【作者】 蔡振刚；

【导师】 王忠裕；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 肝移植已经成为治疗终末期肝病、急性肝衰竭的唯一有效的方法。临床肝移植技术的发展有赖于实验动物的研究,大鼠原位肝移植(orthotopic liver transplantation,OLT)模型由于其价格低廉、饲养管理便捷、操作简单、遗传背景清晰等成为众多实验动物模型的首选,是研究肝脏移植器官保存、缺血-再灌注损伤、血流动力学、免疫抑制剂、移植排斥反应及免疫耐受的理想动物模型。经过30多年中外学者的改进,“二袖套法”成为广泛采用的经典术式。国内由于鼠种、价格等诸多因素,广泛选用SD与Wistar大鼠进行移植,认为SD-Wistar是一组高排斥反应的模型。在肝移植治疗方面尽管各种新的免疫抑制剂不断问世,但移植后的排斥反应仍无法完全避免,诱导移植物抗原免疫耐受,成为解决器官移植排斥的根本方法。此外,免疫抑制剂和抑制方案仍存在不少弊端,寻找高效、低毒及价格低廉的免疫抑制药物为临床移植学界日益关注。青藤碱(sinomenine,SIN)是从防己科防己属青风藤中分离出的主要活性成分,分子式为C19H23NO4,具有镇痛、抗炎等作用,临床主要用于类风湿的治疗,疗效确切。近年来经中、日等国学者的研究,青藤碱的免疫抑制作用受到高度重视。在移植肝脏的病理诊断学方面,彩色多普勒是目前最为常用的影像诊断工具,应用彩色多普勒可以对移植肝脏血流进行监测,了解血流动力学变化,但由于二维图像缺乏客观性,影响了其对移植后排斥反应的诊断价值。背向散射积分技术是近年发展起来的组织定征技术,与组织的病理改变有较好的相关性,有望成为又一重要的诊断技术。本实验以大鼠原位肝移植为模型,探讨青藤碱对大鼠原位肝移植急性排斥反应的作用机制,以及联合环孢素A的治疗效果,同时应用彩色多普勒与背向散射积分技术监测治疗后大鼠肝脏的声学特征,为青藤碱的临床应用和背向散射积分技术的术后监测提供理论依据。第一部分大鼠肝移植模型的建立和术式改进目的:探讨两种改进型大鼠肝移植手术术式对大鼠生存率的影响。方法:经典术式组建立SD-Wistar模型70例,改进术式组35例,比较无肝期时间、肝上下腔静脉吻合时间和大鼠一周生存率。观察经典术式组大鼠的生存时间和肝脏病理改变。结果:经典术式组无肝期20.3±1.6min;吻合肝上下腔静脉时间10.7±1.3min。改进术式组无肝期26.7±4.1min;吻合肝上下腔静脉时间15.1±1.7min。一周生存率,经典术式组17.1%,改进术式组91.4%。经典手术组12只手术成功大鼠生存时间11-22天,平均为14.8±2.8天,大鼠死亡后肝脏病理表现为WilliamsⅢ级。结论:SD-Wistar大鼠原位肝移植模型是一组高排斥反应模型。通过改进肝上下腔静脉离断方式和门静脉袖套制作技术,能够提高大鼠血管吻合质量。大鼠术后能否长期存活的关键在于良好的肝上下腔静脉、门静脉吻合,吻合质量较无肝期时间更为重要。第二部分青藤碱对大鼠原位肝移植急性排斥反应影响的实验研究目的:探讨青藤碱、环孢素及两者联合应用对大鼠肝移植模型排斥反应的影响方法:正常组:8只Wistar大鼠为空白对照。建立SD-Wistar OLT模型,分为四组,对照组:8只未予药物干预,CsA组:8只给予环孢素A30㎎/㎏/d,SIN组:8只给予青藤碱40㎎/㎏/d,CsA+SIN组:8只给予青藤碱40㎎/㎏/d+环孢素15㎎/㎏/d。检测肝功、肾功变化,ELISA法比较各组血浆中IL-2、IL-10、sICAM-1情况,免疫组化法分析肝内TGF-β1表达情况, RT-PCR法分析肾脏TGF-β1mRNA表达情况,TUNEL法分析肝脏内淋巴细胞的凋亡情况,HE染色分析肝脏的病理损害。结果:①对照组肝脏功能损害最为明显,谷丙转氨酶、谷草转氨酶升高,总胆红素升高、直接胆红素明显升高。各组间尿素氮和肌酐均无显著性差异。②CsA组、SIN组、SIN+CsA组IL-2明显低于对照组(P<0.05)。③SIN、SIN+CsA明显刺激IL-10分泌(P<0.05),CsA无明显作用。④对照组sICAM-1明显高于其他四组(P<0.05),随着损伤程度减轻sICAM-1呈下降趋势。⑤各组肝脏表达的TGF-β1蛋白和肾脏TGF-β1mRNA表达均有显著性差异(P<0.05),并CsA组表达最高。⑥移植后肝内汇管区淋巴细胞凋亡以SIN+CsA组最为明显。⑦血浆sICAM与肝脏病理损害间呈正相关(r=0.875,P<0.01)。结论:①SIN能够抑制促炎性因子IL-2、sICAM-1的分泌,促进抑炎性因子IL-10、TGF-β1的表达,抑制炎症反应;②SIN与CsA联合作用能够促进汇管区的淋巴细胞凋亡,诱导免疫耐受的形成;③SIN与CsA联合作用能减少CsA的用量,减轻肾脏纤维化因子TGF-β1的表达;④sICAM-1是监测排斥反应发生和评价抗排斥治疗效果的可靠指标。第三部分应用彩色多普勒与背向散射积分技术对大鼠移植肝脏病理损害的评价目的:应用彩色多普勒与背向散射积分技术评价移植后大鼠肝脏病理损害和药物的治疗作用。方法:正常组:8只Wistar为空白对照。建立SD-Wistar OLT模型,分为四组,对照组:8只未予药物干预,CsA组:8只给予环孢素A30㎎/㎏/d,SIN组:8只给予青藤碱40㎎/㎏/d,CsA+SIN组:8只给予青藤碱40㎎/㎏/d+环孢素15㎎/㎏/d。术后4天、10天测量肝脏的IBS和门静脉、下腔静脉的血流速度,术后10天处死大鼠取肝脏组织行病理检查。结果:①门静脉血流速度:术后4天对照组明显低于各手术组,CsA组与SIN+CsA组速度显著增快,SIN组轻度增快。术后10天血流速度普遍下降(P<0.05)。CsA组与SIN+CsA组仍快于正常组(P<0.05),SIN组与正常组无差异(P>0.05)。②IBS对比:术后4天对照组与SIN组较正常组、CsA组、SIN+CsA组增高(P<0.05),CsA组、SIN+CsA组高于正常组(P<0.05)。术后10天组间IBS对比:CsA组、SIN+CsA组、SIN组明显下降(P<0.05),对照组无明显改变(P>0.05)。③相关性:大鼠肝移植后肝脏病理损害程度与IBS呈正相关关系(r=0.814,P<0.01),动物模型组内肝脏病理损害与门静脉血流速度呈负相关关系(r=-0.776,P<0.01)。结论:①门静脉血流速度降低提示移植肝脏可能发生排斥反应;②移植肝脏的IBS测定能够判定移植肝脏损害的程度。更多还原

【Abstract】 Liver transplantation(LT) has become the major treatment for the end-stage chronic liver disease and severe acute failure.Development in LT has depended on researching in small and large animals, the orthotopic liver transplantation (OLT)has become the first choice in lots of experimental an- imal models for low price, feeding and management easier,simple technique and clear genetic background in rats.The OLT model is an ideal animal model for researching preserving transplanted organ,ischemia- reperfusion injury,hemodynamics,immunosuppressant,graft rejective reaction and imm- uneologic tolerance. After more than 30 years improving by the Chinese and foreign scholars, the two-cuff technique has become the most classic tech- nique. Because of absence other varieties(as Lewis or BN ) and low price, SD and Wistar rats are generally selected for transplantation in our coun- try.This SD-Wistar OLT model is considered a high rejection reaction.Although new immunodepressants constantly