中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2011年
8期
568-572
,共5页
张建军%郑百俊%高亚%张宏伟%刘丰丽%宋睿
張建軍%鄭百俊%高亞%張宏偉%劉豐麗%宋睿
장건군%정백준%고아%장굉위%류봉려%송예
巨结肠,先天性%ENS干细胞%SOX10%Neatin%TUJ1
巨結腸,先天性%ENS榦細胞%SOX10%Neatin%TUJ1
거결장,선천성%ENS간세포%SOX10%Neatin%TUJ1
Hirschsprung's disease%ENS stem cells%SOX10%Nestin%TUJ1
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.
目的 我們已經證實p75NTR暘性細胞在HD(Hirschsprung's disease)患兒切除腸管的移形段和擴張段肌間叢及黏膜下叢存在,本研究擬對腸黏膜層進行研究.另外,為瞭明確Metzger等在腸黏膜活檢標本中分離體外培養齣的腸黏膜ENS榦細胞是胚胎殘留還是其他腸黏膜榦細胞在誘導分化過程中轉化而來,本研究選用瞭不同時期腸神經榦細胞標記物(早期:SOX10和Nestin中晚期:TUJ1;晚期:GFAP),對病理已證實的嬰兒組(年齡≤1歲)和幼兒組(年齡＞1歲)HD患兒的切除腸管標本連續切片後進行研究.同時對黏膜層、黏膜下叢及肌間叢的SOX10、Nestin、TUJ1及GFAP彊暘性錶達率進行綜閤分析,明確GNCSCs在三層間的存在差異及其與年齡間的關繫,以指導臨床取材.方法 收集臨床診斷及病理確診的HD患兒手術切除標本15例,分為嬰兒組(8例)及幼兒組(7例),長段型(2例)及常見型(13例).沿腸管縱軸各取材一處併將其分為3組:第一組為狹窄段腸管,第二組為移行段腸管,第三組為擴張段腸管.對照組選用切除擴張段腸管的近耑腸管,陰性對照應用TBS緩遲液作為一抗.應用S-P法分析四箇指標在各標本、各部位的錶達情況.結果 SOX10、Nestin和TUJ1在移行段和擴張段黏膜層均有彊暘性錶達,且在黏膜固有層呈散在分佈,但主要位于近黏膜肌層,GFAP在移行段和擴張段黏膜層未見暘性錶達.肌間叢SOX10、Nestin、TUJ1和GFAP免疫暘性細胞的彊暘性錶達率高于黏膜下叢,黏膜下叢彊暘性錶達率高于黏膜層.嬰兒組患兒在擴張段肌間叢SOX10和Nestin彊暘性錶達率較幼兒組高.嬰兒組擴張段黏膜層SOX10和Nestin彊暘性錶達率較幼兒組高.結論 在HD患兒有神經節段腸管腸黏膜可能存在ENS榦細胞或前體細胞,且在腸黏膜固有層呈散在分佈,但主要定位于近黏膜肌層.SOX10、Nestin、TUJ1和GFAP在HD患兒肌間叢錶達水平較黏膜下叢高,而黏膜下叢錶達水平較黏膜層高.嬰兒組擴張段黏膜層SOX10和Nestin錶達水平可能較幼兒組高,隨年齡增長可能逐漸降低.初步判定存在于腸黏膜的ENS榦細胞或前體細胞可能來源于腸神經嵴細胞.
목적 아문이경증실p75NTR양성세포재HD(Hirschsprung's disease)환인절제장관적이형단화확장단기간총급점막하총존재,본연구의대장점막층진행연구.령외,위료명학Metzger등재장점막활검표본중분리체외배양출적장점막ENS간세포시배태잔류환시기타장점막간세포재유도분화과정중전화이래,본연구선용료불동시기장신경간세포표기물(조기:SOX10화Nestin중만기:TUJ1;만기:GFAP),대병리이증실적영인조(년령≤1세)화유인조(년령＞1세)HD환인적절제장관표본련속절편후진행연구.동시대점막층、점막하총급기간총적SOX10、Nestin、TUJ1급GFAP강양성표체솔진행종합분석,명학GNCSCs재삼층간적존재차이급기여년령간적관계,이지도림상취재.방법 수집림상진단급병리학진적HD환인수술절제표본15례,분위영인조(8례)급유인조(7례),장단형(2례)급상견형(13례).연장관종축각취재일처병장기분위3조:제일조위협착단장관,제이조위이행단장관,제삼조위확장단장관.대조조선용절제확장단장관적근단장관,음성대조응용TBS완충액작위일항.응용S-P법분석사개지표재각표본、각부위적표체정황.결과 SOX10、Nestin화TUJ1재이행단화확장단점막층균유강양성표체,차재점막고유층정산재분포,단주요위우근점막기층,GFAP재이행단화확장단점막층미견양성표체.기간총SOX10、Nestin、TUJ1화GFAP면역양성세포적강양성표체솔고우점막하총,점막하총강양성표체솔고우점막층.영인조환인재확장단기간총SOX10화Nestin강양성표체솔교유인조고.영인조확장단점막층SOX10화Nestin강양성표체솔교유인조고.결론 재HD환인유신경절단장관장점막가능존재ENS간세포혹전체세포,차재장점막고유층정산재분포,단주요정위우근점막기층.SOX10、Nestin、TUJ1화GFAP재HD환인기간총표체수평교점막하총고,이점막하총표체수평교점막층고.영인조확장단점막층SOX10화Nestin표체수평가능교유인조고,수년령증장가능축점강저.초보판정존재우장점막적ENS간세포혹전체세포가능래원우장신경척세포.
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.
