【作者】 崔忠林；

【导师】 周杰；

【作者基本信息】 南方医科大学 ， 外科学， 2014， 博士

【摘要】 在我国,肝病发病率较高,各类肝炎、肝硬化、肝癌、先天性与代谢性肝病等,一般的药物治疗和外科手术治疗难以达到满意的效果。因此,对肝脏移植手术有很大的需求。由于肝脏功能复杂、手术难度大、术后管理困难等因素,但肝脏在移植过程中往往会遭受不同程度的各种损伤,从而导致供肝术后肝功能不全、原发性移植肝无功能(Primary graft non-function, PNF)和移植物功能延迟恢复(delayed graft function, DGF)等。移植后肝功能不良和供肝保存欠佳、缺血再灌损伤(Ischemia-reperfusion, I/R)关系极为密切。研究表明,肝脏缺血再灌注损伤是导致早期移植后肝功能不全的最常见原因之一,同时也是影响肝移植手术后移植物长期存活的重要因素。我国又是世界上终末期肝病发病率最高的地区之一,肝病患者众多,肝脏供需矛盾突出。因此,寻找一种安全有效的移植肝保存法,探究肝移植缺血再灌损伤机制已成为重要的研究课题。肝移植缺血再灌注损伤及相应炎症反应常导致肝细胞损伤及坏死,如何避免肝细胞缺血再灌注损伤对疾病的治疗及预后有着重要的价值。缺血预适应(Ischemic preconditioning, IPC),也叫缺血预适应,是一种通过预先短暂缺血以耐受随后较长时间的缺血损伤,并减轻组织后续缺血再灌注损伤的现象。1986年Murry等首次提出了缺血预处理这一概念。Laskey和Szmagala的临床研究指出,缺血预适应可预防经皮腔内冠状动脉成形术(Percutaneous transluminal coronary angioplasty, PTCA)和心脏手术所致的心肌缺血损伤。Toosy等首次研究发现了肾脏IPC对肾组织学损伤也具有保护作用。研究表明缺血预适应现象在其它许多器官都存在,如脑、肝、小肠、骨骼肌、肾和肺等,而且各组织器官之间存在交叉缺血耐受现象,例如骨骼肌、肾脏的短暂性缺血可有效减轻冠状血管阻塞所致的心肌梗死灶的容积。缺血预适应对大鼠字体肝移植缺血再灌注损伤的保护作用及其机制还不明确,是与临床肝移植手术减少肝组织损伤密切相关的课题。白介素-6(Interleukin-6,IL-6)是细胞因子的核心成员之一,是广泛表达于活化的B细胞、静止的T细胞、骨髓瘤细胞、肝细胞、自然杀伤细胞、髓样白血病细胞等细胞表面的一种具有多种生物学活性的刺激因子。IL-6主要由单核巨噬细胞和成纤维细胞产生,人类IL-6是由213个氨基酸组成的,可由肿瘤坏死因子(Tumor necrosis factor,T]NF)诱导产生并可增加TNF的有害作用,它具有诱导T、B淋巴细胞分化、刺激肝细胞合成急性期反应蛋白、催化和放大炎性反应和毒性等作用,对组织和细胞产生损害。IL-6作为一种多效性细胞因子,是参与免疫调节和炎性反应的重要因子之一,是机体对感染和组织损伤所引起应答反应的主要介质,能够和集落刺激因子(Colony-stimulating factor, CSF)协同作用,促进骨髓源细胞的生长和分化,增强自然杀伤细胞的裂解功能。病毒、细菌感染均能诱导体内IL-6表达增加,在多种自身免疫疾病和肿瘤疾病中也表达异常。作为机体的保护机制,血清中IL-6水平升高对机体清除感染的病毒起到重要作用,此外,研究指出IL-6的表达水平与感染的严重程度、炎症反应的程度呈正相关,可作为判断病情严重程度的灵敏指标。本研究首先构建大鼠自体肝移植缺血再灌注损伤模型,对缺血预适应组和对照组大鼠肝组织受损情况进行病理分析,然后对相关指标血浆谷草转氨酶(Alanine aminotransferase, ALT)和谷丙转氨酶(Aspartate aminotransferase,AST)的含量、IL-6的表达、超氧化物歧化酶(SuperoxideDismutase, SOD)活性和丙二醛(Malondialdehyde, MDA)含量进行检测,分析血浆中IL-6、TNF-α和IL-1p的表达在缺血预适应组与对照组的差异,以及与肝组织损伤程度的相关性和可能的作用机制。本文旨在对肝移植缺血再灌注损伤的机制和保护性预适应进行研究,为肝移植缺血再灌注损伤的临床治疗和保护提供新的依据。方法首先建立大鼠门静脉灌注的自体肝移植缺血再灌注模型,健康、成年、雄性SPF级纯系SD大鼠72只,体重约280-300g,由南方医科大学实验动物中心提供。大鼠常规饲养,术前禁食12h,不禁水。将72只SD大鼠随机分为3组：正常大鼠对照组(A组)、大鼠肝移植组(B组)和缺血预适应大鼠肝移植组(C组)。B组大鼠直接行肝移植术。C组大鼠术前行肝脏缺血预适应,即夹闭肝门部肝动脉及门静脉15min,后回复肝脏供血,20min后行大鼠自体肝移植。采用乙醚棉球对准大鼠口鼻行开放式吸入麻醉。常规备皮、消毒后,取腹正中切口入腹。进腹后先用棉签将整个肠道翻出腹腔外以利于术野暴露。然后用组织剪游离肝镰状韧带后切断,向右翻动肝左叶和中叶,暴露肝食管韧带,结扎、切断肝食管韧带内血管,并以此为起点逆时针游离肝脏。首先沿食管向上分离,切断左三角韧带,缝扎、切断左膈血管。然后向左翻转肝脏,切断右三角韧带。再继续向上游离肝上下腔静脉(Suprahepatic vena cava,SVC)至膈静脉裂孔,然后向下游离腔静脉至右肾静脉水平,期间结扎、切断腰静脉。游离肝肾韧带,结扎切断右肾上腺静脉。肝脏复位,游离肝下下腔静脉(Infrahepatic vena cava,IVC),将其分离至左肾静脉水平以上。打开肝十二指肠韧带,找出肝动脉(Hepatic artery,HA),将其分离并于动脉后方过线以备阻断。继续从肠系膜下静脉和脾静脉汇合处游离门静脉(Portal vein,PV)至肝门部,期间需结扎幽门静脉。最后钝性分离肝胃韧带,将肝两片叶游离出来。至此肝脏全部游离完毕。将稀释后的肝素注射液(35U/L)3mL通过穿刺针灌入门静脉,使其完全肝素化。在髂总动脉与左肾动脉之间向近心端穿刺腹主动脉,用血管夹固定穿刺针,在腹腔干以上和穿刺点以下上血管夹阻断。同时在SVC、IVC右肾静脉水平上血管夹,并在IVC血管夹稍上方纵行剪开约1mm静脉壁作为流出道。然后以2.5mL/min速度通过输液泵同时经PV、腹主动脉穿刺处灌注4℃含肝素(12.5U/mL)的乳酸林格氏液20mL,在此同时不断用4℃乳酸林格氏液浇注肝脏表面迅速降温。当肝脏颜色变为均匀土黄色时,灌注结束。灌注完成后拔出穿刺针,修补PV和腹主动脉穿刺点,用8-0prolene缝线修补IVC流出道,在肝动脉预留线处阻断肝动脉并上血管夹,然后松开PV、SVC、IVC、腹主动脉的血管夹,恢复肝脏血供,结束无肝期。并同时用38℃生理盐水浇注肝脏表面,快速复温。肝动脉开放后,观察腹腔内无出血点,连续缝合关腹。术后对大鼠进行灯照复温,直至麻醉清醒可以站立。各组大鼠再灌注2h、6h、12h和24h后,将大鼠麻醉,从下腔静脉采血5ml,离心后吸取血清,-80℃冰箱保存,用于ALT.AST检测。切取被阻断的肝组织,林格氏液洗净血液,修成小块(厚度不超过0.5cm),一部分用10%中性甲醛固定,一部分保存于-80℃冰箱。