【作者】 林艺雄；

【导师】 周杰；

【作者基本信息】 南方医科大学 ， 普通外科学， 2010， 博士

【摘要】 背景肝脏缺血再灌注损伤是肝移植的常见问题之一,缺血后的再灌注不仅不能使肝功能恢复,反而引起移植肝功能受损,严重时导致原发性移植物无功能,直接影响肝移植的效果。随着手术技术的成熟和免疫排斥研究的深入,肝移植发展迅速,供体需求量激增,边缘供体渐为各大器官移植中心所接受。边缘供体耐受缺血缺氧能力较正常供体差,导致移植肝无功能的机会也相对增加。近年来针对缺血再灌注损伤的一些研究发现,TLR4启动了宿主天然免疫系统,从而引起心、肺、肝、肾的缺血再灌注损伤。TLR4是Toll样受体家族(TLRs)重要成员,机体可通过宿主TLRs识别病原体,从而启动先天性免疫系统。灯盏花素(Breviscapine)又名灯盏细辛,是从菊科飞蓬属植物短亭飞蓬中提取出来的黄酮类有效成分,主要成分是灯盏乙素(含量占95%以上)。研究发现灯盏花素有改善心脑血管血流量、抗血小板凝聚、抗氧自由基等作用。临床主要治疗闭塞性脑血管病及其后遗症。因其还能降低血浆纤维蛋白原含量和促进纤溶酶活性、改善微循环、清除氧自由基,因此也有保肝作用。但尚未发现其在肝移植冷保存缺血再灌注损伤中作用的研究报道。鉴于此,本课题采用SD大鼠建立原位肝移植模型,研究灯盏花素对肝脏冷保存缺血再灌注损伤是否具有保护作用；并进一步研究TLR4介导的信息通路是否参与其中。第一部分大鼠原位肝移植模型的建立目的建立大鼠原位肝移植模型,以便进行肝移植冷保存缺血再灌注损伤的研究。方法健康、成年、SPF级纯系雄性SD大鼠(Sprague-Dawley)168只,体重260-320g。实验分为两个阶段进行,其中预实验108只,系手术技巧及方法的练习；正式实验60只。手术操作步骤如下：①游离供体肝脏周围韧带、肝上、肝下下腔静脉,门静脉、肝动脉、肝外胆道及右肾静脉。②穿刺门静脉并肝素化。③使用4℃含肝素12.5U／ml的乳酸钠林格氏液行腹主动脉冷灌注,灌注满意后切取供肝。④在修肝容器中进行供肝修整和袖套安装,必须保证全程浸泡在装4℃乳酸林格氏液的修肝容器中。供肝修整完毕后,置4℃冰箱中保存待用。⑤游离受体肝脏周围韧带、肝上、肝下下腔静脉,门静脉、肝动脉、肝外胆道及右肾静脉。⑥阻断受体肝下下腔静脉、门静脉主干、肝上下腔静脉,切除受体肝脏。⑦将供肝从4℃的保存液中取出,原位置入受体腹腔,重建腔静脉、门静脉和胆管,恢复血流。检查腹腔内无出血后关腹。结果1、共进行SD-SD大鼠原位肝移植84次,其中前期预实验54次,后期定型手术30次。2、在正式实验中,供体手术时间为28.42±1.83min,供肝修整时间为36.58±3.31min,受体手术时间47.25±1.59min,无肝期为21.45±1.57min。3、受体门静脉恢复灌注后未见血栓形成,血液复流和肝脏灌注情况良好。4、54次预实验死亡23例,死亡率为42.59%(23／54)。30次定型手术3例死亡,死亡率为10%(3／30)。死亡原因包括无肝期过长、麻醉过深、空气栓塞、腹腔内出血,等。第二部分灯盏花素保护大鼠肝移植冷缺血再灌注损伤的实验研究目的探讨灯盏花素是否对大鼠肝脏冷保存缺血再灌注损伤具有保护作用。方法1.实验动物分组设计健康、成年、SPF级纯系雄性SD大鼠42只,体重260-320g,随机分为假手术组、生理盐水对照组、低剂量灯盏花素治疗组和高剂量灯盏花素治疗组。将供肝在4℃保存液保存4小时后原位植入受体。按照“二袖套”法进行大鼠原位肝移植术。于供肝恢复灌注6小时后取材。2.观察指标及方法(1)测定各组大鼠血清丙氨酸氨基转移酶及天冬氨酸氨基转移酶。(2)检测各组大鼠肝组织匀浆超氧化物歧化酶、丙二醛和谷胱甘肽过氧化物酶。(3)制作石蜡切片,HE染色,于光镜下观察肝组织病理形态学表现,并进行Suzuki病理学评分。(4)制作电镜切片,在透射电镜下观察细胞的超微结构。(5)TUNEL法检测肝细胞的凋亡情况。计算凋亡指数(AI)。3..统计学分析计量资料采用均数±标准差(i±s)表示,数据用SPSS13.0软件进行统计学分析,四组均数比较采用单向方差分析(One-Way ANOVA),组间两两比较采用最小差异t检验(LSD-t)或Tamhane’s T2检验,相关性分析采用双变量相关分析。Suzuki病理评分采用Kruskal-Wallis H-test.P<0.05为差异有统计学意义。结果1、血清肝功能检测结果生理盐水对照组、灯盏花素低剂量治疗组、灯盏花素高剂量治疗组的ALT、AST水平显著高于假手术组(P<0.05)；灯盏花素治疗后ALT、AST水平较生理盐水对照组降低,其中高剂量治疗组较生理盐水对照组显著下降(P<0.05)；高剂量治疗组ALT、AST水平较低剂量治疗组降低,其中门冬氨酸氨基转移酶的降低为显著性(P<0.05)。2.肝组织匀浆SOD、MDA、GSH-Px检测结果假手术组MDA、GSH-Px的表达水平与其它三组差别显著(P<0.05)；灯盏花素治疗后MDA、GSH-Px的表达水平较生理盐水对照组差别显著(P<0.05)；高剂量治疗组MDA较低剂量治疗组有显著降低(P<0.05)。生理盐水对照组SOD表达较假手术组显著下降(P<0.05)；灯盏花素治疗组SOD较生理盐水对照组高但低于假手术组,其中高剂量治疗组相对于生理盐水对照组和低剂量治疗组均有显著升高(P<0.05)。3.肝组织病理学观察和评分假手术组：肝小叶结构大体正常,镜下肝细胞排列规则,可见双核肝细胞。生理盐水对照组：肝细胞索紊乱,并呈现多灶性的肝细胞水泡样变性、凋亡和坏死,肝窦内可见散在脱落的肝细胞核,病变分布没有特异性。低剂量组：肝细胞轻度水样变性,组织改变较生理盐水对照组轻。高剂量组：肝细胞索清晰可辨,肝细胞结构完整,排列尚规则。通过病理学评分分析,各组肝脏淤血、空泡样变、坏死差异不具有统计学意义(P>0.05),总分比较有统计学意义(P<0.05)。4.透射电镜观察假手术组：肝细胞结构正常。细胞核近圆形,核膜完整,核仁居中；细胞浆内可见线粒体、糖原颗粒、内质网、脂滴结构,其中线粒体由不同切面切成柱形、椭圆形、圆形外观,具有双层界膜,内膜内可见线粒体嵴,基质密度较高。生理盐水对照组：显示肝细胞早期凋亡的结构特点。细胞核皱缩,核内染色质固缩并凝结成块,聚集在核膜周边呈新月状或环状小体；内质网变疏松,线粒体数量明显减少,体积明显增大,线粒体内基质密度明显降低,但保持完整而清晰的双层单位膜。灯盏花素低剂量组：细胞核近圆形,核膜完整,核内染色质有向核膜边集趋势；线粒体数量减少,体积增大,基质密度降低,总体结构改变较B组轻。灯盏花素高剂量组：细胞核近圆形,核膜完整,已出现染色质颗粒状聚集；细胞质中线粒体数量没有明显变化,但分布不均,三五聚集,嵴减少,内质网扩张。结构改变程度介于C组和A组之间。5.凋亡指数(AI)大鼠肝移植后6小时各组肝细胞凋亡指数比较差异显著(P<0.001),假手术组凋亡最轻,生理盐水对照组凋亡最严重；灯盏花素治疗组凋亡较生理盐水对照组轻但比假手术组严重,且高剂量组显著低于低剂量组(P<0.05)。