Jin Zhu,Shang Qing,Liu Yuanmei..Establishment and identification of experimental rat model for aganglionosis.[J].Journal of Clinical Pediatric Surgery,2018,17(01):54-59.
无神经节细胞动物模型的建立及鉴定
- Title:
- Establishment and identification of experimental rat model for aganglionosis.
- 关键词:
- Ｈｉｒｓｃｈｓｐｒｕｎｇ病; 动物模型; 大鼠; 苯扎铵化合物
- Keywords:
- Hirschsprung Disease; Models; Animal; Rats; Benzalkonium Compounds
- 文献标志码:
- A
- 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；ｑＲＴＰＣＲ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。ｑＲＴＰＣＲ：２、４周时两组ＧＤＮＦｍＲＮＡ、ｎＮＯＳｍＲＮＡ均无明显差异（Ｐ＞０．０５），６、８周时
实验组ＧＤＮＦｍＲＮＡ比对照组明显降低（０．０６±０．０３ｖｓ１．０５±０．３２；０．３９±０．２４ｖｓ１．０２±０．２２），经统
计学分析差异有意义（Ｐ＜０．００１），８周时ＧＤＮＦｍＲＮＡ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ｎＮＯＳｍＲＮＡ比对照明显降低（０．５４±０．３３ｖｓ１．１４±０．５０；
０．４０±０．２４ｖｓ１．０３±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发生有关。
- Abstract:
- ObjectiveTo establish a simple and valid rat model of aganglionosis.MethodsAmong 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 rectal morphology were observed at Weeks 2,4,6,8 postinjection.The stains of hematoxylineosin (HE) and HuD protein immunofluorescence (IF) were used for counting the number of ganglion cells in mesenteric nervous plexus of rectum.And quantitative real timepolymerase chain reaction (qRTPCR) was employed for detecting the expressions of glial fibrillary acidic protein (GFAP) and neuronal nitric oxide synthase (nNOS) in rectum.ResultsSurvival status and rectal morphology:Varying degrees of diarrhea in two groups disappeared at Week 1.Treatment group showed mild abdominal 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 stricture 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 cells of two groups were 3 to 6 at Week 6 (P<0.05).And mesenteric nervous plexus became smaller and more distorted in treatment group.At 8 weeks postinjection,mesenteric nervous plexus was absent and control group had no abnormities.Immunofluorescence (IF) stain:mesenteric nervous plexus was positive for yellow green fluorescence.Nucleus was obvious while cytoplasm remained obscured.Nerve fiber was nonstained and the boundary of staining was distinct.No intergroup difference existed in mesenteric nervous plexus at Weeks 2 & 4.Smaller size and more irregular shape existed in treatment group versus control group at Week 6 and yellow green fluorescence disappeared at Week 8 postinjection.Control group had no abnormities.Quantitative real timepolymerase chain reaction (qRTPCR):The expressions of GDNF mRNA and nNOS mRNA showed no significant intergroup difference at Weeks 2 & 4 (P>0.05).At Weeks 6 & 8,the expressions of GDNF mRNA and nNOS mRNA were obviously lower in treatment group than those in control group (GDNF:0.06±0.03 vs 1.05±0.32,0.39±0.24 vs 1.02±0.22;nNOS:0.54±0.33 vs 1.14±0.50,0.40±0.24 vs 1.03±0.26).In treatment group,it was obviously higher at Week 8 than that at Week 6.ConclusionAbsence of ganglion cells in rectum may be successfully induced by an injection of BAC into rectum.And abnormal expressions of GDNF and nNOS are probably correlated with an occurrence of aganglionosis.
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