来自先天性巨结肠症患者的肠神经细胞在自体无神经节结肠中形成神经节。
Enteric Neural Cells From Hirschsprung Disease Patients Form Ganglia in Autologous Aneuronal Colon.
作者信息
Rollo Benjamin N, Zhang Dongcheng, Stamp Lincon A, Menheniott Trevelyan R, Stathopoulos Lefteris, Denham Mark, Dottori Mirella, King Sebastian K, Hutson John M, Newgreen Donald F
机构信息
Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.
Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Victoria, Australia.
出版信息
Cell Mol Gastroenterol Hepatol. 2015 Oct 23;2(1):92-109. doi: 10.1016/j.jcmgh.2015.09.007. eCollection 2016 Jan.
BACKGROUND & AIMS: Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells.
METHODS
Cells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2'-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed.
RESULTS
ENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia.
CONCLUSIONS
NC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient.
背景与目的
先天性巨结肠症（HSCR）是由于神经嵴（NC）来源的细胞在胚胎发育早期未能定植于远端肠道，导致出生后肠神经系统（ENS）缺失及肠道运输功能障碍。治疗方法为远端肠切除，但神经细胞替代可能是一种替代方案。我们测试了患者的无神经节（神经节缺失）结肠组织是否可被自体ENS来源的细胞定植。
方法
从31例HSCR患者的结肠近端切除边缘获取细胞并冻存，使用流式细胞术针对NC标志物p75分离ENS细胞（9例患者）。从远端切除边缘获取无神经节结肠组织（23例患者）。通过免疫组织化学和定量逆转录聚合酶链反应评估ENS细胞的NC标志物，并通过乙炔基-2'-脱氧尿苷标记检测有丝分裂。通过与鸡胚肠道进行器官共培养证明人HSCR出生后ENS来源的细胞定植胚胎肠道的能力，并通过与胚胎小鼠ENS细胞进行器官共培养测试人出生后HSCR无神经节结肠肌肉支持ENS形成的能力。最后，评估HSCR患者ENS细胞定植自体无神经节结肠肌肉组织的能力。
结果
患者来源的经p75分选的ENS细胞表达多种NC祖细胞和分化标志物，并在模拟Wnt信号的条件下在培养中增殖。在器官培养中，患者ENS细胞在无神经的鹌鹑胚胎肠道中适当迁移，小鼠胚胎ENS细胞在患者无神经节结肠肌肉组织中迅速扩散、分化并延伸轴突。HSCR患者来源的出生后ENS细胞在共培养中定植于自体无神经节结肠组织，作为神经元和神经胶质细胞增殖并分化。
结论
可从HSCR患者结肠中获取NC谱系细胞，并且这些细胞可在同一患者的无神经节结肠肌肉中形成ENS样结构。
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