【作者】 郎洁；

【导师】 张培建； 刘歆农；

【作者基本信息】 扬州大学 ， 外科学， 2010， 硕士

【摘要】 目的和意义:肝脏缺血再灌注损伤(hepatic ischemia reperfusion injury, HIRI)存在于任何术式肝移植手术过程中,是造成原发性肝无功能的重要原因之一。导致HIRI的原因很多,目前多数认为其损伤机理主要与微循环障碍、氧自由基的产生、能量物质耗竭、钙离子超载、线粒体功能受损等因素有关。其中微循环障碍起重要作用,并决定最终肝组织的损伤程度。一氧化氮(nitric oxide, NO)和内皮素-1(endothelin-1, ET-1)是两种效用相反的血管活性物质,二者浓度失衡是HIRI微循环障碍的一个重要原因。因此,本实验在建立大鼠自体原位肝移植模型的基础上,探讨板蓝根多糖(indigowoad root polysaccharide,IRPS)对移植肝的影响及NO、ET-1含量变化与其关系,从而为临床实践提供参考,减轻术后的并发症,提高术后的生存质量和生存率。方法:SD大鼠,随机分为3组,NC组:正常对照组(n=32);AT组:自体肝移植组(n=32),术前2周持续给予蒸馏水30mg·kg-1·d-1灌胃处理;IRPS组:板蓝根多糖+自体肝移植组(n=32),术前2周持续给予板蓝根多糖30mg·kg - 1·d– 1灌胃处理。三组大鼠分别于术后1h、6h、12h、24h各取8只处死,经门静脉采血作血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、一氧化氮(NO)、内皮素-1(ET-1)的检测;光镜下观察肝细胞显微结构变化,透射电镜下观察肝细胞及线粒体的超微结构改变。结果:(1)大鼠手术成功的标准:大鼠即刻苏醒、翻身,并能维持有效的呼吸、心跳。无肝期控制为(20±2.2)min。(2)血清ALT、AST的情况:与NC组比较,AT组和IRPS组术后各时相点ALT及AST都有不同程度的升高,差异有统计学意义(P<0.05);但AT组与IRPS组相比较,AT组各时相点升高更加显著,差异有统计学意义(P<0.05)。(3)血清NO、ET-1的情况:与NC相比,AT和IRPS两组术后各时相点血清NO含量都有不同程度的降低,ET-1含量都有不同程度的升高,差异有统计学意义(P <0.05);但AT组与IRPS组相比较,AT组各时相点NO降低及ET-1升高更加显著,差异有统计学意义(P <0.05)。(4)肝细胞显微结构:AT组术后1h,6h,12h,肝细胞中重度水肿,肝窦明显淤血狭窄,胞浆疏松,炎症细胞轻度增多,小叶结构基本完整;与AT组相比,IRPS组肝细胞仅表现为轻度水肿。24h后,AT组肝组织灶状坏死,汇管区及坏死灶淋巴细胞浸润;IRPS组24h仅见肝细胞弥漫性中度水肿,未见明显坏死灶,肝小叶结构完整。(5)肝细胞线粒体超微结构:AT组各时段肝细胞浓缩,细胞间隙变大,电子密度增高,细胞核皱缩变形,染色质粗糙,细胞器明显密集化,线粒体肿胀明显,膜模糊不清,部分膜破裂,线粒体嵴疏松溶解,有大量空泡形成;IRPS组肝细胞形态基本正常,线粒体仅表现为轻度肿胀,线粒体排列整齐。结论:板蓝根多糖可以减轻肝移植过程中的HIRI,其可能与提高NO水平、降低ET-1水平,减轻微循环障碍有关。更多还原

【Abstract】 OBJECTIVE:Hepatic ischemia-reperfusion injury (hepatic ischemia reperfusion injury, HIRI) has existed in any surgical procedure of liver transplantation, which is the one of the important reason of primary nonfunction. There are many facts that leads to HIRI, it is generally accepted that the mechanism is related to microcirculatory disturbances, oxygen free radical generation, depletion of energy, calcium overload, dysfunction of mitochondria. In recent years, studies suggest that microcirculatory disturbances play an important role which can determine the final liver tissue injury. NO and ET-1 are two opposite utility of vasoactive substances, the concentration disequilibrium of which is the significance reason for microcirculation disturbances. Therefore, this experiment is based on the model of orthopotic liver autotransplantation of rats. To investigate the effect on the transplanted liver mediated by indigowoad root polysaccharide (IRPS) after the operation and the change of NO, ET-1 content, which may offer the information for clinical practice, lessen postoperative complications, and elevate the postoperative quality of life and the survival rate.METHODS:SD rats were assigned into three groups randomly, including NC group: normal control group (n=32); AT Group: autotransplantation group (n = 32), before the operation the rats were administer distilled water (30mg/kg) through intragastric administration once per day and continued 14 days; IRPS Group: indigowoad root polysaccharide group (n = 32), the rats in this group were received indigowoad root polysaccharide (30mg/kg) at the same method. Blood samples were collected at 1, 6, 12, 24 hours after operation of three groups for examination of the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), nitric oxide (NO), and endothelin -1 (ET-1); the micro-structure of liver cell under light microscope and the ultrastructural changes of liver cell mitochondria were determined in three groups.RESULTS:(1) Rats surgical success criteria: Rats instantly awake, stand up, and can maintain an effective breathing, heartbeat. anhepatic phase was controlled in 20±2.2 minutes.(2) The status of the serum ALT and AST: As compared with NC group, the ALT and AST was increased at whole process in AT and IRPS group (p<0.05), but compared with IRPS group, the data increased more obviously at whole process in AT group (p<0.05).(3) The status of the serum NO and ET-1: As compared with NC group, the ET-1 was increased at whole process in AT and IRPS group (p<0.05), but compared with IRPS group, the data increased more obviously at whole process in AT group (p<0.05). the serum content of NO was decreased in AT and IRPS group, compared with NC group, but the content of NO was more decreased in AT group than that in IRPS group at whole process.(4) The micro-structure of hepatocytes: In postoperative 1h, 6h and 12h the hepatocytes of AT group was severe edema, sinus hepaticus congestion and narrow significantly, endochylema raritas, and mild increase in inflammatory cells. Compared with the AT group, IRPS group of liver cells showed only mild edema. In postoperative 24h, the hepatocytes of AT group was focal necrosis, and periportal lymphocyte infiltration and necrosis; compared with AT group, the IRPS group only show moderate diffuse edema of liver cells, no significant necrosis, and hepatic lobule structural integrity.(5) The ultrastructural changes of hepatocytes and mitochondria: the hepatocytes in the AT group had concentrated; increased electron density as the cells began to condense, condensed or split nuclei and nucleoli, swollen mitochondria, unclear mitochondrial membranes, some disrupted membranes and mitochondrial cristae which became solubilized. Nevertheless, the appearance of hepatocytes in the IRPS group was much better than that in the AT group. The structure of the mitochondria in the IRPS group was normal and was much better than that in the AT group.CONCLUTION:Indigowoad root polysaccharide on hepatic ischemia reperfusion injury has a protective effect after orthotopic liver autotransplantation, which may be related to increase NO levels and lower levels of ET-1 to reduce the microcirculation disturbance.更多还原