应用血凝酶与电灼对兔去外膜颈动脉出血模型止血及修复的研究

发布时间：2018-05-18 21:04

本文选题：血管外膜 + 血凝酶　；参考：《安徽医科大学》2014年硕士论文

【摘要】：目的围手术期血管渗血及术后创口渗血是外科手术过程中常见的并发症，因而减少术中及术后失血在临床上越来越受到重视。目前临床上常用的止血方法有药物止血法和理化止血法等，，但目前关于这两种止血方法对组织修复长期观察的研究较少。本实验旨在通过对新西兰大白兔颈总动脉外膜剥离的方法造成血管渗血，继而用血凝酶以及电灼两种方法止血，以观察血凝酶（hemocoagulase atrox, HA）与电灼(electric cauterization, EC)局部止血对动脉修复的影响，用以指导临床应用。方法健康纯种新西兰大白兔27只，均约20周龄，雌雄各半，体重2.5~3.0kg，按观察时间随机分为3组：0d组（n=9）、14d组（n=9）、28d组（n=9），采用颈动脉外膜剥离的方法造成血管渗血模型，每组再根据止血方法不同随机分成3个亚组：对照组（control，CON组，n=3）、血凝酶组（HA组，n=3）和电灼组（EC组，n=3）。CON组家兔两侧颈总动脉剥离外膜后用生理盐水（normal saline, NS）纱布止血，HA组和EC组在外膜剥离后分别予以HA（浓度为2U/ml）沾湿的纱布及电灼（功率为40w）止血。0d组在术后直接取出两侧颈总动脉，14d组和28d组分别术后正常饮食相应天数后麻醉家兔，活体、无菌条件下取出两侧动脉。将取出的血管标本分为三段：①约0.5cm标本制成蜡块，用HE染色和MASSON三色胶原染色观察不同亚组动脉修复及重塑的病理学改变，免疫组化染色检测动脉单核细胞趋化蛋白-1（MCP-1）、转化生长因子-β1（TGF-β1）及血管内皮生长因子（VEGF）蛋白的表达情况；②约1cm标本用透射电镜观察不同组别动脉血管细胞分化情况；③约1cm标本用逆转录-聚合酶链反应（RT-PCR）法检测不同组别标本中MCP-1、TGF-β1及VEGF的mRNA表达情况。结果 1、所有家兔均成功造成渗血模型，各组动脉渗血程度基本相当，各组家兔一般状况及手术情况差异均无统计学意义。 2、NS、HA及EC止血对动脉外膜剥离后血管重塑的影响病理学观察显示0d CON组及HA组HE及Masson染色见外膜已被去除，内膜中膜形态完好，而EC组见中膜弹力纤维及胶原纤维结构疏松，偶见内皮细胞且形态不规则。术后14d可见三组血管外膜增生活跃，内膜增厚，管腔面积缩小，胶原纤维密度明显增大。HA组动脉中、内膜厚度、胶原纤维相对密度（collagen fibers density，CFD）及狭窄率(stenotic ratio, SR)均小于EC组（P0.05），管腔面积及血管重塑指数(remodeling index, RI)均大于EC组（P0.05），而HA组同CON组相比无统计学意义（P0.05）；28d HA组各指标均显著优于EC组（P0.05），而与CON组无明显差别。 3、生理盐水、血凝酶及电灼止血对动脉细胞增值、分化的影响透射电镜观察显示0d CON组及HA组见内皮细胞完整，平滑肌细胞形态正常；而EC组见内皮细胞空泡样变性，平滑肌细胞水肿严重；术后14d各组新生外膜可见大量微丝及粗面内质网扩张的成纤维细胞（Fibroblasts, F），呈现出肌成纤维细胞（Myofibroblasts, MF）特征，并有向内膜迁徙的趋势；28dCON组及HA组外膜F已逆转为MF，而EC组仍可见F。 4、生理盐水、血凝酶及电灼止血对动脉MCP-1、TGF-β1及VEGF表达的影响免疫组化染色及RT-PCR结果显示：0、14及28d CON组和HA组MCP-1、TGF-β1及VEGF阳性细胞及mRNA表达无明显差异，且28d两组表达已下降近0d水平（P0.05）；而EC组同CON组相比，术后14及28d MCP-1、TGF-β1及VEGF蛋白及mRNA表达差异均有统计学意义（P0.05）。结论在动脉外膜剥离渗血模型中，血凝酶止血可有效减轻损伤血管的炎症反应，降低血管重塑程度和狭窄情况，与单纯的盐水纱布止血血管修复结果一致，是一种安全、有效、更接近自然状况的局部止血药物。
[Abstract]:objective
Perioperative vascular osmotic blood and postoperative wound oozing are common complications in the surgical procedure, thus reducing blood loss in the operation and post operation is becoming more and more important. Currently, the commonly used hemostatic methods are drug hemostasis and physicochemical hemostasis, but the two methods of hemostasis have been observed for a long time. The aim of this study was to investigate the effects of hemostasis on the arterial repair by two methods: hemocoagulase atrox (HA) and electrocautery (electric cauterization, EC).
Method
27 healthy and pure New Zealand white rabbits were divided into 3 groups of 20 weeks old and half female and male and body weight 2.5~3.0kg. According to the observation time, they were randomly divided into 3 groups: group 0d (n=9), group 14d (n=9) and group 28d (n=9). The blood vessel infiltration model was caused by exfoliation of carotid artery, and each group was randomly divided into 3 subgroups according to the different hemostasis methods: the control group (control, CON group, n=3), The hemagglutinin group (group HA, n=3) and electrocautery group (group EC, n=3).CON group of rabbits were treated with normal saline (normal saline, NS) gauze after exfoliation of the lateral common carotid artery in group.CON. The HA group and EC group were treated with HA (concentration for 2U/ml) wet gauze and electric cauterization after exfoliation of the outer membrane. The two arteries were taken out of the rabbits, the living body and the aseptic condition after the normal diet of the 28d group. The blood vessel specimens were divided into three segments: 1. About 0.5cm specimens were made of wax blocks. The