膽堿能α7受體激動(dòng)劑對(duì)燒沖復(fù)合傷休克期血管內(nèi)皮細(xì)胞的保護(hù)作用及機(jī)制研究
發(fā)布時(shí)間：2018-10-26 13:31
【摘要】：目的燒沖復(fù)合傷引起的低血容量休克,是傷員發(fā)生早期死亡和后期并發(fā)癥的主要原因之一,而血管內(nèi)皮細(xì)胞損傷則是休克期血流動(dòng)力學(xué)紊亂和臟器功能損害的病理生理基礎(chǔ)。本文擬采用重度燒沖復(fù)合傷犬和犬臍靜脈血管內(nèi)皮細(xì)胞為模型,研究膽堿能N樣α7受體激動(dòng)劑對(duì)犬燒沖復(fù)合傷血管內(nèi)皮細(xì)胞的保護(hù)作用及其機(jī)制,為臨床燒沖復(fù)合傷早期抗休克治療提供新的思路和實(shí)驗(yàn)依據(jù)。 方法一：1.選取Beagle犬,采用黑索金爆炸致沖擊傷,而后用凝固汽油燃燒造成35%TBSAⅢ°.燒傷,建立中度沖擊傷+重度燒傷的犬重度燒沖復(fù)合傷模型。2.將實(shí)驗(yàn)動(dòng)物隨機(jī)化分為：假傷對(duì)照組；燒傷組；沖擊傷組；燒沖復(fù)合傷組；燒沖復(fù)合傷不復(fù)蘇組和燒沖復(fù)合傷PNU282987組。測(cè)定血流動(dòng)力學(xué)指標(biāo)、血?dú)�、血炎癥介質(zhì)和粘附因子的水平、肝腎功能以及24小時(shí)尿量；計(jì)算氧輸量和氧耗量；測(cè)定血漿容量及血管通透性的變化。傷后24h采用放血法處死犬,測(cè)定臟器含水量,取肺組織行病理診斷和微血管內(nèi)皮細(xì)胞連接蛋白免疫組化。 方法二：1.取新生犬臍帶,采用膠原酶分別消化不同時(shí)間,提取臍靜脈血管內(nèi)皮細(xì)胞。采用臺(tái)盼藍(lán)染色進(jìn)行細(xì)胞計(jì)數(shù)。而后經(jīng)細(xì)胞培養(yǎng),取第3代細(xì)胞進(jìn)行鑒定,建立犬臍靜脈血管內(nèi)皮細(xì)胞提取、培養(yǎng)方法。2.酶消化法提取的犬臍靜脈血管內(nèi)皮細(xì)胞,以第3代為實(shí)驗(yàn)細(xì)胞。給予燒沖復(fù)合傷血清刺激,分別給予煙堿和蛋白激酶C、Rho激酶的阻斷劑和激動(dòng)劑干預(yù),測(cè)定其單層血管內(nèi)皮細(xì)胞的通透性；分析各組血管內(nèi)皮細(xì)胞連接蛋白以及上游調(diào)控因子的表達(dá)。 結(jié)果一：1.5g黑索金爆炸,通過(guò)大體解剖及肺臟組織病理HE染色證實(shí)：距離爆炸源50cm的比格犬可被爆炸致中度沖擊傷,結(jié)合凝固汽油燃燒致35%TBSA重度燒傷,結(jié)合上述兩步致傷可建立犬重度燒沖復(fù)合傷模型。2.燒沖復(fù)合傷后血管內(nèi)皮細(xì)胞嚴(yán)重受損,屏障功能失衡,血管通透性增高,組織水腫,血容量驟減,循環(huán)系統(tǒng)呈低排高阻,臟器缺血缺氧更為嚴(yán)重；膽堿能α7受體激動(dòng)劑可以顯著減低血管通透性和組織水腫,提高血容量,改善血流動(dòng)力學(xué)和肺通氣換氣,糾正臟器的缺血缺氧狀態(tài)。 結(jié)果二：1.1%Ⅰ型膠原酶消化7min可從全臍靜脈提取大量多角形細(xì)胞,免疫熒光染色和流式細(xì)胞儀均可以鑒定該細(xì)胞為血管內(nèi)皮細(xì)胞。2.燒沖復(fù)合傷犬血清刺激血管內(nèi)皮細(xì)胞后,細(xì)胞連接蛋白的表達(dá)顯著下降,其上游的調(diào)節(jié)蛋白(蛋白激酶C和ROCK1)的表達(dá)則顯著升高,單層細(xì)胞通透性顯著提高；而加入煙堿或膽堿能受體激動(dòng)劑均可以抑制其過(guò)高的通透性,降低蛋白激酶C和ROCK1,提高連接蛋白的表達(dá)；但給予受體阻斷劑或提高蛋白激酶C和Rho激酶的活性后則可以抑制該的效果。 結(jié)論燒沖復(fù)合傷能造成血管內(nèi)皮細(xì)胞受損,其連接蛋白表達(dá)降低,屏障功能失衡,血管通透性增高,組織水腫,血容量驟減,導(dǎo)致休克；膽堿能α7受體激動(dòng)劑可以通過(guò)抑制蛋白激酶C的活性,從而提高內(nèi)皮細(xì)胞連接蛋白的表達(dá),保護(hù)內(nèi)皮屏障功能,顯著減低血管通透性和組織水腫,提高血容量,改善血流動(dòng)力學(xué)、肺通氣換氣功能以及臟器的缺血缺氧狀態(tài)。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R644
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R644
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