胆碱能α7受体激动剂对烧冲复合伤休克期血管内皮细胞的保护作用及机制研究
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摘要
目的烧冲复合伤引起的低血容量休克,是伤员发生早期死亡和后期并发症的主要原因之一,而血管内皮细胞损伤则是休克期血流动力学紊乱和脏器功能损害的病理生理基础。本文拟采用重度烧冲复合伤犬和犬脐静脉血管内皮细胞为模型,研究胆碱能N样α7受体激动剂对犬烧冲复合伤血管内皮细胞的保护作用及其机制,为临床烧冲复合伤早期抗休克治疗提供新的思路和实验依据。
方法一：1.选取Beagle犬,采用黑索金爆炸致冲击伤,而后用凝固汽油燃烧造成35%TBSAⅢ°.烧伤,建立中度冲击伤+重度烧伤的犬重度烧冲复合伤模型。2.将实验动物随机化分为：假伤对照组；烧伤组；冲击伤组；烧冲复合伤组；烧冲复合伤不复苏组和烧冲复合伤PNU282987组。测定血流动力学指标、血气、血炎症介质和粘附因子的水平、肝肾功能以及24小时尿量；计算氧输量和氧耗量；测定血浆容量及血管通透性的变化。伤后24h采用放血法处死犬,测定脏器含水量,取肺组织行病理诊断和微血管内皮细胞连接蛋白免疫组化。
方法二：1.取新生犬脐带,采用胶原酶分别消化不同时间,提取脐静脉血管内皮细胞。采用台盼蓝染色进行细胞计数。而后经细胞培养,取第3代细胞进行鉴定,建立犬脐静脉血管内皮细胞提取、培养方法。2.酶消化法提取的犬脐静脉血管内皮细胞,以第3代为实验细胞。给予烧冲复合伤血清刺激,分别给予烟碱和蛋白激酶C、Rho激酶的阻断剂和激动剂干预,测定其单层血管内皮细胞的通透性；分析各组血管内皮细胞连接蛋白以及上游调控因子的表达。
结果一：1.5g黑索金爆炸,通过大体解剖及肺脏组织病理HE染色证实：距离爆炸源50cm的比格犬可被爆炸致中度冲击伤,结合凝固汽油燃烧致35%TBSA重度烧伤,结合上述两步致伤可建立犬重度烧冲复合伤模型。2.烧冲复合伤后血管内皮细胞严重受损,屏障功能失衡,血管通透性增高,组织水肿,血容量骤减,循环系统呈低排高阻,脏器缺血缺氧更为严重；胆碱能α7受体激动剂可以显著减低血管通透性和组织水肿,提高血容量,改善血流动力学和肺通气换气,纠正脏器的缺血缺氧状态。
结果二：1.1%Ⅰ型胶原酶消化7min可从全脐静脉提取大量多角形细胞,免疫荧光染色和流式细胞仪均可以鉴定该细胞为血管内皮细胞。2.烧冲复合伤犬血清刺激血管内皮细胞后,细胞连接蛋白的表达显著下降,其上游的调节蛋白(蛋白激酶C和ROCK1)的表达则显著升高,单层细胞通透性显著提高；而加入烟碱或胆碱能受体激动剂均可以抑制其过高的通透性,降低蛋白激酶C和ROCK1,提高连接蛋白的表达；但给予受体阻断剂或提高蛋白激酶C和Rho激酶的活性后则可以抑制该的效果。
结论烧冲复合伤能造成血管内皮细胞受损,其连接蛋白表达降低,屏障功能失衡,血管通透性增高,组织水肿,血容量骤减,导致休克；胆碱能α7受体激动剂可以通过抑制蛋白激酶C的活性,从而提高内皮细胞连接蛋白的表达,保护内皮屏障功能,显著减低血管通透性和组织水肿,提高血容量,改善血流动力学、肺通气换气功能以及脏器的缺血缺氧状态。
Objective The severe shock of burn-blast combined injury was a major cause of deathin the early stage and an important predisposing factor of complication in the later stage.Vascular endothelial cells (VECs) plays a critical role in the shock. In this study, the nicotinicalpha7acetylcholine receptors (α7NAchR) agonist was used in the resuscitation of shock.The therapeutic effects of resuscitation with the agonist was observed. Then in vitro, theeffects and mechanisms of α7NAchR regulating on the junction proteins of umbilical veinendothelial cells (UVECs) were investigated.
