Department of Surgery, University of Nevada School of Medicine, Las Vegas, NV 89102, USA.
J Pediatr Surg. 2010 Jan;45(1):114-20. doi: 10.1016/j.jpedsurg.2009.10.017.
Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis.
Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO(4). Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined.
The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis.
DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model.
由于缺乏合适的动物模型，对先天性胆道闭锁的研究一直受到阻碍。七鳃鳗是具有明显幼体和成体生命周期阶段的原始脊椎动物。在变态过程中，幼体七鳃鳗的胆道系统消失。七鳃鳗的变态过程被提出作为先天性胆道闭锁的模型。我们已经开始通过评估胆管细胞凋亡来探索七鳃鳗变态过程中的细胞事件。
将海七鳃鳗幼体饲养在受控的环境条件下。通过暴露于 0.01%高氯酸钾将前变态期幼体诱导进行变态。每周对动物进行拍照，并根据外部特征分配变态阶段。收获肝脏并进行常规组织学和免疫组织化学处理。使用脱氧核苷酸转移酶介导的 dUTP-生物素缺口末端标记 (TUNEL) 测定法检测 DNA 片段化，并用细胞角蛋白 19 抗体鉴定胆管细胞。在不同变态阶段确定 TUNEL+胆管细胞的百分比。
在未变态（阶段 0）的七鳃鳗（n=6）中，TUNEL+胆管细胞的百分比为 10%，在阶段 1（n=6）为 51%，在阶段 2（n=5）为 40%，在阶段 3（n=5）为 18%，在阶段 4（n=4）为 9%。常规苏木精和伊红染色的石蜡包埋组织切片显示，在变态的第 3 和第 4 阶段，有频繁的凋亡小体，而没有坏死的组织学证据。
在七鳃鳗诱导变态过程中，最早在变态的早期阶段就发现了 DNA 片段化。需要进一步的研究来验证这个潜在有价值的动物模型。