术后使用肝脏石蜡切片进行HE染色和电子显微镜观察大鼠肝脏组织的病理学改变和肝细胞中线粒体显微结构的变化。在各组大鼠缺血再灌注后2h、6h、12h、24h后,检测血清中谷丙转氨酶(Alanine aminotransferase,ALT)、谷草转氨酶(Aspartate aminotransferase,AST)、以及肝脏组织超氧化物歧化酶(SuperoxideDismutase,SOD)的活性及丙二醛(Malondialdehyde,MDA)含量,并采用多聚酶链反应(Polymerase Chain Reaction,PCR)技术检测肝组织中IL-6的表达水平,采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测血清中NF-α和IL-1β的水平。实验数据均用x±s表示,所有数据均采用IBM SPSS20.0统计软件包进行数据分析,统计数据采用析因设计方差分析。统计分析中,检验标准为α=0.05。结果1.大鼠再灌注后血清ALT和AST水平我们检测了各组大鼠血清中ALT和AST的含量,结果表明再灌注2h后,B组和C组的ALT和AST含量最高,术后24h大鼠血清ALT和AST值逐渐降低。在各时间段中,B组和C组的血清ALT和AST水平显著高于A组,但C组的血清ALT和AST水平在个时间段均低于B组,差异均有统计学意义(P<0.05)。2.肝脏组织病理改变HE染色结果显示对照组肝细胞索排列正常、肝窦内皮细胞均正常；B组大鼠再灌注后12h,中央静脉周围的肝细胞明显肿胀,肝血窦变窄,肝小叶结构不明显,伴有红细胞和血栓,周围出现浸润的炎症细胞；C组大鼠肝小叶结构基本正常,肝细胞轻微肿胀,肝组织无明显改变。3.肝细胞的超微结构损伤缺血再灌注后12h,B组大鼠肝细胞线粒体体积大小不一,呈明显肿胀状态,呈类圆形,内有空泡变性,严重者可见嵴减少、断裂或消失,周围内质网结构分别不清；C组大鼠肝细胞线粒体轻微肿胀,呈椭圆形,内嵴部分断裂,多数正常排列,周围内质网结构尚存。4.缺血预适应降低缺血再灌注后肝脏IL-6的表达RT-PCR结果显示：B组各时间点的IL-6表达量均高于A组,C组6h、12h和24hIL-6表达量均高于A组,差异均有统计学意义(P<0.05)。B组中,再灌注后12h IL-6的表达最高,之后呈下降趋势；C组2h、6h和12h的IL-6表达量均低于B组,差异均有统计学意义(P<0.05)。5.各组大鼠血清中TNF-α和IL-1β水平变化ELISA结果显示,与正常对照组相比,缺血再灌注损伤后B组和C组大鼠血清中TNF-a和IL-1p水平有不同程度的升高,差异有统计学意义(P<0.05),在I/R后12h达到峰值；I/R后6h、12h、14h C组大鼠血清中TNF-α和IL-1β水平均低于B组(P<0.05)。6.各组肝脏组织中SOD及MDA的表达变化实验结果发现,B组与C组肝组织中的SOD活性均低于A组。各时间段中,C组SOD的表达略高于B组,其中术后6h和12h的差异有统计学意义(P<0.05)。B组与C组肝组织中的MDA达均高于A组。各时间段中,C组MDA的表达略低于B组,其差异有统计学意义(P<0.05)。与B组相比,C组的肝组织在各时相中的丙二醛呈低水平表达,超氧化物歧化酶活性较高,提示C组的自由基水平低于B组。结论缺血预适应对大鼠肝移植缺血再灌注损伤具有一定的保护作用,能够显著降低IL-6, TNF-a和IL-1p的表达,降低肝细胞线粒体的损伤,在临床中有一定的作用及意义。更多还原

【Abstract】 Morbidity of liver diseases is comparatively high in China, including hepatitis, hepatocirrhosis, carcinoma, Congenital and metabolic liver disease, routine medications and surgreys could not get satisfied endpoint in therapy. Liver transplantation as the only method could cure liver diseases was in ever-growing demand. But owing to the complexity and difficulty in surgrey and perioperative management, donor liver more or less might endure injure in the transplantation procedures that leading to poor or delayed graft function and even primary graft non-function(PNF). Poor graft function was closely relevant to the poor preservation and ischemia-reperfusion (I/R) injure. Literatures indicated ischemia-reperfusion was one of the most important causes that may induce poor early graft function and affect long-time outcomes. The great disparity between the demand of potential transplant patients in need of liver donors and the availability of grafts urgently demand a satisfied method of graft preservation, and the mechanism of ischemia-reperfusion injure spontaneously became a important research point. As ischemia-reperfusion injure could induce damage and apoptosis to hepatocyte, avoiding ischemia-reperfusion injure was important to the therapy and prognosis.Ischemic preconditioning (IPC) was a procedure to decrease tissue ischemia-reperfusion injure in long-time ischemia by pretreatment of short-time ischemia. Murry raised the conception of pretreatment of ischemia for the first time in1986. The research by Laske and Szmagala revealed pretreatment of