摘要
目的建立一种简单、有效的无神经节细胞大鼠模型,为肠神经干细胞移植治疗先天性巨结肠提供可用的动物模型。方法取新生1周龄乳鼠16窝,每窝中随机取4只肛门灌注生理盐水为对照组,4只肛门灌注1%的苯扎氯铵(BAC)作为实验组,灌注后2、4、6、8周观察两组灌注后表现(饮食、活动、腹部及排便等情况)以及直肠形态;HE染色、HuD免疫荧光(IF)染色观察肠神经节形态,并计算各组大鼠肠肌间神经节的数量;qRT-PCR检测胶质细胞源性神经营养因子(GDNF)以及神经型一氧化氮合酶(nNOS)的表达情况。结果灌注后2、4周,两组大鼠无明显异常,6周后实验组出现轻微腹胀,排便减少,至8周时腹胀明显,不排大便,伴精神萎靡,对照组大鼠无异常表现。2周、4周时两组直肠形态无异常,6周时实验组出现轻度狭窄,8周时明显狭窄,对照组未见异常改变。HE染色:术后2、4周两组直肠神经节细胞大小、形状以及数量上无明显异常(P>0.05),6周时两组神经节细胞数量的中位数分别为3.0和6.0,两组比较差异有统计学意义(z=-5.82,P<0.001),至8周时实验组未见肌间神经节细胞,对照组无变化。免疫荧光染色:HuD在两组大鼠肠肌间神经节中均有表达,2、4周两组无明显区别;6周时,实验组神经节细胞较对照组体积小、形态异常,至8周时,实验组已无荧光表达,对照组无改变。qRT-PCR:2、4周时两组GDNF mRNA、nNOS mRNA均无明显差异(P>0.05),6、8周时实验组GDNF mRNA比对照组明显降低(0.06±0.03 vs 1.05±0.32;0.39±0.24 vs 1.02±0.22),经统计学分析差异有意义(P<0.001),8周时GDNF mRNA的表达量较6周时明显升高,经统计学分析差异有意义(P<0.001);6、8周时实验组nNOS mRNA比对照明显降低(0.54±0.33 vs 1.14±0.50;0.40±0.24 vs 1.03±0.26),经统计学分析差异有意义(P<0.001)。结论直肠灌注BAC可以导致大鼠直肠神经节细胞完全缺如,GDNF以及nNOS异常表达可能与HD发生有�
Objective To establish a simple and valid rat model of aganglionosis. Methods Among 128 neonatal rats aged 1 week in 16 litters ,8 rats per litter were randomly divided into control and treatment groups （ n =4 each）. Saline and benzalkonium chloride （BAC） were injected respectively into rectum. Such clinical manifestations as abdominal distention and defecation and recta[ morphology were observed at Weeks 2, 4,6,8 post-injection. The stains of hematoxylin-eosin （HE） and HuD protein immunofluorescence （IF） were used for counting the number of ganglion cells in mesenterie nervous plexus of rectum. And quantitative real time-polymerase chain reaction （qRT-PCR） was employed for detecting the expressions of glial fibrillary acidic protein （GFAP） and neuronal nitric oxide synthase （nNOS） in rectum. Results Survival status and rectal morphology ： Varying degrees of diarrhea in two groups disappeared at Week 1. Treatment group showed mild ab- dominal distention and decreased defecation at Week 6. Abdominal distention worsened and defecation further improved at Week 8. There was an onset of mental depression. And control group had no obvious abnormalities. There were no obvious abnormal changes in neither groups at Weeks 2 ＆ 4. In treatment group, mild rectal stric- ture occurred at Week 6, worsened at Week 8 and proximal intestine had obvious dilation with an accumulation of intestinal contents. Control group showed no change. HE staining： The mean numbers of ganglion cells of treatment and control groups were 6 vs 6 and 6 vs 6 at Weeks 2 ＆ 4 and size or shape showed no difference （ P 〉 0.05 ）. The mean numbers of ganglion ceils of two groups were 3 to 6 at Week 6 （ P 〈 0.05 ）. And mesen- teric nervous plexus became smaller and more distorted in treatment group. At 8 weeks post-injection, mesenter- ic nervous plexus was absent and control group had no abnormities. Immunofluorescence （IF） stain：mesenteric nervous plexus was positive for yellow green fluorescence. Nucle
出处
《临床小儿外科杂志》
CAS
2018年第1期54-59,共6页
Journal of Clinical Pediatric Surgery
基金
贵州省科学技术基金项目(黔科合J字[2012]2364号)
遵义市"15851"人才项目
遵义医学院附属医院硕士科研启动基金(院字(2011)01号)
