卡巴胆碱在大鼠背部随意皮瓣存活的研究
发布时间：2018-08-01 12:28
【摘要】：背景:皮肤是人体最重要且最大的保护器官,但是各种原因所导致的皮肤缺损在现当代社会是相当常见的。随意皮瓣因其简便灵活的优点,通常被整形外科、颌面外科等科室用于修复由外伤、先天畸形或肿瘤等引起的皮肤缺损,这也是临床上最常用的修复皮肤缺损的治疗手段之一[1]。随着现代医学的飞速发展,多数学者也致力于制作各种动物模型来研究皮瓣,同时也发现,皮瓣移植术后影响其存活情况的因素很多。但是研究者们发现,缺血再灌注损伤(IR)是制约皮瓣存活的很常见的一个因素。因随意皮瓣缺乏知名血管,术后皮瓣易发生缺血再灌注损伤致皮瓣部分或全部坏死,影响术后愈合效果。缺血再灌注损伤是jennings首次提出,是指组织器官缺血后恢复血供反而加重组织器官损伤的现象。因缺血再灌注损伤在皮瓣存活中发挥着至关重要的作用,近年来学者们做了大量试验来研究缺血再灌注损伤的相关病理机制,但是具体机制尚不清楚,不过总结发现主要有自由基损伤学说、炎症细胞因子学说、化学因子损伤学说等。由此可见,皮瓣在转移或移植手术后能不能成活的关键环节就在于是否能避免发生缺血再灌注损伤,而多数学者认为炎症反应在缺血再灌注损伤中有着至关重要的作用,因此抑制炎症反应是预防缺血再灌注损伤而增加随意皮瓣存活率重要方法之一[2-4]。多年来人们也一直在研究探讨抑制炎症反应的方法,因炎症反应时,机体活性最高的炎性细胞因子il-6、il-8、tnf-α就成为学者们在实验中主要的检测指标。尽管抗生素抗炎药物等在临床上应用甚广,但是炎症的控制仍不理想,且副作用多。因此,寻找更理想的抗炎药物成为现在的研究热点。研究者们通过实验发现,卡巴胆碱(carbamylchocline,carbachol)虽然是一种胆碱能受体激动剂,但是通过n样胆碱能受体却能发挥抗炎效应。卡巴胆碱是一种人工合成的拟胆碱药物,其作用与乙酰胆碱(ach)极度相似,但其稳定性较乙酰胆碱好,很难被胆碱酯酶水解,因此其作用时间较生理性ach更持久。卡巴胆碱是受体激动剂,能同时激活毒蕈碱样(m)受体和烟碱样(n)受体,但近年来大量体内外实验研究发现,卡巴胆碱兴奋n样受体与抗炎密切相关,其主要作用机制是n样受体激活后明显抑制tnf-α、il-iβ、il-6、高迁移率族蛋白b1(high-mobilitygroupbox1,hmgb1)等炎症介质的过度产生与释放[5]。以往多数实验主要是研究卡巴胆碱对烧烫伤的动物模型各个重要器官缺血再灌注损伤的保护作用,目前关于卡巴胆碱对皮瓣移植疗效观察的应用与研究相对较少,本实验以大鼠为造模对象,观察卡巴胆碱对大鼠背部随意皮瓣存活的影响。目的:卡巴胆碱具有抗炎作用,很大程度上能减缓缺血再灌注损伤,对组织细胞起到保护作用。本研究是制作大鼠蒂在尾端的随意皮瓣模型,对大鼠灌服卡巴胆碱,通过大鼠皮瓣存活情况、组织学观察、血液炎症因子测量情况,探索卡巴胆碱对大鼠随意皮瓣存活的保护作用。方法:本实验将36只sd大鼠分为a(卡巴胆碱)和b(生理盐水)两组,各组18只。首先在大鼠背部设计蒂在尾端的宽2*4cm的随意皮瓣手术范围。卡巴胆碱组在术后灌服卡巴胆碱30ug/100g,生理盐水组灌服等量生理盐水,一天一次,连续七天。术后第七天肉眼观察皮瓣存活情况,抽取腹腔动脉血离心后取血清行elisa检测血中细胞炎性因子白介素6、白介素8、肿瘤坏死因子α含量,并取皮瓣远、中、近端皮瓣组织行he染色观察组织学变化。实验所有数据以均值±标准差(?x±s)表示,采用两个随机独立样本均数的t检验分析两组之间的差异。结果:(1)肉眼观察:术后第7天肉眼大体观测皮瓣存活情况:a组(卡巴胆碱):皮瓣可见毛发生长,色泽佳,弹性较好;皮瓣远端边缘可见少许黑色痂壳,无明显分泌物溢出;将皮瓣掀起,可见皮瓣下肉芽、血管生长良好,血管网形成,出血较多,未见明显脓性分泌物。b组(生理盐水):皮瓣组织近端可见毛发生长,淡红,弹性尚可;皮瓣中段至远端无毛发生长,颜色暗黑,有较多痂壳,无弹性;皮瓣掀起可见少量肉芽组织生长,明显炎性细胞,血管生长少,出血少。(2)镜下观察:a组(卡巴胆碱):皮瓣远端能见到较少量的中性粒细胞、单核细胞、巨噬细胞等炎性细胞,中、近段未见明显炎性细胞;皮瓣组织未见明显水肿带及坏死组织细胞,可见新生血管网。b组(生理盐水):远、中段皮瓣组织可见大量炎性细胞浸润带,近段可见少许炎性细胞;皮瓣组织可见明显坏死组织细胞、水肿明显、血管稀疏、管壁薄。根据术后肉眼及镜下观察见a组皮瓣组织各段的存活均好于b组,a组皮瓣各段的炎性细胞均明显少于b组。(3)血清炎性因子浓度:il-6表达量a组(108.14±5.99)pg/ml,b组(146.21±7.13)pg/ml;IL-8表达量A组(105.12±6.11)pg/ml,B组(140.41±9.78)pg/ml;TNF-α表达量A组(186.75±10.31)pg/ml,B组(246.27±20.69)pg/ml。实验组IL-6、IL-8、TNF-α各表达量均明显低于对照组,且两组间IL-6、IL-8、TNF-α表达量均差异明显,卡巴胆碱在降低炎性因子IL-6、IL-8、TNF-α表达上均有统计学意义(P0.01)。结论:(1)皮瓣移植术后存在炎症反应的损害,表现为IL-6、IL-8、TNF-α表达水平升高。(2)卡巴胆碱灌服能明显抑制IL-6、IL-8、TNF-α的产生和释放,一定程度上能明显抑制炎症反应,改善大鼠背部随意皮瓣的存活,对皮瓣有保护作用。
[Abstract]:Background: skin is the most important and largest protective organ of the human body, but skin defects caused by various causes are quite common in modern society. The random skin flap is usually used for repair of skin defects caused by trauma, congenital deformity or tumor due to its simple and flexible advantages, such as plastic surgery, maxillofacial surgery and other departments. One of the most commonly used treatments for the repair of skin defects is [1]., with the rapid development of modern medicine, most scholars are also committed to making various animal models to study skin flaps. Meanwhile, there are many factors that affect their survival after the flap transplantation. However, researchers have found that ischemia reperfusion injury (IR) is a restriction on the skin flap. It is a common cause of survival. Because of the lack of known blood vessels in the free skin flap, the skin flap is prone to ischemia reperfusion injury, which may cause partial or complete necrosis of the skin flap, which affects the effect of postoperative healing. The ischemia reperfusion injury is first proposed by Jennings, which means that the recovery of blood supply after the ischemia of tissues and organs aggravates the injury of tissues and organs. Perfusion injury plays a vital role in the survival of the skin flap. In recent years, a large number of scholars have done a lot of experiments to study the pathological mechanism of