-type calcium channel both increase in RAF group which indicates the function of these two calcium channels is upregulated and may play a role in the intracellular calcium overload. Moreover, in AF patient, the calcium influx enhancement of L-type calcium channel is much higer than T-type calcium channel which indicates that L-type calcium channel makes more important effect in intracellular calcium overloading.3. Using this method of LMP agarose embedding and vibratome slicing, it is available to prepare living atrial myocardium slice of adult human with good morphous and normal electrical mechnial movement.更多还原