摘要
背景和目的:病态窦房结综合征(简称病窦综合征)是临床常见疾病,对人类健康危害很大。目前病窦综合征确切的发病机理尚不清楚,探讨病窦综合征的发病机制,寻求可能的预防及根治措施,是心血管疾病领域中研究的重要内容之一。缝隙连接是心肌细胞间特化的连接方式之一,而缝隙连接蛋白(connexin, Cx)是构成细胞缝隙连接的分子基础,心肌组织中存在大量不同种类的Cx,其介导了心肌细胞间电冲动及化学信号的传递,保证不同心肌细胞间电、机械偶联正常进行,从而维持正常的心脏功能。哺乳动物心脏组织中Cx主要有Cx45、Cx43及Cx40三种。研究表明,Cx的病变与心脏疾病尤其是心律失常关系密切,但有关兔窦房结组织损伤后细胞Cx的表达如何?目前尚未见文献报道。本研究用甲醛湿敷兔窦房结组织后,观察兔窦房结电生理功能变化、细胞形态结构病变及细胞Cx45、Cx43表达的变化规律,探讨用甲醛湿敷法建立兔窦房结组织急性损伤模型的可行性。
方法:取健康成年兔60只,随机分为对照组、损伤后2h、1w、2w、3w及4w组(每组10只)。麻醉后固定,从胸骨正中偏右2～3mm开胸,暴露心脏,用棉签浸湿20%甲醛(对照组用生理盐水)后湿敷兔窦房结区,建立兔窦房结组织急性损伤模型。自麻醉后至实验结束持续观测心率(heart rate, HR)及心律变化;对照组、损伤后2h组兔在模型建立后2h而其余实验组在相应时间点再次麻醉后,经右颈外静脉插入自制3F四极电极导管至右心房,行窦房结功能测定(SNRT、CSNRT及SACT)。电生理检查完毕即处死动物快速切取窦房结组织并分别固定,在光镜、电镜下观察窦房结细胞形态结构的变化,用TUNEL法检测窦房结细胞凋亡情况,用免疫组化SP法检测窦房结细胞Cx45、Cx43的表达。
结果:
1. 对照组兔窦房结组织经生理盐水湿敷前、后其HR及心律均无明显变化。各实验组兔窦房结组织经甲醛湿敷后,HR均明显下降,且与对照组相比有显著性差异(P<0.05);实验组中有80%兔于湿敷后出现窦性停搏、交界性逸搏心律及心房颤动。
2. 实验组兔窦房结组织经甲醛湿敷后测量SNRT、CSNRT及SACT,与对照组比
较,均明显延长,有显著性差异(P<0.05)。
3. 实验组兔窦房结组织经甲醛湿敷后,光镜下观察:早期可见窦房结细胞肿胀、粒细胞浸润及局灶性坏死,后期则可见部分胶原纤维组织增生。电镜下观察:可见窦房结细胞基质疏松变淡,线粒体肿胀及嵴变短、变少甚至消失,内质网解体、离断及空泡变,核膜极不规整,肌原纤维断裂及核仁裂解。
4. 对照组兔窦房结组织中仅见少量的TUNEL阳性细胞。各实验组兔窦房结组织中均见较多的TUNEL阳性细胞,其细胞凋亡率与对照组相比有显著性差异(P<0.01)。
5. 兔窦房结细胞Cx45的表达主要位于窦房结中央区细胞膜表面,表达的Cx45呈点状散乱分布;而兔窦房结细胞Cx43的表达位于窦房结周边区心房肌细胞闰盘处,窦房结中央区细胞则未见其表达。
6.各实验组兔窦房结细胞Cx45的IOD值与对照组相比均显著性减少(P<0.05);各实验组兔窦房结周边区心房肌细胞Cx43的IOD值与对照组相比则均无显著性差异(P>0.05)。
结论:
1. 采用甲醛湿敷法建立兔窦房结组织急性损伤模型方法可靠,操作简便,重复性好,可以满足实验要求。
2. 用甲醛湿敷兔窦房结区可致窦房结电生理功能发生持续性改变,心电图表现为HR减慢、窦性停搏伴交界性逸搏心律及心房颤动等。此特点与病窦综合征患者的心电图改变相似。
3. 甲醛湿敷兔窦房结组织所致的窦房结损伤范围局限,且为不可逆性损伤,早期以细胞坏死及细胞凋亡为主,后期主要表现为纤维组织增生,同时伴有Cx45表达下降,这些改变系兔窦房结电生理异常的病理学基础。
4. 用甲醛湿敷兔窦房结组织所致的电生理功能改变与细胞形态学异常具有良好的相关性,该方法已形成兔窦房结细胞慢性持续性损伤,是较理想的病窦综合征动物模型方法。
Background and objective: Sick sinoatrial node syndrome (SSS) is a common cardiovascular disease which is greatly harmful to human health. To elucidate the pathogenesis of SSS and to find out the possible measures of prevention and radical cure is important in cardiovascular field. Gap junction whose molecular basis is made up of connexin (Cx) is one of the specialized intercellular connection manners. There are many kinds of Cxs in myocardial cells which are crucial for guiding electric and chemical signals amongst myocardial cells and guarantee normal electric-machinery coupling to maintain normal cardiac function. There are different connexins in different mammals as well as one of mammal with different connexins. There are three kinds of connexins (Cx45, Cx43 and Cx40) in mammalian heart. Up to date, there are many reports on the lesions of connexins in myocardial diseases especially arrhythmias, but there are few reports on connexin changes in rabbit sinoatrial node cells when damaged. The purpose of our investigation is to observe the dynamic regulation of electrophysiologic function, cell morphologic changes and expression of connexin of rabbit as damaged by formaldehyde wet dressing method, and discuss the feasibility of establishing the acute damage model of rabbit sinoatrial node by formaldehyde wet dressing method.
