卡巴膽堿在大鼠背部隨意皮瓣存活的研究
發(fā)布時間：2018-08-01 12:28
【摘要】：背景:皮膚是人體最重要且最大的保護器官,但是各種原因所導(dǎo)致的皮膚缺損在現(xiàn)當(dāng)代社會是相當(dāng)常見的。隨意皮瓣因其簡便靈活的優(yōu)點,通常被整形外科、頜面外科等科室用于修復(fù)由外傷、先天畸形或腫瘤等引起的皮膚缺損,這也是臨床上最常用的修復(fù)皮膚缺損的治療手段之一[1]。隨著現(xiàn)代醫(yī)學(xué)的飛速發(fā)展,多數(shù)學(xué)者也致力于制作各種動物模型來研究皮瓣,同時也發(fā)現(xiàn),皮瓣移植術(shù)后影響其存活情況的因素很多。但是研究者們發(fā)現(xiàn),缺血再灌注損傷(IR)是制約皮瓣存活的很常見的一個因素。因隨意皮瓣缺乏知名血管,術(shù)后皮瓣易發(fā)生缺血再灌注損傷致皮瓣部分或全部壞死,影響術(shù)后愈合效果。缺血再灌注損傷是jennings首次提出,是指組織器官缺血后恢復(fù)血供反而加重組織器官損傷的現(xiàn)象。因缺血再灌注損傷在皮瓣存活中發(fā)揮著至關(guān)重要的作用,近年來學(xué)者們做了大量試驗來研究缺血再灌注損傷的相關(guān)病理機制,但是具體機制尚不清楚,不過總結(jié)發(fā)現(xiàn)主要有自由基損傷學(xué)說、炎癥細胞因子學(xué)說、化學(xué)因子損傷學(xué)說等。由此可見,皮瓣在轉(zhuǎn)移或移植手術(shù)后能不能成活的關(guān)鍵環(huán)節(jié)就在于是否能避免發(fā)生缺血再灌注損傷,而多數(shù)學(xué)者認為炎癥反應(yīng)在缺血再灌注損傷中有著至關(guān)重要的作用,因此抑制炎癥反應(yīng)是預(yù)防缺血再灌注損傷而增加隨意皮瓣存活率重要方法之一[2-4]。多年來人們也一直在研究探討抑制炎癥反應(yīng)的方法,因炎癥反應(yīng)時,機體活性最高的炎性細胞因子il-6、il-8、tnf-α就成為學(xué)者們在實驗中主要的檢測指標。盡管抗生素抗炎藥物等在臨床上應(yīng)用甚廣,但是炎癥的控制仍不理想,且副作用多。因此,尋找更理想的抗炎藥物成為現(xiàn)在的研究熱點。研究者們通過實驗發(fā)現(xiàn),卡巴膽堿(carbamylchocline,carbachol)雖然是一種膽堿能受體激動劑,但是通過n樣膽堿能受體卻能發(fā)揮抗炎效應(yīng)。卡巴膽堿是一種人工合成的擬膽堿藥物,其作用與乙酰膽堿(ach)極度相似,但其穩(wěn)定性較乙酰膽堿好,很難被膽堿酯酶水解,因此其作用時間較生理性ach更持久。卡巴膽堿是受體激動劑,能同時激活毒蕈堿樣(m)受體和煙堿樣(n)受體,但近年來大量體內(nèi)外實驗研究發(fā)現(xiàn),卡巴膽堿興奮n樣受體與抗炎密切相關(guān),其主要作用機制是n樣受體激活后明顯抑制tnf-α、il-iβ、il-6、高遷移率族蛋白b1(high-mobilitygroupbox1,hmgb1)等炎癥介質(zhì)的過度產(chǎn)生與釋放[5]。以往多數(shù)實驗主要是研究卡巴膽堿對燒燙傷的動物模型各個重要器官缺血再灌注損傷的保護作用,目前關(guān)于卡巴膽堿對皮瓣移植療效觀察的應(yīng)用與研究相對較少,本實驗以大鼠為造模對象,觀察卡巴膽堿對大鼠背部隨意皮瓣存活的影響。目的:卡巴膽堿具有抗炎作用,很大程度上能減緩缺血再灌注損傷,對組織細胞起到保護作用。