说明灯盏花素能减轻大鼠肝移植术后肝实质细胞的凋亡,且具有剂量依赖关系。结论1、大鼠供肝在冷保存4小时后原位植入受体,术后肝脏恢复灌注后6小时,缺血再灌注损伤明显。血清ALT、AST明显升高；肝组织SOD、GSH-Px降低、脂质过氧化物MDA升高；肝细胞出现凋亡表现。2、受体术前给予灯盏花素预处理后,可以明显减轻移植肝的缺血再灌注损伤。通过抑制氧自由基生成,降低了SOD、GSH-Px的消耗,抑制了脂质过氧化反应,脂质过氧化终产物MDA生成减少,对肝细胞损伤减轻,肝功能好转,肝细胞凋亡减少。第三部分灯盏花素对肝移植冷缺血再灌注损伤TLR4、NF-κB和细胞因子表达的影响目的研究灯盏花素是否通过抑制TLR4启动的天然免疫系统,从而减轻肝移植缺血再灌注损伤。方法1.实验动物分组设计健康、成年、SPF级纯系雄性SD大鼠42只,体重260-320g,随机分为假手术组、生理盐水对照组、低剂量灯盏花素治疗组和高剂量灯盏花素治疗组。将供肝在4℃保存液保存4小时后原位植入受体。按照“二袖套”法进行大鼠原位肝移植术。于供肝恢复灌注6小时后取材。2.观察指标及方法Western Blotting检测TLR4和NF-κB蛋白,免疫组织化学染色观测肝脏TNF-α、IL-1β在组织中的表达情况。3.统计方法计量资料用均数±标准差(x±s)表示,数据用SPSS13.0软件进行统计学分析,多组均数间比较采用方差分析,组间两两比较采用最小差异t检验(LSD-t),P<0.05为差异有统计学意义。结果1. Western Blotting检测结果假手术组肝脏TLR4蛋白的表达量极低,而经缺血再灌注损伤6小时后,生理盐水对照组肝脏TLR4蛋白表达量显著升高(P<0.05)。经灯盏花素处理后TLR4蛋白表达受到抑制(P<0.05),且高剂量组对TLR4的抑制程度较C组显著(P<0.05)。2.免疫组化观测结果假手术组肝细胞内未见TNF-α、IL-1β着色的阳性表达(-)；经冷缺血再灌注损伤6小时后,生理盐水对照组大鼠的肝细胞的细胞浆、细胞核内均出现深棕黄色染色(++～+++),尤以细胞浆内表达明显；经灯盏花素处理后,低剂量组TNF-α的阳性表达强度下降(+～++),阳性表达主要在细胞浆；而高剂量组只表现为细胞浆内轻微黄染(+)。结论1、TLR4介导的天然免疫反应参与了大鼠原位肝移植冷保存再灌注损伤的过程,其信息通路为“LPS→TLR4→核因子κB→细胞因子”。2、采用灯盏花素预处理能够保护大鼠肝移植的冷缺血再灌注损伤,通过抑制TLR4和核因子κB,减少下游TNF-α、IL-1β等炎症性细胞因子的分泌,从而减轻移植肝的缺血再灌注损伤。更多还原

【Abstract】 BackgroundThe damage to the liver caused by ischemia/reperfusion injury (IRI) is one tough problem in liver transplantation. The liver effects anomaly after cold ischemia followed by reperfusion. IRI can even results in primary graft failure(PGF).It affects liver transplantation outcome. With the progress of liver transplantation technology and immunological rejection research, liver transplantation increases rapidly.It demands more and more grafts.Marginal donors are employed.When a patient is dangerously ill because of liver dysfunction,liver transplantation using a marginal donor may save the patient’s life.Ischemia and hypoxia are of great disservice to marginal donors,which lead to primary graft failure easily.A serious of researches have found that TLR4,an important member of TLR family, activates host innate immune system, causing heart, lung, liver, kidney ischemia/reperfusion injury. Recognization pathogens by Toll-like receptor(TLR) of the host is one of the main role of innate immune system.Breviscapine is a flavonoid constituent isolated from a traditional Chinese herb Erigerin breviscapus (Vant.) Hand-Mazz. Scutellarin is the major pharmacologically active compound of breviscapine. Study found that scutellarin known to improve cardiovascular and cerebrovascular blood flow, anti-platelet aggregation, antioxidant free radical. Breviscapine has been widely used in thetreatment of cerebral infarction and its sequela. But there is little research of the breviscapine on protecting donor liver against preservation and reperfusion injury during orthotopic liver transplantation.Based on the conclusions mentioned aboved,we used SD rats to establish orthotopic liver transplantation model to investigate if breviscapine could protect donor liver against