MethodsⅠ：1. A severe burn-blast combined injury animal canine model injuried byhexogen explosion and napalm burning, which was proved by gross anatomy andpathological examination of lung tissue.2. Thirty six canines were randomized to six groups:①Sham group.②Burn group.③Blast group.④Burn-blast combined injury group.⑤Burn-blast combined injury without resuscitation group.⑥Burn-blast combined injury withPNU282987treatment group. Hemodynamics parameters, vascular permeability and PVwere measured. The urine output of24hours was collected. The levels of inflammatorymediators in serum were detected. Canines were sacrificed by phlebotomy at24hours post-injury. Then the visceral water content was measured. Microvascular endothelial cellsconnexin in the lung was examined by pathology and immunohistology
MethodsⅡ：1. The VECs were isolated from these umbilical cords digested bycollagenase A with different time. After cultured, the vascular endothelial cells wereidentified by morphology, immunofluorescence and flow cytometry.2. The UVECs werestimulated bythe serum of burn-blast combined injury canines, and intervened by nicotine,blockers and agonists of protein kinase C or Rho kinase. The permeability of the monolayerof UVECs was measured and the expression of connexin of UVECs and upstream regulatoryfactor were detected by western blotting and immunofluorescence.
ResultⅠ:1. The canine with50cm away from the explosion of hexogen was diagnosedas moderated blast injury by gross anatomy and pathological examination of lung tissue. Asevere burn-blast combined injury animal model was established by the blast injury andsevere burn caused by napalm.2. After burn-blast combined injury, VECs were damaged,which induced the vascular permeability rising, tissue edema, blood volume lowering,circulatory system disequilibrium and organ ischemia and severe hypoxia. The α7NAchRagonist alleviated the vascular permeability and tissue edema, increased the blood volume,enhanced hemodynamics and pulmonary ventilation ventilation function, and improvedorgan ischemia and hypoxia.
ResultⅡ:1. Maximal VECs can be achieved with7-minute digestion.2. After culturedby the serum of canines with burn-blast combined injury, the permeability of the monolayerof UVECs was increased and the expression of connexin was decreased significantly.However, the permeability was reduced and the expression of connexin was increased bynicotines or PNU282987or inhibitor of PKC or Rho kinase, which can be broken down byα-BGT or agonist of PKC or Rho kinase.
Conclusion The VECs were damaged after burn-blast combined injury, and thenexpression of connexin was reduced, barrier functions of VECs were unbalanced, whichled to the increase of vascular permeability, tissue edema, blood volume plummeted, andanoxaemia. By inhibiting protein kinase C, the α7NAchR agonist can increase theexpressions of VECs’connexins, protect its barrier functions, reduce vascular permeabilityand tissue edema significantly, increase blood volume, repair hemodynamics andpulmonary ventilation ventilatory function, and improve the ischemia and hypoxia.
方法一：1.选取Beagle犬,采用黑索金爆炸致冲击伤,而后用凝固汽油燃烧造成35%TBSAⅢ°.烧伤,建立中度冲击伤+重度烧伤的犬重度烧冲复合伤模型。2.将实验动物随机化分为：假伤对照组；烧伤组；冲击伤组；烧冲复合伤组；烧冲复合伤不复苏组和烧冲复合伤PNU282987组。测定血流动力学指标、血气、血炎症介质和粘附因子的水平、肝肾功能以及24小时尿量；计算氧输量和氧耗量；测定血浆容量及血管通透性的变化。伤后24h采用放血法处死犬,测定脏器含水量,取肺组织行病理诊断和微血管内皮细胞连接蛋白免疫组化。
方法二：1.取新生犬脐带,采用胶原酶分别消化不同时间,提取脐静脉血管内皮细胞。采用台盼蓝染色进行细胞计数。而后经细胞培养,取第3代细胞进行鉴定,建立犬脐静脉血管内皮细胞提取、培养方法。2.酶消化法提取的犬脐静脉血管内皮细胞,以第3代为实验细胞。给予烧冲复合伤血清刺激,分别给予烟碱和蛋白激酶C、Rho激酶的阻断剂和激动剂干预,测定其单层血管内皮细胞的通透性；分析各组血管内皮细胞连接蛋白以及上游调控因子的表达。
结果一：1.5g黑索金爆炸,通过大体解剖及肺脏组织病理HE染色证实：距离爆炸源50cm的比格犬可被爆炸致中度冲击伤,结合凝固汽油燃烧致35%TBSA重度烧伤,结合上述两步致伤可建立犬重度烧冲复合伤模型。2.烧冲复合伤后血管内皮细胞严重受损,屏障功能失衡,血管通透性增高,组织水肿,血容量骤减,循环系统呈低排高阻,脏器缺血缺氧更为严重；胆碱能α7受体激动剂可以显著减低血管通透性和组织水肿,提高血容量,改善血流动力学和肺通气换气,纠正脏器的缺血缺氧状态。
结果二：1.1%Ⅰ型胶原酶消化7min可从全脐静脉提取大量多角形细胞,免疫荧光染色和流式细胞仪均可以鉴定该细胞为血管内皮细胞。2.烧冲复合伤犬血清刺激血管内皮细胞后,细胞连接蛋白的表达显著下降,其上游的调节蛋白(蛋白激酶C和ROCK1)的表达则显著升高,单层细胞通透性显著提高；而加入烟碱或胆碱能受体激动剂均可以抑制其过高的通透性,降低蛋白激酶C和ROCK1,提高连接蛋白的表达；但给予受体阻断剂或提高蛋白激酶C和Rho激酶的活性后则可以抑制该的效果。