ischemia may prevent injure of myocardial ischemia in Percutaneous transluminal coronary angioplasty (PTCA) and cardiosurgery. Research from Tossy showed IPC also had a protection in ischemia-reperfusion injure in renal tissue. Literatures showed that IPC could apply to many other organs, such as brain, liver, small intestine, skeletal muscle, kidney and lung, and the existence of cross ischemic tolerance phenomenon between the various tissues and organs. For example, transient ischemic in skeletal muscle or kidney could effectively reduce the volume of myocardial infarction induced by coronary vascular obstruction. Protective effects and mechanism of IPC against I/R in liver autotransplantation in rats transplantation is not clear, it may reveal the way to decrease hepatocyte injure in liver transplantation.Interleukin-6(IL-6) is one of the core members of the cytokine, with many kinds of biological stimulating activity, widely expressed on activated B lymphocyte, resting T lymphocyte, myeloma cell, hepatocyte, natural killer cell and myeloid leukemia cells. Human IL-6is composed of213amino acids, mainly produced by mononuclear macrophage and Fibroblast, could be induced to produce by Tumor necrosis factor (TNF) and enhanced the adverse effect of TNF in return. IL-6can induce T, B lymphocyte differentiation, stimulate the synthesis of acute phase protein in hepatocyte, catalyse and amplify the inflammatory response and toxicity effects and cause damage to tissues.As a pleiotropic cytokine, IL-6is one of the important factors involved in the regulation of immunity and inflammatory reaction, is the main medium of the body reaction to infection and tissue injury, cooperating with colony stimulating factor (CSF), it can promote the growth and differentiation of marrow cells and enhance the cracking function of natural killer cells. Both viral and bacterial infection could induce the increased expression of IL-6, but this increased expression was also observed in a variety of autoimmune diseases and cancers. As part of the immune system, the raised level of serum IL-6play a important role in eliminating virus. Literatures also indicated the level of serum IL-6positively correlated with the degree of infection and inflammatory reaction, that make IL-6a sensitive index for infection and inflammatory reaction.This study firstly constructed the model of liver autotransplantation of ischemia reperfusion injury in rats. Histologic examination for liver tissue injure degree, laboratory tests for serum Alanine aminotransferase(ALT), Aspartate aminotransferase(AST), Superoxide Dismutase(SOD), Malondialdehydem(MDA) and IL-6, were both conducted in experiment and control group. Serum level of IL-6, TNF-a and IL-1β were compared between two groups, explored its correlation to histologic degree of liver tissue injure and the potential action mechanism. By exploring the mechanism of I/S injure and the injure protection method, this study aimed to supply a evidence for therapy and protection in I/S injure in liver transplantation.Objective