ischemia reperfusion injury, but the specific mechanism is still not clear, but the main findings are the theory of free radical damage, the theory of inflammatory cytokines, the theory of chemical factor damage, etc. The key to the survival of the flap after the transfer or transplantation is to prevent the occurrence of ischemia-reperfusion injury, and many mathematicians believe that inflammation plays a vital role in ischemia reperfusion injury. Therefore, inhibition of inflammation is an important way to prevent ischemia reperfusion injury and increase the survival rate of random flaps. For more than [2-4]. years, people have been studying the methods of inhibiting inflammatory reactions. The inflammatory cytokine IL-6, IL-8, and tnf- alpha, which have the highest activity in the body, have become the main detection indexes in the experiment. Although the antibiotic anti inflammatory drugs should be widely used in the clinic, the control of inflammation is still not ideal, and the side of the inflammation is not ideal. So, the search for more ideal anti-inflammatory drugs is now a hot spot. Researchers have found that, through experiments, Kaba choline (Carbamylchocline, carbachol) is a cholinergic receptor agonist, but it can play an anti-inflammatory effect through the n like cholinergic receptor. Kaba choline is an artificial synthetic choline drug, which is a kind of cholinergic receptor. The effect is extremely similar to acetylcholine (ACh), but its stability is better than acetylcholine, and it is difficult to be hydrolyzed by cholinesterase, so its action time is more persistent than that of biological ACh. Kaba choline is a receptor agonist and can activate muscarinine like (m) receptor and nicotinic (n) receptor at the same time. But in recent years, a large number of experiments in vitro and in vivo have found that Kaba choline is in favor of choline. The n like receptor is closely related to anti-inflammatory, its main mechanism is that n like receptor activates the inhibition of tnf- a, il-i beta, IL-6, high mobility group protein B1 (high-mobilitygroupbox1, HMGB1) and other inflammatory mediators, the excessive production and release of [5]. most of the previous experiments are mainly to study the various important organs of the animal model of burned and scalded animal models. The protective effect of blood reperfusion injury, at present, the application and Research on the effect of Kaba choline on skin flap transplantation is relatively small. In this experiment, the effect of Kaba choline on the survival of the dorsal flap of the rat was observed. Objective: the effect of Kaba choline with anti inflammation was used to reduce the injury of ischemia-reperfusion to a large extent. The tissue cell played a protective role. This study was to make a random flap model of the rat tail at the end of the rat. The rat perfusion of Kaba choline, the survival of the rat skin flap, the histological observation, the measurement of blood inflammatory factors, and the protective effect of Kaba choline on the survival of the random skin flap in rats were explored. Methods: this experiment divided 36 SD rats into a (card). Two groups of B (normal saline) and 18 groups. First, the wide 2*4cm free flap operation was designed on the back of the rat on the back of the rat. The Kaba choline group was given the Kaba choline 30ug/100g after operation, and the saline group was given the same amount of saline, once a day for seven days. The survival of the flap was observed by the naked eye seventh days after the operation and the abdomen was taken out of the abdomen. The serum levels of leukin 6, IL-8 and TNF - alpha in blood were detected by ELISA after the blood centrifugation. The histological changes were observed