cold preservation and reperfusion injury during orthotopic liver transplantation.If the answer was ’yes’,whether the breviscapine initiated by inhibiting TLR4 innate immune system, thereby reducing ischemia reperfusion injury of liver transplantation.Part One Model construction of orthotopic liver transplantation in ratsObjectiveTo construct an orthotopic liver transplantation model in rats for researching the cold preservation ischemia-reperfusion injury of OLT.Methods168 Sprague-Dawley rats (SPF grade) of male and weighing 260～320g were selected for operation. The research was divided into two steps:preliminary experiment 108 rats) for exploring operation skills, and formal experiment(60 rats). The operation was conducted as follows:①Dissected the liver ligament, suprahepatic inferior vena cava, infrahepatic inferior vena cava, portal vein, hepatic artery, biliary tract and right renal vein of the donor.②Heparinization.③Perfused Lactated Ringer’S Solution(4℃, heparin 12.5U/ml) via abdominal aorta puncture points and removed donor liver.④Donor liver preparation and cuffs were performed in container,ensuring the liver container fully immersed in Ringer’s solution (4℃) Then preserved the donor liver in refrigerator at 4℃.⑤Dissected the liver ligament, suprahepatic inferior vena cava, infrahepatic inferior vena cava, portal vein, hepatic artery, biliary tract and right renal vein of the recipient.⑥Blocking infrahepatic inferior vena cava, portal vein and suprahepatic inferior vena cava followed by removing the liver of the recipient.⑦Placing the donor liver into the the original location of the recipient’s abdomin. Reconstruction of superior vena cava, portal vein and bile duct.Ensuring without hemorrhage and closing the abdomen of the recipient.Results1.84 cases of SD-SD rat orthotopic liver transplantation were performed totally. 54 cases of OLT were performed in preliminary experiment and 30 cases of OLT were performed in formal experiment.2. In formal experiment, the average time of donor operation was 28.42±1.83min, the average time of donor liver preparation was 36.58±3.31min, the average time of recipient operation was 47.25±1.59min, unhepatic phase was 21.45±1.57min.3. No thrombosis was found after reperfusion of portal vein.Blood flow was good and liver was perfused well.4.23 cases of recipients died in preliminary experiment and mortality was 42.59 %.3 cases of recipients died in formal experiment and mortality was 10%. The causes of failure in operation included long unhepatic phase, deep anesthesia, air embolism or massive hemorrhage in abdomen. Part Two Experimental study of breviscapine protects donor liver against preservation and reperfusion injury during orthotopic liver transplantation in ratsObjectiveTo explore whether breviscapine protects donor liver against preservation and reperfusion injury during orthotopic liver transplantation in rats.Methods1. Animals and experimental