To investigate the effect of ischemic preconditioning on ischemia-reperfusion (I/R) injury in orthotopic rat liver transplantation and the changes of Interleukin-6(IL-6), Tumor necrosis factor-a(TNF-a) and Interleukin-1β(IL-1β) levels in liver, we discussed the pathological changes and protection mechanisms of ischemic preconditioning on ischemia-reperfusion injury in orthotopic rat liver transplantation.Methods The portal vein infusion of autologous liver transplantation model was established in rats.72rats were randomly divided into three groups:(1) normal rats control group (group A);(2) the rats liver transplantation group (group B):derectly recept autogenous liver transplantation.(3) the ischemic preconditioning rats liver transplantation group (group C):clip rats liver hepatic artery, portal vein hepatic vein and portal vein in15min, reply liver blood flow, autogenous liver transplantation was performed1h later. Cotton with ether was used for open inhalation anesthesia for rats. Conventional skin preparation and sterilization were conducted before making the median incision. Bowel was moved inside out with a cotton swab to get a better surgrey field. Isolated and cut falciform ligament with tissue shear, raised the left and middle lobe of liver rightward, identified, cut and ligatured the esophageal ligament of liver, isolated liver anticlockwise from here. Firstly separated upward along the esophagus, cut the left triangle ligament, suture ligated and cut the left phrenic vessels. Then raised the right lobe of liver and cut the right triangle ligament. Isolated upward along Suprahepatic vena cava(SVC) to phrenic vein hiatus, then separated downward along cava to the level of left renal vein after ligaturing and cutting the lumbar vein. Separated the ligamentum hepatorenale, ligtured and cut right suprarenal vein. Reseted the liver and isolated the Infrahepatic vena cava(IVC) to the above level of left renal vein. Open the hepatoduodenal ligament and indentified the hepatic artery(HA), isolated it and embeded the silk prepared to block the artery. Continue to free portal vein(PV) from confluence of inferior mesenteric vein and splenic venous to the hilar, pylorus vein was ligatured meanwhile. Finally, hepatogastric ligament was bluntly separated to free each lobe of liver, and the procesure of free of liver accomplished. The diluted heparin(35U/L)3mL were irrigatted into portal vein by puncture needle to sustain fully heparinization. Puncture abdominal aortic proximally between common iliac artery and the left renal artery, vascular clamp was used to fix the needle, then blocked the artery above celiac trunk and below the puncture site. with vascular clamp. Then blocked the SVC and IVC at the level of right renal vein with vascular clamp, cut the vein wall about lmm on the IVC in the longitudinal direction as the outflow tract. Infused4℃heparin (12.5U/mL) in Ringer’s lactate20mL through the PV and abdominal aortic puncture site, at the same time,4℃Ringer’s lactate was pouring the liver