【作者】 陈小波；

【导师】 闵家新；

【作者基本信息】 第三军医大学 ， 外科学， 2011， 硕士

【摘要】 随着道路交通的发展,交通事故已日益成为威胁人类生命安全的世界性公害,全世界的交通事故平均每年造成120万人死亡,中国一年内因车祸丧生的人数已超过十万,成为交通事故多发国家。在交通事故中,单纯胸部创伤与复合伤中的胸部创伤总的发生率超过1/3,是临床仅次于颅脑损伤的第二位外伤致死因素。交通事故中对胸部的高速撞击常造成心脏损伤,钝性胸部创伤（Blunt chest trauma,BCT）所致的心脏损伤临床发生率约8-76%,其中以心肌挫伤（Myocardial contusion,MC）最常见。在临床上,MC因其发病隐秘,且缺乏诊断的“金标准”,特别是在患者合并其他系统的严重伤情时,往往容易忽略心脏损伤的严重性。目前对心脏撞击伤的研究主要针对胸部撞击后心脏损伤的诊断及严重损伤致心脏大血管破裂急救方面的研究,而对撞击后中至重度的心肌挫伤及其继发的心脏功能障碍方面的研究较少。动物实验研究发现中至重度MC后可出现心功能障碍,伤后2-4h尤为明显,至伤后24h左心室收缩压已可恢复至伤前水平,舒张功能的恢复时间还要延长,同时研究还发现,MC后受损的心肌细胞出现钙超载现象,钙超载尤以伤后4⁸h为明显,伤后24h胞内游离钙离子([Ca²⁺]i)浓度仍高于伤前。MC后心脏功能障碍与钙超载的发生机制目前尚不明了。本实验在建立大鼠胸部撞击伤致心脏中至重度损伤动物模型的基础上,应用钙通道阻滞剂地尔硫卓进行干预,观察其在伤后不同时相点减轻细胞钙超载、保护心功能方面的作用,并进一步从细胞骨架结构的破坏方面探讨MC后心功能障碍的发生机制,为临床诊断和治疗此类患者提供理论和实验依据。主要方法:因为MC患者伤情多较重,不适宜行临床研究,本实验应用自制的BIM-Ⅲ小型多功能生物撞击机建立大鼠胸部撞击致中至重度心肌挫伤动物模型,应用大体解剖AIS评分系统+左心室置管心功能检测综合评价致伤效果。于伤后应用钙通道阻滞剂地尔硫卓进行干预,通过监测伤后心功能的变化情况及细胞内[Ca²⁺]i浓度,评价MC后应用地尔硫卓在改善心功能、保护受损心肌方面的作用;应用Western blot和RT-PCR方法对心肌细胞结蛋白及结蛋白mRNA表达进行检测,评估心肌细胞骨架结构的破坏情况,通过伤后用药组与非用药组的比较,进一步探讨其与伤后心肌细胞钙超载及心脏收缩舒张功能障碍发生的关系。主要结果:①应用BIM-Ⅲ小型多功能生物撞击机以220Kpa驱动压驱动弹头对大鼠胸部剑突左侧0.5cm处撞击,所建立的大鼠胸部撞击致心脏损伤动物模型在伤后2h出现心功能下降、血流动力学紊乱,心脏损伤的AIS评分4-5分,符合胸部撞击伤中至重度心肌挫伤的临床特征。②大鼠伤后心肌细胞内[Ca²⁺]i显著升高,伤后4h达峰值后开始下降,但24h后仍高于正常（P<0.05）,伤后应用地尔硫卓（D组）可显著降低伤后4-12h细胞内[Ca²⁺]i浓度;动物致伤初期出现心率下降,而后逐步升高,伤后8h心率最快、24h仍低于正常,D组显著降低动物伤后4-12h心率（P<0.01）;单纯致伤（T组）左心室收缩期末压（LVESP）明显降低,4h达最低值后开始上升,伤后24h恢复至伤前水平, D组LVESP与T组对比无明显变化;T组左心室舒张期末压（LVEDP ）伤后升高,4h达峰值,此后下降,但伤后24h仍未恢复至伤前水平,D组伤后24h降至正常范围。③T组心肌细胞结蛋白的表达降低,伤后8h最低,但伤后24h结蛋白表达增高明显,至伤后48h其表达量高于伤前（P<0.05）;结蛋白mRNA伤后呈逐步增高趋势,伤后24h达峰值,D组结蛋白在伤后8h表达高于T组（P<0.01）、48h低于T组（P<0.05）,结蛋白mRNA在伤后24-48h低于T组（P<0.05）;心肌组织结蛋白免疫组化显示,伤后心肌细胞结蛋白表达在伤后初期呈灶状缺失,且随时间延长其表达降低,但伤后48h结蛋白表达明显增强出现杂乱聚集。结论:①本实验建立的大鼠胸部撞击致心脏损伤动物模型具有模拟伤情稳定,操作简便,可重复性良好等特点;②MC后早期应用钙通道阻滞剂地尔硫卓能显著降低伤后心肌细胞钙超载,有效保护受损心肌细胞,改善心功能;③MC后早期心肌细胞出现明显的结蛋白破坏,且随着伤后胞内[Ca²⁺]i浓度增高,其介导的心肌结蛋白降解增加,通过伤后应用钙通道阻滞剂降低[Ca²⁺]i浓度可有效减缓解伤后结蛋白的降解破坏;结蛋白基因在伤后出现明显的表达增高,结蛋白降解产物是否是上调结蛋白基因表达的原因尚需进一步研究。更多还原