Groups42 Sprague-Dawley rats (SPF grade) of male and weighing 260～320g were randomly divided into four groups(n=6).Group A was sham-operation group. GroupB, saline control group. Group C, low-dose treatment group. Group D, high-dose treatment group.Donor liver was preserved at 4℃for 4 hours and then was implanted into recipient. Orthotopic liver transplantation was performed In accordance with the "two-cuff" method. Specimens were harvested at 6 hours after the donor liver was reperfused.2. Observation indexes and the methodsMeasuring serum Alanine transaminase(ALT) and aspartate aminotransferase (AST) of the recipients.Detecting superoxide dismutase, malondialdehyde and glutathione peroxidase on liver tissue of the recipients.The donor liver was made into paraffin section and HE stained, and observed with light microscope. Suzuki pathological score was executed too.The donor liver was made into electrical microscope slice to observe the ultrastructure of the liver cells.Apoptosis of hepatocytes was detected with TUNEL. Apoptotic cells were examined at original magnification×400 in 5 randomly and blindly chosen fields per section. The apoptotic index is defined as the percentage of apoptosis cellular score to total cellular score.3. Statistical analysisThe experimental data was expressed as mean±SD with statistical analysis conducted by using the SPSS 13.0 for Windows. The data comparision of 4 groups was performed in One-Way ANOVA. Least-significant Difference test or Tamhane’s T2 test for significance of comparisons between groups were applied for multiple comparisons. Only Suzuki pathological score was was performed in Kruskal-Wallis H-test. Differences at P less than 0.05 are considered statistically significant.Results1. serum Alanine transaminase(ALT) and aspartate aminotransferase (AST)Serum ALT, AST of saline control group, low-dose treatment group and high-dose treatment group were significantly higher than which of the sham group(P <0.05). Serum ALT, AST of breviscapine treatment group decreased than which of saline control group. high dose treatment group decreased significantly than which of saline control group. (P<0.05). ALT, AST of high dose treatment group decreased than which of low-dose treatment group and aspartate aminotransferase was significantly lower (P<0.05).2. Superoxide dismutase, malondialdehyde and glutathione peroxidaseMDA, GSH-Px of the sham group were different significantly with the other three groups(P<0.05). MDA, GSH-Px of breviscapine treatment group were different significantly with saline group (P<0.05). MDA of high-dose treatment group was significantly lower than which of lower-dose group (P<0.05). SOD of saline control group was significantly lower than which of the sham group (P<0.05). SOD of breviscapine treatment group was higher than which of saline group but lower than which of the sham group. SOD of high-dose treatment group significantly higher than which