surface to get rapid cooling. When the liver turned into the color of yellow soil, ended the perfusion and pull out the needle, repaired PV and abdominal aortic puncture point. The outflow tract on IVC was sutured with8-0Prolene. Blocked the hepatic artery on the lighted line and release the vascular clamps on PV, SVC, IVC and abdominal aortic to regain the blood supply to liver and finish the anhepatic phase. At the same time,38℃saline was pouring the liver surface for rapid rewarming. After opening the hepatic artry, continuous abdominal closure was conducted if there was no intra-abdominal hemorrhage. When the surgrey accomplished, lights were used to rewarm the rats until they got through anesthesia recovery period and arisesed.Anaesthesia was conducted in rats of each group2h,6h,12h and24h after reperfusion,5ml blood was collected through the inferior vena cava. Blood was centrifuged and serum was preserved in-80℃refrigerator for later test of ALT and AST. Cut off liver tissue and wash away the blood with Ringer’s solution, and tissue was sliced into a thickness less than0.5cm, partly fixed in10%neutral formalin, the others preserved in-80℃refrigerator.HE staining was performed with paraffin section of the rats liver12h after operation to observe the pathology changes of liver and changes in the microstructure of the hepatocyte mitochondria by electron microscopy.2h,6h,12h and24h after ischemia reperfusion in each groups, the level of serum Alanine aminotransferase (ALT), aspertate aminotransferase (AST) and liver tissue superoxide dismutase (SOD) activity and Malondialdehyde, Malondialdehyde (MDA) content in rats were detected. The level of interleukin-6expression after ischemia-reperfusion Injury in orthotopic rat liver transplantation was detected by polymerase chain reaction. The levels of TNF-a and IL-1β following I/R injury in rat serum were measured by enzyme linked immunosorbent assay.Data was analyzed using IBM SPSS20.0for Windows software. Data were expressed as mean and standard deviation. The statistical significance of differences between groups was analyzed using factorial design ANOVA. P value<0.05was considered to be statistically significant.Results1. The rat serum ALT and AST levels after reperfusion. We tested ALT and AST level of the rats serum in each groups.The results show that the content of ALT and AST level in group B and group C reach the top at2h after reperfusion.The ALT and AST levels of rat serum were gradually reducing in24h after operation.In each period,the levels of serum ALT and AST in group B and group C is obviously higher than that in group A. AST and AST levels in group C were lower compared thanaaaa that in group B at each time point, whose differences were statistically significant.