中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
Chinese Journal of Pediatric Surgery
2015年
9期
661-665
,共5页
陈绪勇%夏雪%韦佳%张洪毅%焦春雷%冯杰雄
陳緒勇%夏雪%韋佳%張洪毅%焦春雷%馮傑雄
진서용%하설%위가%장홍의%초춘뢰%풍걸웅
巨噬细胞活化%Hirschsprung病%小肠结肠炎
巨噬細胞活化%Hirschsprung病%小腸結腸炎
거서세포활화%Hirschsprung병%소장결장염
macrophage activation%Hirschsprung disease%Enterocolitis
目的 探讨巨噬细胞活化在先天性巨结肠相关性小肠结肠炎发病中的作用.方法 饲养并繁殖SPF级Ednrb基因敲除小鼠(先天性巨结肠小鼠模型鼠).选择21日龄EdnrB-/-小鼠作为实验组(6只),相应日龄野生型小鼠作为对照组(6只).收集肠管标本,实验组小鼠结肠肠管依据形态学分为结肠狭窄、移行、扩张段,对照组小鼠肠管相应分为结肠远、中、近段.HE染色法评估各段肠管炎症损伤程度,CD68和TNF-α免疫荧光双染法评估肠管巨噬细胞活化状况,Western blot检测肠管TNF-α蛋白表达水平.结果 HE染色显示EdnrB-/-小鼠结肠扩张段肠管均有不同程度的炎性细胞浸润.免疫荧光染色显示,HD模型小鼠扩张、移行、狭窄段CD68阳性细胞百分数分别为(10.81±2.38)％、(8.33±1.78)％和(4.28±1.25)％;野生型小鼠结肠近、中、远段CD68阳性细胞百分数分别为(2.15±1.17)％、(1.89±0.98)％和(1.97±1.32)％.HD模型小鼠扩张、移行、狭窄段TNF-α阳性细胞百分数分别为(6.81±2.35)％、(5.00±1.41)％和(2.80±0.98)％;野生型小鼠结肠近、中、远段TNF-α阳性细胞数量百分数分别为(1.21±0.56)％、(0.89±0.46)％和(1.07±0.32)％.HD模型小鼠结肠扩张段肠管CD68及TNF-α阳性细胞数量明显增多,与HD模型小鼠移行段、狭窄段及野生型各段肠管相比,差异有统计学意义(P＜0.05).Western blot结果发现EdnrB-/-小鼠结肠扩张段肠管TNF-α表达水平明显升高,与移行段、狭窄段、及对照组各段肠管相比较,差异有统计学意义(P＜0.05);移行段TNF-α表达水平较扩张段降低,但与狭窄段及野生型小鼠肠管相比较仍然升高,差异有统计学意义(P＜0.05).对照组三段肠管均未见明显TNF-α表达.结论 先天性巨结肠小鼠模型中扩张段肠管组织损伤可能与肠壁巨噬细胞活化及分泌的促炎因子有关.
目的 探討巨噬細胞活化在先天性巨結腸相關性小腸結腸炎髮病中的作用.方法 飼養併繁殖SPF級Ednrb基因敲除小鼠(先天性巨結腸小鼠模型鼠).選擇21日齡EdnrB-/-小鼠作為實驗組(6隻),相應日齡野生型小鼠作為對照組(6隻).收集腸管標本,實驗組小鼠結腸腸管依據形態學分為結腸狹窄、移行、擴張段,對照組小鼠腸管相應分為結腸遠、中、近段.HE染色法評估各段腸管炎癥損傷程度,CD68和TNF-α免疫熒光雙染法評估腸管巨噬細胞活化狀況,Western blot檢測腸管TNF-α蛋白錶達水平.結果 HE染色顯示EdnrB-/-小鼠結腸擴張段腸管均有不同程度的炎性細胞浸潤.免疫熒光染色顯示,HD模型小鼠擴張、移行、狹窄段CD68暘性細胞百分數分彆為(10.81±2.38)％、(8.33±1.78)％和(4.28±1.25)％;野生型小鼠結腸近、中、遠段CD68暘性細胞百分數分彆為(2.15±1.17)％、(1.89±0.98)％和(1.97±1.32)％.HD模型小鼠擴張、移行、狹窄段TNF-α暘性細胞百分數分彆為(6.81±2.35)％、(5.00±1.41)％和(2.80±0.98)％;野生型小鼠結腸近、中、遠段TNF-α暘性細胞數量百分數分彆為(1.21±0.56)％、(0.89±0.46)％和(1.07±0.32)％.HD模型小鼠結腸擴張段腸管CD68及TNF-α暘性細胞數量明顯增多,與HD模型小鼠移行段、狹窄段及野生型各段腸管相比,差異有統計學意義(P＜0.05).Western blot結果髮現EdnrB-/-小鼠結腸擴張段腸管TNF-α錶達水平明顯升高,與移行段、狹窄段、及對照組各段腸管相比較,差異有統計學意義(P＜0.05);移行段TNF-α錶達水平較擴張段降低,但與狹窄段及野生型小鼠腸管相比較仍然升高,差異有統計學意義(P＜0.05).對照組三段腸管均未見明顯TNF-α錶達.結論 先天性巨結腸小鼠模型中擴張段腸管組織損傷可能與腸壁巨噬細胞活化及分泌的促炎因子有關.