of saline group and low-dose treatment group (P<0.05).3. Pathological observation and scoring of liver tissueSham group:Hepatic lobules are generally normal. Hepatocytes are arranged well. There are several dual-core liver cells. Saline control group:Hepatocytes are in great disorder. Vesicles, apoptosis and necrosis can be easily found in iver tissue. Nucleus of hepatocyte can be seen scattered off in sinusoids. Low-dose group:Mild hydropic degeneration of liver cells. The damage is slighter than which in saline control group. High-dose group:Hepatocytes are arranged clearly. Hepatocytes are in integrity. Pathology scores of congestion, vacuolar degeneration and necrosis are not statistically significant (P> 0.05), but total pathology score is statistically significant (P<0.05).4. transmission electron observationSham operation group:Structure of hepatocytes is normal. Nucleus is nearly round and nuclear membrane is integrity. Mitochondria, glycogen granules, endoplasmic reticulum and lipid droplets can be found in cytoplasm.Mitochondria are Cylindrical, oval or round and of double membrane.Saline control group:The liver cells show the structural features of early apoptosis. Nuclear shrinkage, chromatin condensation and clot can be observed. Endoplasmic reticulum become loose. Mitochondria decreased significantly and showed enlarged obviously. The matrix density decreased within mitochondria.Low-dose group:Nucleus is nearly round and nuclear membrane is integrity. The chromatin aggregate to the nuclear membrane. Mitochondria decreased and showed enlarged. The matrix density decreased within mitochondria. The structural change is slighter than which of saline control group.High-dose group:Nucleus is nearly round and nuclear membrane is integrity. The chromatin aggregate into particle. The number of mitochondria doesn’t change, but unevenly distributed. Endoplasmic reticulum. The structural change is between sham operation group and between low-dose group.5. Apoptosis index (AI)Apoptosis index of hepatocyte in every group were significantly different (P <0.001). The apoptosis of sham group was the slightest and that of saline control group was the most serious. The apoptosis of breviscapine treatment group was slighter than saline control group, but was serious than which of the sham group(P <0.05). So breviscapine was thought to reduce the hepatocyte apoptosis after OLT in rats.Conclusions1. Donor liver was preserved at 4℃for 4 hours and then was implanted into recipient. Hepatic ischemia-reperfusion injury was significant 6 hours after the liver was reperfused. Serum ALT, AST increased significantly; SOD and GSH-Px decreased; Lipid peroxide MDA increased. Apoptosis of the hepacyte can be found easily.2. Breviscapine can significantly reduce the hepatic ischemia-reperfusion injury. Which reduces the depletion of SOD and GSH-Px by inhibiting oxygen free radicals. Breviscapine can also inhibits lipid