先天性巨结肠患儿切除肠管肠黏膜ENS干细胞的初步鉴定

目的 我们已经证实p75NTR阳性细胞在HD(Hirschsprung's disease)患儿切除肠管的移形段和扩张段肌间丛及黏膜下丛存在,本研究拟对肠黏膜层进行研究.另外,为了明确Metzger等在肠黏膜活检标本中分离体外培养出的肠黏膜ENS干细胞是胚胎残留还是其他肠黏膜干细胞在诱导分化过程中转化而来,本研究选用了不同时期肠神经干细胞标记物(早期:SOX10和Nestin中晚期:TUJ1;晚期:GFAP),对病理已证实的婴儿组(年龄≤1岁)和幼儿组(年龄＞1岁)HD患儿的切除肠管标本连续切片后进行研究.同时对黏膜层、黏膜下丛及肌间丛的SOX10、Nestin、TUJ1及GFAP强阳性表达率进行综合分析,明确GNCSCs在三层间的存在差异及其与年龄间的关系,以指导临床取材.方法 收集临床诊断及病理确诊的HD患儿手术切除标本15例,分为婴儿组(8例)及幼儿组(7例),长段型(2例)及常见型(13例).沿肠管纵轴各取材一处并将其分为3组:第一组为狭窄段肠管,第二组为移行段肠管,第三组为扩张段肠管.对照组选用切除扩张段肠管的近端肠管,阴性对照应用TBS缓冲液作为一抗.应用S-P法分析四个指标在各标本、各部位的表达情况.结果 SOX10、Nestin和TUJ1在移行段和扩张段黏膜层均有强阳性表达,且在黏膜固有层呈散在分布,但主要位于近黏膜肌层,GFAP在移行段和扩张段黏膜层未见阳性表达.肌间丛SOX10、Nestin、TUJ1和GFAP免疫阳性细胞的强阳性表达率高于黏膜下丛,黏膜下丛强阳性表达率高于黏膜层.婴儿组患儿在扩张段肌间丛SOX10和Nestin强阳性表达率较幼儿组高.婴儿组扩张段黏膜层SOX10和Nestin强阳性表达率较幼儿组高.结论 在HD患儿有神经节段肠管肠黏膜可能存在ENS干细胞或前体细胞,且在肠黏膜固有层呈散在分布,但主要定位于近黏膜肌层.SOX10、Nestin、TUJ1和GFAP在HD患儿肌间丛表达水平较黏膜下丛高,而黏膜下丛表达水平较黏膜层高.婴儿组扩张段黏膜层SOX10和Nestin表达水平可能较幼儿组高,随年龄增长可能逐渐降低.初步判定存在于肠黏膜的ENS干细胞或前体细胞可能来源于肠神经嵴细胞.

Abstract：

Objective To identify the enteric nervous system (ENS) stem cells in intestinal mucosa of the patients with Hirschsprung's disease (HD). Methods Fifteen patients were pathologically diagnosed with Hirschsprung's disease. They underwent surgery to remove the aganglionic intestines.The removed intestines were collected for this study. Of the 15 patients, 8 were infants (age≤1 year)and 7 were toddlers (age＞ 1year). Two were long-segment HD, and 13 were short-segment HD.Three pieces of tissues were collected from the stenotic segment, transitional zone and dilated segment of the removed intestines. The intestines proximate to the dilated segment were selected as controls.Immunohistochemical staining of stem cell markers including SOX10, Nestin, TuJ1 and GFAP was performed to indentify the ENS stem cells in intestinal mucosa. The TBS buffer was used as the negative control of the primary antibody. The S-P method was applied to analyze the expressions of the 4stem cell markers through the intestine. Results The expressions of SOX10, Nestin, and TUJ1 in the mucosa of the transitional zone and dilated segment were strong. And scattered SOX10, Nestin, and TUJ1 positive cells were observed in the lamina propria. But most of the SOX10, Nestin, and TUJ1 positive cells located in mucosa closed to the muscularis mucosa. The expression of GFAP in the mucosa of the transitional zone and dilated segment was negative. The expressions of SOX10, Nestin,TUJ1 and GFAP in myenteric plexus were stronger than those of submucosal plexus (SOX10 expression, infant 50% vs 31.3%, toddler 35. 7% vs 21.4%; Nestin expression, infant 43. 8% vs 37. 5%,toddler 28. 6% vs 14. 2%; TUJ1 expression, infant 37. 5% vs 18. 5%; toddler 28. 6% vs 28. 6%;GFAP expression 25.0 % vs 18. 5 % ; toddler 57. 1% vs 35. 7 % ; P ＜ 0. 05 ). The expressions of SOX10, Nestin and TUJ1 were stronger in submucosal plexus than those in mucosa (SOX10 expression, infant 31.3% vs 25. 0%, toddler 21.4% vs 7. 1% ; Nestin expression, infant 25. 0% vs 18. 8%,toddler 14. 3% vs 14. 3%; TUJ1 expression, infant 18. 5% vs 12. 5 %, toddler 28. 6% vs 7. 1%; P＜0. 05). The expressions of SOX10 and Nestin in myenteric plexus of the dilated segment in infants were stronger than thoset in toddler (SOX10, 75. 0% vs 42. 8% ; Nestin, 62. 5% vs 42. 8%, P＜0. 05). The expressions SOX10 and Nestin in mucosa of the the dilated segment in infants were stronger than those in toddler (SOX10, 37. 5% vs 14. 3%; Nestin, 25. 0% vs 14. 3%, P＜0. 05). Conclusions The ENS stem cells may exist in the mucosa of the ganglionic intestines of HD patients, whichare scattered and mostly located in the mucosa near muscularis mucosa. In HD patients, the expressions of SOX10, Nestin, TUJ1 and GFAP in myenteric plexus are higher than those in submucosal plexus. However, the expressions of SOX10, Nestin, TUJ1 and GFAP in the submucosal plexus are higher than those in the mucous layer. The expressions of SOX10 and Nestin in mucosa of the dilated segment in infants are higher than those in toddlers, and it decreases with age. These observations suggest the ENS stem cells or precursor cells in the gut mucosa may be derived from the gut neural crest cells.

