先天性巨结肠的持久模型。
A durable model of Hirschsprung's colon.
作者信息
Wagner Justin P, Sullins Veronica F, Khalil Hassan A, Dunn James C Y
机构信息
Department of Surgery, Division of Pediatric Surgery, University of California, Los Angeles, Los Angeles, CA 90095-1749, USA.
Department of Surgery, Division of Pediatric Surgery, University of California, Los Angeles, Los Angeles, CA 90095-1749, USA; Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA 90095-7098, USA.
出版信息
J Pediatr Surg. 2014 Dec;49(12):1804-8. doi: 10.1016/j.jpedsurg.2014.09.024. Epub 2014 Oct 3.
INTRODUCTION
Hirschsprung's disease is characterized by colonic aganglionosis, curable only by surgical correction. Stem cells may offer regenerative benefits while preventing surgical risks. Existing Hirschsprung's model systems are limited by alimentary compromise and spontaneous ganglionic reconstitution. We endeavored to generate a model of permanent colonic aganglionosis to support longitudinal cell therapy studies.
METHODS
Among adult female Lewis rats (n=11), laparotomy was performed and one-centimeter segments of descending colon were isolated from continuity and denervated by trans-serosal benzalkonium chloride (BAC) exposure. Postoperative weights were plotted. The colon segments were retrieved after 50 or 100days. Immunohistochemical staining (IHC) for beta-III tubulin (TUJ1) and glial fibrillary acid protein (GFAP) revealed colonic ganglia. Muscle layer diameter and the presence of ganglia were contrasted between normal and denervated segments.
RESULTS
All animals survived, experienced 5% weight loss after one week, and then consistently gained weight. Isolated segments had significantly hypertrophied smooth muscle layers compared to normal colon. Ganglia were identified by IHC in normal colonic segments, and denervated colonic segments had no IHC evidence of myenteric ganglia.
CONCLUSION
Colonic segmental isolation and denervation result in an effective model of irreversible colonic aganglionosis. Animals retain alimentary function. Muscularis hypertrophy, myenteric denervation, and normal animal longevity are suitable for long-term studies of cell therapy.
引言
先天性巨结肠症的特征是结肠神经节细胞缺失，仅通过手术矫正才可治愈。干细胞可能带来再生益处，同时避免手术风险。现有的先天性巨结肠症模型系统受限于消化功能受损和神经节细胞的自发重建。我们致力于构建一种永久性结肠神经节细胞缺失模型，以支持纵向细胞治疗研究。
方法
选取成年雌性刘易斯大鼠（n = 11），进行剖腹手术，分离出1厘米长的降结肠段，使其与肠管其余部分断开连接，并通过经浆膜暴露苯扎氯铵（BAC）使其去神经支配。绘制术后体重变化曲线。50天或100天后取出结肠段。对β-III微管蛋白（TUJ1）和胶质纤维酸性蛋白（GFAP）进行免疫组织化学染色（IHC）以显示结肠神经节。对比正常段和去神经支配段的肌层直径及神经节的存在情况。
结果
所有动物均存活，术后一周体重减轻5%，随后体重持续增加。与正常结肠相比，分离出的结肠段平滑肌层明显肥厚。通过免疫组织化学染色在正常结肠段中鉴定出神经节，而去神经支配的结肠段没有肌间神经节的免疫组织化学证据。
结论
结肠段分离和去神经支配可形成一种有效的不可逆结肠神经节细胞缺失模型。动物保留消化功能。肌层肥厚、肌间神经去支配以及动物正常寿命适合用于细胞治疗的长期研究。
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