peroxidation and the product of lipid peroxidation, MDA decreased. Liver function was improved. Apoptosis of the hepacyte reduced. Part Three The effect of breviscapine on TLR4, NF-κB and cytokine expression against cold ischemia-reperfusion injury in OLT ObjectiveTo explore whether the breviscapine initiated by inhibiting innate immune system via TLR4, thereby reducing ischemia reperfusion injury of liver transplantation.Methods1. Animals and experimental Groups42 Sprague-Dawley rats (SPF grade) of male and weighing 260～320g were randomly divided into four groups(n=6).Group A was sham-operation group. GroupB, saline control group. Group C, low-dose treatment group. Group D, high-dose treatment group.Donor liver was preserved at 4℃for 4 hours and then was implanted into recipient. Orthotopic liver transplantation was performed In accordance with the "two-cuff" method. Specimens were harvested at 6 hours after the donor liver was reperfused.2. Observation indexes and the methodsWestern Blotting was used to evaluated the express of the TLR4 and NF-κB. Immunohistochemical staining was used to observe the TNF-α, IL-1βexpression.3、Statistical analysisThe experimental data was expressed as mean±SD with statistical analysis conducted by using the SPSS 13.0 for Windows. The data comparision of 4 groups was performed in factorial design’s double-factors variance analysis. Fisher’s posthoc least significant difference test or Tamhane’s T2 test for significance of comparisons between groups were applied for multiple comparisons between the groups. Differences at P less than 0.05 are considered statistically significant. Results1. The results from western blottingTLR4 and NF-κB expression in the liver of sham group is low. Both of saline control group was significantly increased After 6 hours of IRI (P<0.05). Both expression of breviscapine treatmet group was inhibited (P<0.05). Both expression between the high-dose group and the low-dose group was significant (P<0.05).2. The results from immunohistochemical stainingSham group:TNF-αand IL-1βexpression in hepatocytes was negative (-).Saline control group:TNF-αand IL-1βin cytoplasm and nucleus were stained dark brown(++～+++) after 6 hours of ischemia and reperfusion injury, especially in the cytoplasm.Low-dose breviscapine group:TNF-αand IL-1βin cytoplasm and nucleus were brown(+～++), mainly in the cytoplasm.High-dose group:a slight yellow staining in cytoplasm (+).Conclusion1. Innate immune via TLR4 was involved in cold ischemia and reperfusion injury in orthotopic liver transplantation in rats. The pathway was "LPS→TLR4→nuclear factorκB→cytokines."2. Breviscapine, pharmacological preconditioning, protect cold ischemia-reperfusion injury in orthotopic liver transplantation in rats. It reduce the secretion of downstream inflammatory cytokines such as TNF-α, IL-1βby inhibiting TLR4 and nuclear factorκB, thereby reducing the graft ischemia-reperfusion injury.更多还原