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
发布时间：2018-10-26 13:31
【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。
[Abstract]:Objective To investigate the causes of low blood volume shock caused by compound injury, which is one of the main causes of early death and late complications of the wounded, while vascular endothelial cell injury is the pathophysiological basis of hemodynamic disturbance and organ dysfunction in shock phase. In this paper, we study the protective effect and mechanism of cholinergic N-like substance 7 receptor agonist on vascular endothelial cells in dogs by using severe burn-in compound injury dog and canine umbilical vein endothelial cell as model. To provide new thought and experimental basis for early anti-shock treatment of clinical burn-in compound injury. Method I: 1. Beaver dog was selected, shock injury was induced by black cord gold explosion, and then 35% TBSA was caused by napalm combustion A model of severe burn-and-shoot composite injury in dogs with moderate impact injury and severe burn.. 2. Randomization of experimental animals was divided into two groups: sham injury control group, burn group, impact injury group, burn-in compound injury group, burn-in composite injury non-resuscitation group and burn-in composite injury PNU28298. 7. Determination of blood flow dynamics index, blood gas, blood inflammatory medium and adhesion factor levels, liver and kidney function and 24-hour blood flow rate; calculate oxygen loss and oxygen consumption; determine plasma volume and vascular permeability. 24 h after injury, the dog was sacrificed by exsanguination, the water content of organs was determined, the pathological diagnosis of lung tissue and the expression of connexin in microvascular endothelial cells were measured. packetization. Methods: 1. Fresh dog umbilical cord was taken and different time was digested with collagenase to extract umbilical vein blood. Endovascular endothelial cells stained with trypan blue performing cell culture, performing identification on the third generation cell, and establishing canine umbilical vein endothelial cell extraction and culture; Method 2. Endovascular endothelial cells of canine umbilical vein extracted by enzymatic digestion, for 3rd generation To determine the permeability of monolayer vascular endothelial cells, the connexin and upstream regulation of vascular endothelial cells in each group were analyzed. Results One: 1. 5g of black cord gold exploded, confirmed by gross anatomy and pathological HE staining of lung tissue: the dogs with a distance of 50cm from the explosion source could be struck with moderate shock, combined with 35% TBSA severe burns due to the burning of napalm, combined with the two-step injury to establish severe burn in dogs. The vascular endothelial cells were severely damaged, the barrier function was unbalanced, the vascular permeability was increased, the tissue edema and the volume of blood were decreased, and the circulatory system was in a low-row and high resistance. Ischemia and hypoxia are more serious; the cholinergic receptor 7 receptor agonist can significantly reduce vascular permeability and tissue edema, improve blood volume, improve hemodynamics and pulmonary ventilation, and correct the contamination. The results are as follows: 1. 1% collagenase digestion for 7min can extract a large amount of polygonal cells from the whole umbilical vein, and the immunofluorescent staining and flow cytometry can be used for identification. Cells were vascular endothelial cells. The expression of connexin was significantly decreased following the stimulation of vascular endothelial cells in canine serum with burn-and-shoot composite injured dogs, and the upstream regulatory proteins (protein kinase C and ROCK1) increased significantly. Addition of nicotine or cholinergic receptor agonists can inhibit their hyperpermeability, decrease protein kinase C and ROCK1, and increase the expression of connexin, but give receptor blockers or improve the activity of protein kinase C and Rho kinase. Conclusion It can be used to inhibit the damage of vascular endothelial cells, the expression of connexin, the imbalance of barrier function, the increase of vascular permeability, the edema of tissue, the sudden decrease of blood volume, and the cause of shock. can inhibit the activity of protein kinase C, thereby improving the expression of connexin in endothelial cells, protecting endothelial barrier function, remarkably reducing vascular permeability and tissue edema, improving blood volume, improving hemodynamics, and pulmonary ventilation.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R644
本文编号：2295875
[Abstract]:Objective To investigate the causes of low blood volume shock caused by compound injury, which is one of the main causes of early death and late complications of the wounded, while vascular endothelial cell injury is the pathophysiological basis of hemodynamic disturbance and organ dysfunction in shock phase. In this paper, we study the protective effect and mechanism of cholinergic N-like substance 7 receptor agonist on vascular endothelial cells in dogs by using severe burn-in compound injury dog and canine umbilical vein endothelial cell as model. To provide new thought and experimental basis for early anti-shock treatment of clinical burn-in compound injury. Method I: 1. Beaver dog was selected, shock injury was induced by black cord gold explosion, and then 35% TBSA was caused by napalm combustion A model of severe burn-and-shoot composite injury in dogs with moderate impact injury and severe burn.. 2. Randomization of experimental animals was divided into two groups: sham injury control group, burn group, impact injury group, burn-in compound injury group, burn-in composite injury non-resuscitation group and burn-in composite injury PNU28298. 7. Determination of blood flow dynamics index, blood gas, blood inflammatory medium and adhesion factor levels, liver and kidney function and 24-hour blood flow rate; calculate oxygen loss and oxygen consumption; determine plasma volume and vascular permeability. 24 h after injury, the dog was sacrificed by exsanguination, the water content of organs was determined, the pathological diagnosis of lung tissue and the expression of connexin in microvascular endothelial cells were measured. packetization. Methods: 1. Fresh dog umbilical cord was taken and different time was digested with collagenase to extract umbilical vein blood. Endovascular endothelial cells stained with trypan blue performing cell culture, performing identification on the third generation cell, and establishing canine umbilical vein endothelial cell extraction and culture; Method 2. Endovascular endothelial cells of canine umbilical vein extracted by enzymatic digestion, for 3rd generation To determine the permeability of monolayer vascular endothelial cells, the connexin and upstream regulation of vascular endothelial cells in each group were analyzed. Results One: 1. 5g of black cord gold exploded, confirmed by gross anatomy and pathological HE staining of lung tissue: the dogs with a distance of 50cm from the explosion source could be struck with moderate shock, combined with 35% TBSA severe burns due to the burning of napalm, combined with the two-step injury to establish severe burn in dogs. The vascular endothelial cells were severely damaged, the barrier function was unbalanced, the vascular permeability was increased, the tissue edema and the volume of blood were decreased, and the circulatory system was in a low-row and high resistance. Ischemia and hypoxia are more serious; the cholinergic receptor 7 receptor agonist can significantly reduce vascular permeability and tissue edema, improve blood volume, improve hemodynamics and pulmonary ventilation, and correct the contamination. The results are as follows: 1. 1% collagenase digestion for 7min can extract a large amount of polygonal cells from the whole umbilical vein, and the immunofluorescent staining and flow cytometry can be used for identification. Cells were vascular endothelial cells. The expression of connexin was significantly decreased following the stimulation of vascular endothelial cells in canine serum with burn-and-shoot composite injured dogs, and the upstream regulatory proteins (protein kinase C and ROCK1) increased significantly. Addition of nicotine or cholinergic receptor agonists can inhibit their hyperpermeability, decrease protein kinase C and ROCK1, and increase the expression of connexin, but give receptor blockers or improve the activity of protein kinase C and Rho kinase. Conclusion It can be used to inhibit the damage of vascular endothelial cells, the expression of connexin, the imbalance of barrier function, the increase of vascular permeability, the edema of tissue, the sudden decrease of blood volume, and the cause of shock. can inhibit the activity of protein kinase C, thereby improving the expression of connexin in endothelial cells, protecting endothelial barrier function, remarkably reducing vascular permeability and tissue edema, improving blood volume, improving hemodynamics, and pulmonary ventilation.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R644
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