结论烧冲复合伤能造成血管内皮细胞受损,其连接蛋白表达降低,屏障功能失衡,血管通透性增高,组织水肿,血容量骤减,导致休克；胆碱能α7受体激动剂可以通过抑制蛋白激酶C的活性,从而提高内皮细胞连接蛋白的表达,保护内皮屏障功能,显著减低血管通透性和组织水肿,提高血容量,改善血流动力学、肺通气换气功能以及脏器的缺血缺氧状态。
Objective The severe shock of burn-blast combined injury was a major cause of deathin the early stage and an important predisposing factor of complication in the later stage.Vascular endothelial cells (VECs) plays a critical role in the shock. In this study, the nicotinicalpha7acetylcholine receptors (α7NAchR) agonist was used in the resuscitation of shock.The therapeutic effects of resuscitation with the agonist was observed. Then in vitro, theeffects and mechanisms of α7NAchR regulating on the junction proteins of umbilical veinendothelial cells (UVECs) were investigated.
MethodsⅠ：1. A severe burn-blast combined injury animal canine model injuried byhexogen explosion and napalm burning, which was proved by gross anatomy andpathological examination of lung tissue.2. Thirty six canines were randomized to six groups:①Sham group.②Burn group.③Blast group.④Burn-blast combined injury group.⑤Burn-blast combined injury without resuscitation group.⑥Burn-blast combined injury withPNU282987treatment group. Hemodynamics parameters, vascular permeability and PVwere measured. The urine output of24hours was collected. The levels of inflammatorymediators in serum were detected. Canines were sacrificed by phlebotomy at24hours post-injury. Then the visceral water content was measured. Microvascular endothelial cellsconnexin in the lung was examined by pathology and immunohistology
MethodsⅡ：1. The VECs were isolated from these umbilical cords digested bycollagenase A with different time. After cultured, the vascular endothelial cells wereidentified by morphology, immunofluorescence and flow cytometry.2. The UVECs werestimulated bythe serum of burn-blast combined injury canines, and intervened by nicotine,blockers and agonists of protein kinase C or Rho kinase. The permeability of the monolayerof UVECs was measured and the expression of connexin of UVECs and upstream regulatoryfactor were detected by western blotting and immunofluorescence.
ResultⅠ:1. The canine with50cm away from the explosion of hexogen was diagnosedas moderated blast injury by gross anatomy and pathological examination of lung tissue. Asevere burn-blast combined injury animal model was established by the blast injury andsevere burn caused by napalm.2. After burn-blast combined injury, VECs were damaged,which induced the vascular permeability rising, tissue edema, blood volume lowering,circulatory system disequilibrium and organ ischemia and severe hypoxia. The α7NAchRagonist alleviated the vascular permeability and tissue edema, increased the blood volume,enhanced hemodynamics and pulmonary ventilation ventilation function, and improvedorgan ischemia and hypoxia.
ResultⅡ:1. Maximal VECs can be achieved with7-minute digestion.2. After culturedby the serum of canines with burn-blast combined injury, the permeability of the monolayerof UVECs was increased and the expression of connexin was decreased significantly.However, the permeability was reduced and the expression of connexin was increased bynicotines or PNU282987or inhibitor of PKC or Rho kinase, which can be broken down byα-BGT or agonist of PKC or Rho kinase.
Conclusion The VECs were damaged after burn-blast combined injury, and thenexpression of connexin was reduced, barrier functions of VECs were unbalanced, whichled to the increase of vascular permeability, tissue edema, blood volume plummeted, andanoxaemia. By inhibiting protein kinase C, the α7NAchR agonist can increase theexpressions of VECs’connexins, protect its barrier functions, reduce vascular permeabilityand tissue edema significantly, increase blood volume, repair hemodynamics andpulmonary ventilation ventilatory function, and improve the ischemia and hypoxia.
引文
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