by HE staining in the distal, middle and proximal flap tissues of the skin flap. All the experimental data were expressed with mean mean standard deviation (? X + s), and two groups were analyzed by two random sample averages. Results: (1) naked eye observation: seventh days after the operation, the naked eye observed the survival of the skin flap: a group (Kaba choline): the skin flaps showed hair growth, good color and good elasticity; a few black scab shells were seen on the distal edge of the flap, and there were no obvious secretions spillover; the flap was lifted, the skin flaps were visible, the vascular growth was good, and the vascular network formed, There were more bleeding and no obvious purulent secretion.B group (physiological saline): hair growth in the proximal end of the flap was visible, light red and resilient, and the skin flap had no hair growth in the middle to distal end, dark dark, more eschar shell, and inelastic; the skin flap raised a small number of granulation tissue growth, obvious inflammatory cells, less vascular growth and less bleeding. (2) a Group (Kaba choline): a small amount of neutrophils, mononuclear cells, macrophages and other inflammatory cells were seen in the distal part of the flap. There was no obvious inflammatory cells in the proximal segment. There was no obvious edema zone and necrotic tissue in the flap tissue, and the.B group (physiological salt water) in the new vascular network (physiological salt water): a large number of inflammatory cells were seen in the middle segment of the flap. A few inflammatory cells were visible in the segment, and the necrotic tissue cells of the skin flap were visible, the edema was obvious, the blood vessels were sparse, and the wall of the tube was thin. The survival of all segments of the flap tissue in group A was better than that of group B according to the naked eye and microscope. (3) the inflammatory cells in each segment of the a group were less than that of the B group. (3) the serum inflammatory factor concentration was (108.14 + 5.99) P in the IL-6 expression (108.14 + 5.99) P. G/ml, group B (146.21 + 7.13) pg/ml, IL-8 expression in A group (105.12 + 6.11) pg/ml, B group (140.41 + 9.78) pg/ml, TNF- alpha expression in A group (186.75 + 10.31) pg/ml, B group (246.27 + 20.69), all expressions were significantly lower than those of the control group, and both of the two groups were significantly different, and Kaba choline was reduced to inflammation The expression of factor IL-6, IL-8, TNF- alpha had statistical significance (P0.01). Conclusion: (1) there was an increase in the expression level of IL-6, IL-8 and TNF- alpha after the flap transplantation. (2) Kaba choline irrigation could obviously inhibit the production and release of IL-6, IL-8, TNF- a, to a certain extent, inhibit the inflammatory reaction and improve the random back of the rat. The survival of the flap has a protective effect on the skin flap.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622
本文编号：2157580
[Abstract]:Background: skin is the most important and largest protective organ of the human body, but skin defects caused by various causes are quite common in modern society. The random skin flap is usually used for repair of skin defects caused by trauma, congenital deformity or tumor due to its simple and flexible advantages, such as plastic surgery, maxillofacial surgery and other departments. One of the most commonly used treatments for the repair of skin defects is [1]., with the rapid development of modern medicine, most scholars are also committed to making various animal models to study skin flaps. Meanwhile, there are many factors that affect their survival after the flap transplantation. However, researchers have found that ischemia