목적 탐토거서세포활화재선천성거결장상관성소장결장염발병중적작용.방법 사양병번식SPF급Ednrb기인고제소서(선천성거결장소서모형서).선택21일령EdnrB-/-소서작위실험조(6지),상응일령야생형소서작위대조조(6지).수집장관표본,실험조소서결장장관의거형태학분위결장협착、이행、확장단,대조조소서장관상응분위결장원、중、근단.HE염색법평고각단장관염증손상정도,CD68화TNF-α면역형광쌍염법평고장관거서세포활화상황,Western blot검측장관TNF-α단백표체수평.결과 HE염색현시EdnrB-/-소서결장확장단장관균유불동정도적염성세포침윤.면역형광염색현시,HD모형소서확장、이행、협착단CD68양성세포백분수분별위(10.81±2.38)％、(8.33±1.78)％화(4.28±1.25)％;야생형소서결장근、중、원단CD68양성세포백분수분별위(2.15±1.17)％、(1.89±0.98)％화(1.97±1.32)％.HD모형소서확장、이행、협착단TNF-α양성세포백분수분별위(6.81±2.35)％、(5.00±1.41)％화(2.80±0.98)％;야생형소서결장근、중、원단TNF-α양성세포수량백분수분별위(1.21±0.56)％、(0.89±0.46)％화(1.07±0.32)％.HD모형소서결장확장단장관CD68급TNF-α양성세포수량명현증다,여HD모형소서이행단、협착단급야생형각단장관상비,차이유통계학의의(P＜0.05).Western blot결과발현EdnrB-/-소서결장확장단장관TNF-α표체수평명현승고,여이행단、협착단、급대조조각단장관상비교,차이유통계학의의(P＜0.05);이행단TNF-α표체수평교확장단강저,단여협착단급야생형소서장관상비교잉연승고,차이유통계학의의(P＜0.05).대조조삼단장관균미견명현TNF-α표체.결론 선천성거결장소서모형중확장단장관조직손상가능여장벽거서세포활화급분비적촉염인자유관.
Objective To explore the role of macrophage activation in a murine model of Hirschsprung-associated enterocolitis.Methods The Ednrb knockout mice were bred and fed at a SFP level laboratory.Experimental group: 21-day-old EdnrB-/-mice were selected (n =6) and comparableage wild-type mice as control group (n =6).Intestinal samples were collected.The experimental colons were divided into narrow, transitional and expansion sections according to their morphological changes.The control colons were divided into distal, middle and proximal sections.Hematoxylin & eosin (HE) staining was used for assessing the degree of bowel inflammation injury.The expressions of CD68 and tumor necrosis factor-alpha (TNF-α) were detected by immunofluorescent staining for assessing the activation level of intestinal macrophage.And the expression of TNF-α protein was measured by Western blot.Results HE staining showed that dilated bowel of EdnrB-/-mice had varying degrees of inflammatory injury.Immunofluorescent staining showed that the numbers of CD68 positive cells in narrow, transitional and expansion sections were (10.81 ± 2.38) ％, (8.33 ± 1.78) ％ and (4.28± 1.25)％ respectively.However those numbers in distal, middle and proximal sections of control group were (2.15 ± 1.17)％, (1.89± 0.98)％ and (1.97 ± 1.32)％ respectively.The TNFαpositive cells in narrow, transitional and expansion sections were (6.81 ± 2.35) ％, (5.00 ± 1.41) ％ and (2.80 ± 0.98) ％ respectively.In control groups, those numbers were (1.21 ± 0.56) ％, (0.89 ± 0.46) ％ and (1.07 ± 0.32) ％ respectively.Compared with wild-type murine intestines and narrow and transitional sections, the positive cells of CD68 and TNF-α were more intensely stained in dilated colonic segments (P ＜ 0.05).Western blot showed that the expression of TNF-α significantly increased in dilated colonic segments (P＜ 0.05).However, the level of TNF-α was lower in transitional section than that in expansion section (P＜0.05).However, it was still higher than that in narrow section in EdnrB-/-and wide-type mice (P ＜ 0.05).The control group showed no significant expression of TNF-α.Conclusions The injury of expansion section of bowel tissue is serious in a murine model of Hirschsprung's disease.And it may be associated with intestinal macrophage activation and secretion of proinflammatory cytokines.