(P<0.05).2. The liver pathological tissue changes stain in HE. The result show that both the arrangement of liver cell line and liver sinus endothelial cells are normal.In12h after reperfusion,the liver cells around the central vein become markedly swollen,liver blood sinus become narrow,and the structure of hepatic lobule appears unclear,which is company with red blood cells and blood clots and infiltrated by inflammatory cells in group B.In the roup C,the structure of hepatic lobule is essentially normal,the liver cells appears slight swell,the liver tissue has no obvious change.3. The injury in ultrastructure of liver cell12h after ischemia reperfuson in group B,the mitochondria volume appears to be different size and markedly swollen state,which is round like with degeneration in vacuolar.It shows that crest reduction,fault and disappearance can be found in some serious ones,endoplasmic reticulum around structures were not clear.The mitochiondria volume is a little swollen, which appears to be oval and rupture in internal crest,which is most normal arrangement.The structure of endoplasmic reticulum is still exsit.4. Ischemic preconditioning reduce the expression of IL-6in liver after ischemia-reperfusion. The result of RT-PCR shows that,a small amount of expression of IL-6could be found in group A,little fluctuation appears in each time. Expression of IL-6in group B and group C were higher than that in group A,whose differences have statistically significant (P<0.05).In group B, the expression of IL-6reachs the highest level in12h after reperfusion,and a downward trend appears after that. Expression of IL-6in all period of group A were lower than group B,whose difference at2h,6h,12h were statistically significant (P<0.05).5. The changes of rats serum TNF-α and IL-10level in each group The result of ELISA shows that,compared with normal control group,there is a different degree of rise in the rats serum TNF-α and IL-1β in group B and group C after ischemia-reperfusion injury,whose differences have statistically significant,the level reach the peak in12h after I/R, the level of rats serum TNF-α and IL-1β at6h、12h and14h in group C is lower than that in group B.6.The experiment conclusion of change in expression of SOD and MDA in liver tissue in each groups shows that,SOD activity of liver tissue in group B and group C were lower than that of group A.Each time,the expression of SOD in group C is slightly higher than that of group B,the differences of expression at6h and12h after operation were statistically significant (P<0.05).The expression of MDA in liver tissue in group B and group C were higher than that in group A.In each period, the expression of MDA in group C is slightliy lower than group B,whose difference was statistically significant (P<0.05).compared with group B, the expression of malondialdehyde is in the low lever in all period of liver tissue in group C,the activity of superoxide dismutase (sod) is higher,which prompt the level of free radical in group C is lower than that in group C.ConclusionsIschemic preconditioning has certain protection effect on ischemia-reperfusion injury of rat liver transplantation. Ischemic preconditioning can significantly reduce the expression of il-6, alleviate mitochondria damage in liver cell and has important potential clinical effect.更多还原