pathological changes of the repair and remodeling of different subgroups were observed by HE staining and MASSON trichromatic collagen staining, and the arterial monomer was detected by immunohistochemical staining. Nuclear cell chemoattractant protein -1 (MCP-1), transforming growth factor - beta 1 (TGF- beta 1) and vascular endothelial growth factor (VEGF) protein expression. (2) about 1cm specimens were observed by transmission electron microscopy to observe the differentiation of vascular cells in different groups of arteries; and about 1cm specimens were detected by reverse transcription polymerase chain reaction (RT-PCR) method for the detection of MCP-1, TGF- beta 1 in different groups. And the mRNA expression of VEGF.
Result
1, all rabbits were successful in oozing the blood model. The degree of arterial bleeding in each group was basically the same.
2, the effects of NS, HA and EC hemostasis on vascular remodeling after epicardial detachment.
Pathological observation showed that 0d CON group and HA group HE and Masson staining showed that the outer membrane had been removed, the membrane morphology of the intima was intact, and the middle membrane elastic fibers and collagen fibers were loose in the EC group, and the endothelial cells were irregular in morphology. After the operation, the three groups of vascular adventitial hyperplasia were active, the inner membrane was thickened, the area of the lumen was reduced, and the collagen fiber density was obvious In the.HA group, the thickness of the intima, the relative density of collagen fiber (collagen fibers density, CFD) and the stenosis rate (stenotic ratio, SR) were all smaller than that of the EC group (P0.05), and the lumen area and the vascular remodeling index (remodeling index) were all larger than those in the group. It was better than group EC (P0.05), but no significant difference from group CON.
3, the effects of physiological saline, hemocoagulase and electrocautery on the proliferation and differentiation of arterial cells.
The transmission electron microscope showed that the endothelial cells were intact in 0d CON group and HA group, and the morphology of smooth muscle cells was normal, while in group EC, the endothelial cells were vacuolated and the smooth muscle cells were edema. After the operation, a large number of microfilaments and dilated fibroblasts of rough endoplasmic reticulum (F) were found in all 14d groups, and myofibroblast (Myofi) was presented. Broblasts, MF) had the characteristics of migration to the intima, while the outer membrane F of group 28dCON and group HA had been reversed to MF, while F. was still visible in EC group.
4, the effects of normal saline, hemagglutinase and electrocautery on the expression of MCP-1, TGF- beta 1 and VEGF in arteries
Immunohistochemical staining and RT-PCR results showed that there was no significant difference between 0,14 and 28d CON group and HA group MCP-1, TGF- beta 1 and VEGF positive cells and mRNA expression, and the expression of 28d two decreased near 0d level (P0.05).
conclusion
In the exfoliation model of epicardial exfoliation, hemostasis can effectively reduce the inflammatory reaction of damaged vessels, reduce the degree of vascular remodeling and stenosis, and is consistent with the results of simple saline gauze repair blood vessel repair. It is a safe, effective, and closer to the natural state of the local hemostatic drugs.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R619.1

【参考文献】