emerged, the rejection reaction was still inevitable after transplantation.Inducing immunotolerance of transplanted antigen might be an essential method to solve this problem. In addition, it was deficiency of drug treatment and treatment plans of immune tolenranc in clinical.Therefore, it was becoming the hot issue in clinical transplantation field to look for effective, low-toxicity and cheap immunodepressants.Recently some Chinese herbal medicines has been fou- nd to have the function of immunodepression.The medicinal plant sinom- enium acutum Rehd. Et Wils.has been used in china for more than 2000 years,for the treatment of various diseases.The main active constituent has been identified as the alkaloid sinomenine (7,8-didehydro-4-hydro-xy- 3,7-dimethoxy-17-methyl-9α13α,14α-morphinan-6-one).Sinomenine (SIN) was in clinical used in China and Japan due to analgetic and anti-inflannatory activities.In the pathology diagnostics field of liver transplantation, the color dopplor flow image(CDFI) has been the most used technique of imaging diagnosis.Because two-dimension is lack of objectivity, the diagnostic value of CDFI become weaken.The integrated backscatter(IBS) is a novel technique of tissue charac- terization, and related to pathological change.IBS is expected to be another important technology.In this study, we investigated the functional mechanisms of SIN against acute rejection reaction in the model of OLT, and observed the effect of SIN combining with sub-dose cyclosporine A(CsA), at the same time detecting the acoustic characteristics of liver by color Doppler flow image and IBS.In order to provide experimental evidence for clinical drug administration and application of ultrasonic technology.PartⅠEstablished and modified orthotopic liver transplantation in ratsObjective:To investigate modified technique and observed survival rate of orthotopic liver transplantation(OLT) in rats.Method:Classic operative technique was used in establishing of 70 OLT models in SD-Wistar rats, then established of 35 cases with modified operative technique.Compared the anhepatic phase, anastomosis time of suprahepatic cena cave and one-week survival rate.Observed the survival rate of classic opration group and the change of liver parenchyma.Result: The time of anhepatic phase and anastomosising the supra- hepatic inferior vene cavum (SVC) was 20±1.6min and 10.7±1.3min for using classic technique,26.7±4.1min and 15.1±1.7min for using modified technique.One-week survival rate after one week is 17.1% and 91.4% respecttively .There were 12 survivals in classic operation group,and the survival time was 11-22days(mean14.8±2.8days).After the death of those rats, liver pathology coincided toⅢof Williams system.Conclusion:The OLT model of SD-Wistar consistented with severe rejection reaction.It could improve the stability of OLT by ameliorating the way of retouching SVC and cuff technique.The key point of long-term surviving was favourable anastomoses of the SVC and PV, and the anastomoses quality was more important than anhepatic phase.PartⅡAn experimental study on the effect of sinomenine on the acute rejection reaction after liver transplantation in rats.Objective:To compare the effect of CsA,SIN and their combination on the rejection reaction after the orthotopic liver transplantation in rats.Method: we established the stable orthotopic liver transplantation model.The rat models were randomly divided into four groups,and every group was eight SD-Wistar rats:control group,CsA treated group(30㎎/㎏/d),SIN treated group(40㎎ /㎏ /d),SIN and half-value dose CsA treated group(SIN 40㎎/㎏/d+CsA 15㎎/㎏/d).The blank group was eight Wistar rats.Detected the hepatic function and renal function,Enzyme-linked immu- nosorbent assays was carried out to detect the level of IL-2、IL-10、sICAM-1.The expression of TGF-β1 protein in liver and TGF-β1mRNA in kidney used immunohistochemisty staining and RT-PCR,respectivly.The lymphocyte apoptotic index in header district of liver was detected using the TUNEL.The liver pathological lesion was scored with Williams system after HE staining.Result:⑴The damage of liver function in the control group was the most serious.The level of ALT、AST、TBIL、DBIL in this group were higher than other groups,but the level of BUN CRE in every group had no signif- icant difference.