reperfusion injury (IR) is a restriction on the skin flap. It is a common cause of survival. Because of the lack of known blood vessels in the free skin flap, the skin flap is prone to ischemia reperfusion injury, which may cause partial or complete necrosis of the skin flap, which affects the effect of postoperative healing. The ischemia reperfusion injury is first proposed by Jennings, which means that the recovery of blood supply after the ischemia of tissues and organs aggravates the injury of tissues and organs. Perfusion injury plays a vital role in the survival of the skin flap. In recent years, a large number of scholars have done a lot of experiments to study the pathological mechanism of ischemia reperfusion injury, but the specific mechanism is still not clear, but the main findings are the theory of free radical damage, the theory of inflammatory cytokines, the theory of chemical factor damage, etc. The key to the survival of the flap after the transfer or transplantation is to prevent the occurrence of ischemia-reperfusion injury, and many mathematicians believe that inflammation plays a vital role in ischemia reperfusion injury. Therefore, inhibition of inflammation is an important way to prevent ischemia reperfusion injury and increase the survival rate of random flaps. For more than [2-4]. years, people have been studying the methods of inhibiting inflammatory reactions. The inflammatory cytokine IL-6, IL-8, and tnf- alpha, which have the highest activity in the body, have become the main detection indexes in the experiment. Although the antibiotic anti inflammatory drugs should be widely used in the clinic, the control of inflammation is still not ideal, and the side of the inflammation is not ideal. So, the search for more ideal anti-inflammatory drugs is now a hot spot. Researchers have found that, through experiments, Kaba choline (Carbamylchocline, carbachol) is a cholinergic receptor agonist, but it can play an anti-inflammatory effect through the n like cholinergic receptor. Kaba choline is an artificial synthetic choline drug, which is a kind of cholinergic receptor. The effect is extremely similar to acetylcholine (ACh), but its stability is better than acetylcholine, and it is difficult to be hydrolyzed by cholinesterase, so its action time is more persistent than that of biological ACh. Kaba choline is a receptor agonist and can activate muscarinine like (m) receptor and nicotinic (n) receptor at the same time. But in recent years, a large number of experiments in vitro and in vivo have found that Kaba choline is in favor of choline. The n like receptor is closely related to anti-inflammatory, its main mechanism is that n like receptor activates the inhibition of tnf- a, il-i beta, IL-6, high mobility group protein B1 (high-mobilitygroupbox1, HMGB1) and other inflammatory mediators, the excessive production and release of [5]. most of the previous experiments are mainly to study the various important organs of the animal model of burned and scalded animal models. The protective effect of blood reperfusion injury, at present, the application and Research on the effect of Kaba choline on skin flap transplantation is relatively small. In this experiment, the effect of Kaba choline on the survival of the dorsal flap of the rat was observed. Objective: the effect of Kaba choline with anti inflammation was used to reduce the injury of ischemia-reperfusion to a large extent. The tissue cell played a protective role. This study was to make a random flap model of the rat tail at the end of the rat. The rat perfusion of Kaba choline, the survival of the rat skin flap, the histological observation, the measurement of blood inflammatory factors, and the protective effect of Kaba choline on the