【Abstract】 Background: With the development of road traffic all over the world, traffic accident has been becoming one of the most dangerous threated to human life on the worldwide. More than 1.2 million people on average had passed out owing to traffic accidents every year over the world, and the number of the this in China was more than 100 000, the incidence of simply chest injury and chest injury in combined injuries were over one third of those traffic accidents, so, the chest injury was the second death reason next to traumatic brain injuries. Blunt cardiac injury （BCI）, which was caused by high-speed impact on the chest, could result in heart injuries at the probability of 8-76%, and the myocardial contusion （MC） was the most common kind in those injuries. The results show that cardiac dysfunction occurred after MC, which progress in the most serious on 2-4h after injury, then recovery in 24h, the left ventricular diastolic pressure recover need more time. At the same time, research reports the phenomenon of calcium overload in cardiac cells after injury, which occured most singnificantly on 4-8h, and higher than normal level in 24h after injury, but we had little to know about the Mechanism of cardiac dysfunction and calcium overload after MC. In clinical practices, we also had not a gold standard to definite diagnos MC, especially in some complicated traumatic conditions. So, in this study, we build a new rat model to learn how cardiac injury taken place by blunt chest trauma. With intraperitoneal injection of Diltiazem and observed its effect of protect heart function and reduced the calcium overload in cardiac cells after injury, then further we studied the relationship of desmin degradation and Ca²⁺ overload after the Myocardial Contusion （MC） in aspects of the cytoskeleton dilapidated, and the results could provide some theoretical basis to diagnosis and treat to this kind of patients.Methods: As it is hard to collect cases and to make experiment on those patients for the crisis pathogenesis conditions, we established a model of MC caused by chest impact from BIM-Ⅲ, and we injected Diltiazem, a calcium channel blocker, to intervene the injurie’s progression. After this, we monitored heart function and the variation of intracellular calcium concentration, so, we could evaluated the effect of Diltiazem to protect myocardial cells and improve heart function. Then we detected expressions of myocardial cytoskeletal protein and desmin, through western-blot and RT-PCR, evaluated damage of cytoskeleton. Furthermore, we explored the relationship between the calcium overload in cardiac cells and cardiac dysfunction.Results:①This animal model made by BIM-ⅢWith 220Kpa driving pressure crash animal chest target, which marked by the left of xiphoid. Cardiac dysfunction occurred after injury on this model. Furthermore, the cardiac injury has 4-5 AIS. Death rat had no significant in this model. In thus condition, we can use for subsequent experiments by this animal model.②W e can record increased of Ca²⁺ accumulation intra-cellular Obviously after trauma. The max concentration appeared in 4h after and release later. And higher than 24h after Compared with model group （P<0.05）, Diltiazem can anti-calcium overloading after injury, speeding up the recovery of diastolic function of left ventricular, had no effect in contractile function of left ventricular.③The protein expression of desmin was lower than control group after injury （P<0.01）, diminishing degree of desmin was significant in 8h after injury, the protein were aggregated random in 48h after injury. Decreased free Ca²⁺ content 4h、8h after injury compare with trauma group （P<0.01）, and desmin expression was no significant difference compared with the trauma group after injury 2h、4h, only 8h after injury, Diltiazem Group was higher than trauma group （P<0.05）. Desmin mRNA was overexpression and the highest in 24h after injury, but the expression of trauma group less than Diltiazem group especially 8h after injury.Conclusion:①This animal model with trauma of stable degree can been repeated easily and meet the needs of early pathophysiological study of MC.②Diltiazem reduced cellular calcium overload significantly after MC, and improved heart function and protected damaged myocardial cells.③The desmin disrupt were caused directly by mechanical injury in the early stage of MC, Raised of the intracellular Ca²⁺ after injury could increased desmin protein degradation, reduced Ca²⁺ concentration by Diltiazem after injury could reduce the degradation of desmin damage effectively.更多还原