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当前位置: 代写毕业论文 >> 临床医学 >> 大鼠肝移植重建肝动脉对胆管超微结构和并发症的影响

大鼠肝移植重建肝动脉对胆管超微结构和并发症的影响

来源:华世论文网
 

          作者:邱明链 曾永毅 林科灿 张国寿 刘景丰 

【摘要】  目的 观察大鼠原位肝移植重建肝动脉对肝内胆管上皮细胞缺血再灌注损伤后超微结构及术后胆道并发症的影响。方法 228只SD大鼠分为假手术组(8只)、肝移植重建肝动脉组(55对)和未重建肝动脉组(55对)。重建肝动脉组和未重建肝动脉组分别于肝脏复流后0.5、3、6、12、24、36、48 h取材,用透射电镜观察肝内胆管上皮细胞的超微结构,通过计算机图像分析系统对线粒体形态计量分析;观察术后胆道并发症。结果 两组肝内胆管上皮细胞损伤均有加重,表现为线粒体肿胀、嵴模糊或消失、微绒毛减少等超微结构改变,至24 h达高峰,以后逐渐恢复。术后两组线粒体平均面积和周径随时间的延长逐渐增大,线粒体数密度随时间延长而减少。在24 h,两组缺血再灌注损伤最显著,之后均开始缓解。在24、36、48 h,两组线粒体平均面积、平均周径比较,差异均有统计学意义(t=-3.566,-7.780,-4.730,-4.610,-2.599,-5.370,P<0.05);在36、48 h,两组线粒体平均数密度比较,差异有统计学意义(t=-4.619,4.000,P<0.05)。重建肝动脉组的胆道并发症发生率低于未重建肝动脉组(χ2=4.286,P<0.05)。结论 大鼠肝移植重建肝动脉对肝内胆管上皮细胞缺血再灌注损伤后的超微结构具有保护作用,有利于术后恢复和减少胆道并发症的发生。

【关键词】  肝移植; 肝动脉重建; 胆道并发症; 大鼠; 超微结构

  Effects of hepatic artery reconstruction in rat with orthotopic liver transplantation on ultrastructure of intrahepatic biliary epithelial cells and biliary complications 

    【Abstract】  Objective  To observe the effects of hepatic artery reconstruction in rat with orthotopic liver transplantation (OLT) on ultrastructure changes of intrahepatic biliary epithelial cells after ischemia reperfusion injury and postoperative biliary complications. Methods  Male SD rats were divided into sham operation group(SO), OLT with hepatic artery reconstruction group (HA) and OLT without hepatic artery reconstruction group (NA). The liver tissue samples were collected at 0.5, 3, 6, 12, 24, 36, 48 hours after ischemia reperfusion in both HA group and NA group. The ultrastructure of intrahepatic biliary epithelial cells was observed by transmission electron microscope, and the morphometric analysis by the computer image analysis system. The postoperative biliary complications were also observed. Results  In HA group and NA group, the injury of intrahepatic biliary epithelial cells aggravated gradually along with the reperfusion. The ultrastructure changes, such as enlarged mitochondria, blurred or disappeared cristae and decreased number of microvilli, were most significant at 24 hours and then recovered gradually. The mitochondrial average area and average perimeter were increased and the mitochondrial numerical density was decreased gradually along with the reperfusion. At 24, 36, 48 hours, the mitochondrial average area and average perimeter in NA group were significantly greater than those in HA group (t=-3.566, -7.780, -4.730, -4.610, -2.599, -5.730, P<0.05). The average numerial density of mitochondia in NA group was significantly less than that in HA group at 36, 48 hours(t=-4.619, 4.000, P<0.05). The incidence rate of biliary complications in HA group is significantly lower than that in NA group (χ2=4.286, P<0.05). Conclusions  Hepatic artery reconstruction in rat with OLT has an protective effect on the ultrastructure of intrahepatic biliary epithelial cells after ischemia reperfusion. It is beneficial to the recovery of intrahepatic bile duct epithelial cells and can reduce the incidence rate of biliary complications.

    【Key words】  Liver transplantation;  Hepatic artery reconstruction;  Biliary complication;  Rat;
Ultrastructure

    大鼠肝移植是肝移植基础研究的常用模型。由于大鼠肝动脉血流量较少,未重建肝动脉并不影响大鼠肝移植术后生存率。因此,在该模型中是否重建肝动脉一直存在争论。本实验通过对大鼠肝内胆管上皮细胞缺血再灌注损伤的变化进行电镜超微结构定量分析,探讨大鼠肝移植重建肝动脉对术后肝内胆管上皮细胞缺血再灌注损伤及胆道并发症的影响。

  1  材料与方法

  1.1  实验分组及肝移植模型的建立

    雄性SD大鼠228只,体质量240~260 g,由福建医科大学实验动物中心提供。分为假手术组(8只)、肝移植重建肝动脉组(55对)和未重建肝动脉组(55对)。其中重建肝动脉组和未重建肝动脉组又根据肝脏再灌注后0.5、3、6、12、24、36、48 h分为7小组,每小组5只。两组各留20只观察术后胆道并发症。
   
  未重建肝动脉组按改良“二袖套”法建立大鼠肝移植模型[1-2]。重建肝动脉组则在未重建肝动脉的基础上,保留供肝的肝固有动脉、肝总动脉及腹腔干,分别结扎、远端离断脾动脉和胃左动脉;修肝时于腹腔干置一自制内支架并结扎固定;植肝时结扎受体左肾动脉远端,近端动脉夹暂时钳夹阻断血流,将受体腹腔干近腹主动脉处剪一楔形小口,置入位于供肝腹腔上的内支架,结扎固定,移去动脉夹,开放血流,即完成重建肝动脉的肝移植模型。未重建肝动脉组供体手术时间为(19.6±2.4)min,无肝期时间为(17.5±2.5)min;重建肝动脉组供体手术时间为(21.3±2.9)min,无肝期时间为(17.9±2.3)min, 肝动脉重建时间为(4.2±1.7)min。两组热缺血和冷缺血时间分别控制在5 min和60 min。假手术组只开腹游离肝周韧带后取标本。

  1.2  术后标本的处理和胆道并发症的观察

    两组分别于术后0.5、3、6、12、24、36、48 h取材。重建肝动脉组先观察肝动脉重建处及供肝肝固有动脉的搏动及充血情况,搏动、充血均良好者,予以取材,否则剔除。标本用3%戊二醛-1.5%多聚甲醛前固定,1%锇酸-1.5%亚铁氰化钾后固定,酒精-丙酮脱水,环氧树脂618包埋剂包埋;超薄切片,醋酸铀、柠檬酸铅染色,在飞利浦208型透射电镜下观察肝内胆管上皮细胞并采集图像,每张切片选出5个肝内胆管上皮细胞,描出其内所有线粒体,计算肝内胆管上皮细胞线粒体平均面积、平均周径和数密度。术后30 d解剖未死亡的大鼠,观察是否存在胆漏、肝外胆管梗阻、肝内小胆管阻塞(肝叶片状黄染)等并发症及重建肝动脉组动脉的通畅情况。

  1.3  统计学分析

    胆管上皮细胞线粒体平均面积、平均周径和数密度的数据采用x±s表示,不同组间均数的比较采用方差分析。胆道并发症发生率采用χ2检验。应用SPSS 13.0进行统计分析。P<0.05为差异有统计学意义。

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