⑵The level of IL-2 of CsA-treated group、SIN-treated gro- up、SIN+CsA-treated group was lower than control group (P<0.05).⑶The SIN-treated group and SIN+CsA-treated group could stimulate the secretion of IL-10, but the CsA had no effect.⑷The level of sICAM-1 in the control group was the highest, and accompanied by less injury, sICAM-1 decreased gradually.⑸The expression of TGF-β1 protein in liver and TGF-β1mRNA in kidney had significant difference in five groups,and the expression of CsA-treated group was the highest.⑹The lymphocyte apoptotic index in header district of SIN+CsA-treated group was the most obvious than other groups(P<0.05).⑺The correlation was positive bet- ween the level sICAM-1 and liver pathological damage(r=0.875,P<0.01).Conclusion:⑴SIN could inhibit the Pro-inflammatory factor IL-2 ICAM-1,and stimulate the inhibition of inflammatory factor.SIN could restrain the inflammatory reaction.⑵SIN and CsA combination could enhance the lymphocyte apoptosis in header district, induced the immune- ological tolerance.⑶SIN and CsA combination could reduce CsA-reduced renal injury, and protected the kidney.⑷The level of sICAM-1 was a relia- ble indicator to monitor the rejection reaction and evaluate the antirejection therapy.PartⅢEvaluatated the hepatic pathological lesion of OLT by CDFI and IBS in ratsObjectie:Evaluated the hepatic pathological lesion and pharmacal therapeutical effect by CDFI and IBS after OLT in ratsMethod:We established the stable orthotopic liver transplantation model. The rat models were randomly divided into four groups,and every group was eight SD-Wistar rats:control group,CsA treated group(30㎎/㎏ /d),SIN treated group(40㎎ /㎏ /d),SIN and half-value dose CsA treated group(SIN 40㎎/㎏/d+CsA 15㎎/㎏/d).The blank group was eight Wistar rats. Measured hepatic IBS and the blood flow velocity of portal vein and inferior vena cava in the fourth and tenth day after OLT in rats.These rats sacrificed,then cut part of liver for pathological examination.Result:⑴In the fourth day:The portal venous flow velocity of control group was lower than other operative groups.The PV-velocity of CsA- treat- ed group and SIN+CsA-treated group were significantly faster,and the PV-velocity of SIN was slightly faster also.In the tenth day:the PV-velocity of all of operative groups slowed down significantly(P<0.05),but the velocity of CsA-treated group and SIN+CsA-treated group were still higher than blank control.The SIN-treated group and the blank group had no difference.⑵In the fourth day: the parameter of IBS in the control and SIN groups were higher than the other groups.And the CsA-treated and SIN+ CsA-treated groups were higher than blank group(P<0.05).In the tenth day:the parameter of IBS were lower than a week ago(P<0.05),but the co- ntrol group had no change(P>0.05).⑶Correlation analysis:The correlation was positive between IBS and liver pathological damage(r=0.814, P<0.01),but between liver pathological damage and portal vena velocity were negative correlation(r=-0.776,P<0.01).Conclusion:⑴Lower velocity of the portal venous could prompt the existence of acute rejection of OLT.⑵Detecting hepatic AII could diagnosis the level of liver damage after OLT.更多还原