Preliminary identification of enteric nervous system stem cells in intestinal mucosa of the patients with Hirschsprung' s disease

Objective To identify the enteric nervous system (ENS) stem cells in intestinal mucosa of the patients with Hirschsprung's disease (HD). Methods Fifteen patients were pathologically diagnosed with Hirschsprung's disease. They underwent surgery to remove the aganglionic intestines.The removed intestines were collected for this study. Of the 15 patients, 8 were infants (age≤1 year)and 7 were toddlers (age＞ 1year). Two were long-segment HD, and 13 were short-segment HD.Three pieces of tissues were collected from the stenotic segment, transitional zone and dilated segment of the removed intestines. The intestines proximate to the dilated segment were selected as controls.Immunohistochemical staining of stem cell markers including SOX10, Nestin, TuJ1 and GFAP was performed to indentify the ENS stem cells in intestinal mucosa. The TBS buffer was used as the negative control of the primary antibody. The S-P method was applied to analyze the expressions of the 4stem cell markers through the intestine. Results The expressions of SOX10, Nestin, and TUJ1 in the mucosa of the transitional zone and dilated segment were strong. And scattered SOX10, Nestin, and TUJ1 positive cells were observed in the lamina propria. But most of the SOX10, Nestin, and TUJ1 positive cells located in mucosa closed to the muscularis mucosa. The expression of GFAP in the mucosa of the transitional zone and dilated segment was negative. The expressions of SOX10, Nestin,TUJ1 and GFAP in myenteric plexus were stronger than those of submucosal plexus (SOX10 expression, infant 50% vs 31.3%, toddler 35. 7% vs 21.4%; Nestin expression, infant 43. 8% vs 37. 5%,toddler 28. 6% vs 14. 2%; TUJ1 expression, infant 37. 5% vs 18. 5%; toddler 28. 6% vs 28. 6%;GFAP expression 25.0 % vs 18. 5 % ; toddler 57. 1% vs 35. 7 % ; P ＜ 0. 05 ). The expressions of SOX10, Nestin and TUJ1 were stronger in submucosal plexus than those in mucosa (SOX10 expression, infant 31.3% vs 25. 0%, toddler 21.4% vs 7. 1% ; Nestin expression, infant 25. 0% vs 18. 8%,toddler 14. 3% vs 14. 3%; TUJ1 expression, infant 18. 5% vs 12. 5 %, toddler 28. 6% vs 7. 1%; P＜0. 05). The expressions of SOX10 and Nestin in myenteric plexus of the dilated segment in infants were stronger than thoset in toddler (SOX10, 75. 0% vs 42. 8% ; Nestin, 62. 5% vs 42. 8%, P＜0. 05). The expressions SOX10 and Nestin in mucosa of the the dilated segment in infants were stronger than those in toddler (SOX10, 37. 5% vs 14. 3%; Nestin, 25. 0% vs 14. 3%, P＜0. 05). Conclusions The ENS stem cells may exist in the mucosa of the ganglionic intestines of HD patients, whichare scattered and mostly located in the mucosa near muscularis mucosa. In HD patients, the expressions of SOX10, Nestin, TUJ1 and GFAP in myenteric plexus are higher than those in submucosal plexus. However, the expressions of SOX10, Nestin, TUJ1 and GFAP in the submucosal plexus are higher than those in the mucous layer. The expressions of SOX10 and Nestin in mucosa of the dilated segment in infants are higher than those in toddlers, and it decreases with age. These observations suggest the ENS stem cells or precursor cells in the gut mucosa may be derived from the gut neural crest cells.