survival of the random skin flap in rats were explored. Methods: this experiment divided 36 SD rats into a (card). Two groups of B (normal saline) and 18 groups. First, the wide 2*4cm free flap operation was designed on the back of the rat on the back of the rat. The Kaba choline group was given the Kaba choline 30ug/100g after operation, and the saline group was given the same amount of saline, once a day for seven days. The survival of the flap was observed by the naked eye seventh days after the operation and the abdomen was taken out of the abdomen. The serum levels of leukin 6, IL-8 and TNF - alpha in blood were detected by ELISA after the blood centrifugation. The histological changes were observed by HE staining in the distal, middle and proximal flap tissues of the skin flap. All the experimental data were expressed with mean mean standard deviation (? X + s), and two groups were analyzed by two random sample averages. Results: (1) naked eye observation: seventh days after the operation, the naked eye observed the survival of the skin flap: a group (Kaba choline): the skin flaps showed hair growth, good color and good elasticity; a few black scab shells were seen on the distal edge of the flap, and there were no obvious secretions spillover; the flap was lifted, the skin flaps were visible, the vascular growth was good, and the vascular network formed, There were more bleeding and no obvious purulent secretion.B group (physiological saline): hair growth in the proximal end of the flap was visible, light red and resilient, and the skin flap had no hair growth in the middle to distal end, dark dark, more eschar shell, and inelastic; the skin flap raised a small number of granulation tissue growth, obvious inflammatory cells, less vascular growth and less bleeding. (2) a Group (Kaba choline): a small amount of neutrophils, mononuclear cells, macrophages and other inflammatory cells were seen in the distal part of the flap. There was no obvious inflammatory cells in the proximal segment. There was no obvious edema zone and necrotic tissue in the flap tissue, and the.B group (physiological salt water) in the new vascular network (physiological salt water): a large number of inflammatory cells were seen in the middle segment of the flap. A few inflammatory cells were visible in the segment, and the necrotic tissue cells of the skin flap were visible, the edema was obvious, the blood vessels were sparse, and the wall of the tube was thin. The survival of all segments of the flap tissue in group A was better than that of group B according to the naked eye and microscope. (3) the inflammatory cells in each segment of the a group were less than that of the B group. (3) the serum inflammatory factor concentration was (108.14 + 5.99) P in the IL-6 expression (108.14 + 5.99) P. G/ml, group B (146.21 + 7.13) pg/ml, IL-8 expression in A group (105.12 + 6.11) pg/ml, B group (140.41 + 9.78) pg/ml, TNF- alpha expression in A group (186.75 + 10.31) pg/ml, B group (246.27 + 20.69), all expressions were significantly lower than those of the control group, and both of the two groups were significantly different, and Kaba choline was reduced to inflammation The expression of factor IL-6, IL-8, TNF- alpha had statistical significance (P0.01). Conclusion: (1) there was an increase in the expression level of IL-6, IL-8 and TNF- alpha after the flap transplantation. (2) Kaba choline irrigation could obviously inhibit the production and release of IL-6, IL-8, TNF- a, to a certain extent, inhibit the inflammatory reaction and improve the random back of the rat. The survival of the flap has a protective effect on the skin flap.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622
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