Methods: Sixty rabbits were randomly divided into 6 groups (10 for each group): the control group and groups of after operation 2h, 1w, 2w, 3w and 4w. The rabbits were anesthetized with sodium pentobarbital and the chest was opened through the right lateral line away 2 to 3 millimeters from the sternum. The sinoatrial node area of rabbits was damaged by 20% formaldehyde wet dressing in the experimental groups and by saline wet dressing in the control group. 3F tetra-electrode catheter was inserted into the right atrium through right external jugular vein in the control group and 2h group after wet dressing for 2h, in the other groups at the corresponding times of re-operation respectively; electrocardiogram of body surface and endocardium were recorded synchronically; then the right atrium was stimulated, and sinus node recovery time (SNRT), corrected SNRT and sino-atrial conduction time (SACT) were measured respectively. The rabbits were sacrificed
after electrophysiological study and the tissue of SAN was cut and fixed respectively. Light and electronic microscope was used to examine the pathological changes of SAN cells. Apoptosis of sinoatrial node cells was detected by TUNEL staining and expression of Cx45 and Cx43 detected by immunohistochemical staining.
Results:
1. The heart rate (HR) and cardiac rhythm of the control group dressed by saline didn’t change distinctly while that of the experimental groups dressed by formaldehyde were slower than before dressing. The change of HR in experimental groups was significant compared to that of control group (P<0.05). Sinus arrest, junctional escape rhythm and atrial fibrillation were present in 80% rabbits of experimental groups.
2. The SNRT、corrected SNRT and SACT of experimental groups were all significantly longer than those of the control group (P<0.05) .
3. There were cell swelling, granulocytic cell infiltration and focal necrosis found by light microscopy in experimental groups at the early stage, and late part of collagen fiber proliferation was found. Under electron microscope, the sinoatrial nodal matrix became sparse and lightly-stained, and mitochondria swelling, cristae disorganization or break, endoplasmic reticulum disintegration or break as well as the nucleolus splitting was also found in experimental groups.
4. There were few TUNEL positive cells in sinoatrial node of rabbits in the control group while there were lots of TUNEL positive cells in sinoatrial nodes of all experimental groups. The ratios of apoptosis in all experimental groups were significantly higher than that of the control group (P<0.01).
5. The Cx45 of sinoatrial node cells of rabbits expressed in the center of the sinoatrial nodal area wa
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