本研究是制作大鼠蒂在尾端的隨意皮瓣模型,對大鼠灌服卡巴膽堿,通過大鼠皮瓣存活情況、組織學(xué)觀察、血液炎癥因子測量情況,探索卡巴膽堿對大鼠隨意皮瓣存活的保護作用。方法:本實驗將36只sd大鼠分為a(卡巴膽堿)和b(生理鹽水)兩組,各組18只。首先在大鼠背部設(shè)計蒂在尾端的寬2*4cm的隨意皮瓣手術(shù)范圍�？ò湍憠A組在術(shù)后灌服卡巴膽堿30ug/100g,生理鹽水組灌服等量生理鹽水,一天一次,連續(xù)七天。術(shù)后第七天肉眼觀察皮瓣存活情況,抽取腹腔動脈血離心后取血清行elisa檢測血中細胞炎性因子白介素6、白介素8、腫瘤壞死因子α含量,并取皮瓣遠、中、近端皮瓣組織行he染色觀察組織學(xué)變化。實驗所有數(shù)據(jù)以均值±標準差(?x±s)表示,采用兩個隨機獨立樣本均數(shù)的t檢驗分析兩組之間的差異。結(jié)果:(1)肉眼觀察:術(shù)后第7天肉眼大體觀測皮瓣存活情況:a組(卡巴膽堿):皮瓣可見毛發(fā)生長,色澤佳,彈性較好;皮瓣遠端邊緣可見少許黑色痂殼,無明顯分泌物溢出;將皮瓣掀起,可見皮瓣下肉芽、血管生長良好,血管網(wǎng)形成,出血較多,未見明顯膿性分泌物。b組(生理鹽水):皮瓣組織近端可見毛發(fā)生長,淡紅,彈性尚可;皮瓣中段至遠端無毛發(fā)生長,顏色暗黑,有較多痂殼,無彈性;皮瓣掀起可見少量肉芽組織生長,明顯炎性細胞,血管生長少,出血少。(2)鏡下觀察:a組(卡巴膽堿):皮瓣遠端能見到較少量的中性粒細胞、單核細胞、巨噬細胞等炎性細胞,中、近段未見明顯炎性細胞;皮瓣組織未見明顯水腫帶及壞死組織細胞,可見新生血管網(wǎng)。b組(生理鹽水):遠、中段皮瓣組織可見大量炎性細胞浸潤帶,近段可見少許炎性細胞;皮瓣組織可見明顯壞死組織細胞、水腫明顯、血管稀疏、管壁薄。根據(jù)術(shù)后肉眼及鏡下觀察見a組皮瓣組織各段的存活均好于b組,a組皮瓣各段的炎性細胞均明顯少于b組。(3)血清炎性因子濃度:il-6表達量a組(108.14±5.99)pg/ml,b組(146.21±7.13)pg/ml;IL-8表達量A組(105.12±6.11)pg/ml,B組(140.41±9.78)pg/ml;TNF-α表達量A組(186.75±10.31)pg/ml,B組(246.27±20.69)pg/ml。實驗組IL-6、IL-8、TNF-α各表達量均明顯低于對照組,且兩組間IL-6、IL-8、TNF-α表達量均差異明顯,卡巴膽堿在降低炎性因子IL-6、IL-8、TNF-α表達上均有統(tǒng)計學(xué)意義(P0.01)。結(jié)論:(1)皮瓣移植術(shù)后存在炎癥反應(yīng)的損害,表現(xiàn)為IL-6、IL-8、TNF-α表達水平升高。(2)卡巴膽堿灌服能明顯抑制IL-6、IL-8、TNF-α的產(chǎn)生和釋放,一定程度上能明顯抑制炎癥反應(yīng),改善大鼠背部隨意皮瓣的存活,對皮瓣有保護作用。
[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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622
本文編號：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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622
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