本文選題：全反式維甲酸 + 腸神經(jīng)系統(tǒng)　； 參考：《寧夏醫(yī)科大學》2012年碩士論文

【摘要】：目的利用全反式維甲酸（all-trans retinoic acid, ATRA）制作大鼠腸神經(jīng)系統(tǒng)（Enteric nervous system）發(fā)育異常的動物模型，應用蛋白基因產(chǎn)物9.5（protein geneproduct9.5, PGP9.5）及突觸素(synaptophysin, SYP)動態(tài)觀察ENS在胚胎發(fā)育過程的陽性表達及變化并進行透射電鏡超微結構研究，以期了解ENS發(fā)育及ATRA是否誘導ENS異常發(fā)育，為臨床防治ENS發(fā)育異常誘發(fā)的疾病提供實驗基礎。 方法 1.實驗一 40只健康成熟未孕的SD雌鼠，體重250～300g，由寧夏醫(yī)科大學實驗動物中心提供。雌雄大鼠按1：1比例于午夜合籠，上午8:10-9:10時檢測有陰道栓排出的雌鼠，即認為已交配，此日標為妊娠第0天（Embryonic day0，E0），正常飼養(yǎng)。將已孕雌鼠30只隨機分為2組，每組15只。實驗組于E10將ATRA按100mg/kg一次性灌胃；對照組于E10給予體積(2.5mL/kg)橄欖油，繼續(xù)飼養(yǎng)。兩組分別于E14-20腹腔麻醉下行剖宮術。檢查判定所有胚胎存活數(shù)量及畸形并記錄。制作結直腸段標本，行HE常規(guī)染色及PGP9.5和SYP免疫組化。已知陽性切片作陽性對照，磷酸鹽緩沖液代替一抗作空白對照。圖像分析、采圖、數(shù)據(jù)記錄分析。 2.實驗二 已孕母鼠8只隨機分為2組，每組4只。實驗組于E10將ATRA按100mg/kg灌胃；對照組于E10給予體積(2.5mL/kg)橄欖油，繼續(xù)飼養(yǎng)。兩組于E21腹腔麻醉下行剖宮術。制作結直腸電鏡標本，預切片定位，超薄切片，透射電鏡觀察，拍片記錄分析。 結果 1、免疫組織化學： （1）對照組: E16，在直腸腸壁內可見散在稀疏分布的棕色PGP9.5陽性細胞，直腸上端結腸壁內未能見SYP陽性細胞。從E16-18，PGP9.5環(huán)肌漿膜側出現(xiàn)陽性細胞，成散在單個出現(xiàn)，粘膜下陽性細胞少見。直腸壁內可見SYP棕黃色成條索狀的陽性表達，主要分布在腸壁漿膜下層。E20， PGP9.5陽性細胞呈黃褐色團簇狀，環(huán)肌層和縱肌層結構清晰，排列整齊，粘膜下可見陽性細胞。SYP在E20陽性表達增強。 （2）實驗組: E16直腸肌間組織開始分化，結構整齊，PGP9.5在直腸中可顯示散在分布的陽性細胞，與正常相比無明顯改變，直腸大部分未見SYP陽性細胞。E18開始，近端直腸可見PGP9.5陽性表達，直腸壁內可見棕黃色成條索狀的SYP陽性表達，但是與正常相比，，發(fā)育滯后，體積較小，分布稀疏。E18-E20陽性細胞較正常組密度減低。 2、透射電鏡超微結構觀察： （1）對照組：神經(jīng)叢主要分布在環(huán)形肌和縱行肌之間，肌間結構排列整齊，細胞間質內可見微管、微絲、線粒體和密集的核糖體，有無髓神經(jīng)纖維分布，軸突終末都有突觸聯(lián)系。 （2）實驗組：肌層肌間結構紊亂，可見軸突變性或偶見不典型突觸結構，未見典型無髓神經(jīng)纖維，微管、微絲顯示不清，各種細胞器結構模糊，線粒體腫脹。不成熟神經(jīng)膠質細胞分布，膠質細胞核膜有不規(guī)則突起，核固縮、染色質聚集，胞體相對較小，胞漿少。 結論 1、ATRA可誘導大鼠ENS發(fā)育異常，致畸率高。 2、ENS的發(fā)育是一個逐漸成熟的過程。E16實驗組和對照組形態(tài)無明顯差異，隨著胎齡的增加，實驗組的ENS發(fā)育出現(xiàn)異常，且逐漸顯著。 3、E21實驗組胚胎發(fā)育過程中其超微結構與正常組織結構不同。
[Abstract]:Objective to make use of all-trans retinoic acid (ATRA) to make an animal model of the dysplasia of the intestinal nervous system (Enteric nervous system) in rats. The positive expression and change of the protein gene product 9.5 (protein geneproduct9.5, PGP9.5) and synaptophysin (synaptophysin, SYP) were used to observe and change the positive expression and change of the embryonic development process. The ultrastructural study of transmission electron microscopy is conducted in order to understand whether the development of ENS and the abnormal development of ENS induced by ATRA provide an experimental basis for the clinical prevention and control of the disease induced by ENS.
Method
A 1. experiment
40 SD female mice, healthy and unpregnant, weighed 250 to 300g, were provided by the experimental animal center of Ningxia Medical University. The female and male rats were caged at midnight in the middle of the night, and the female rats with vaginal suppositories were detected at 8:10-9:10 a.m., that was, the day was marked as zeroth days of pregnancy (Embryonic day0, E0) and normal feeding. 30 pregnant female rats were randomly divided. In the 2 group, 15 in each group. The experimental group was given a one-time gavage of ATRA by 100mg/kg at E10, the control group was given a volume (2.5mL/kg) olive oil and continued to be fed in the control group. The two groups were subjected to caesarean section under the E14-20 abdominal anesthesia. The number of survival and deformities of all embryos were checked and recorded. The specimens were made with HE routine staining and PGP9.5 and SYP immunity. Histochemical analysis. Positive positive sections were used as positive control. Phosphate buffer was used instead of single antibody as blank control. Image analysis, image analysis, data recording and analysis were performed.
2. experiment two
8 pregnant female rats were randomly divided into 2 groups, each group was 4. The experimental group was given ATRA by 100mg/kg at E10; the control group was given E10 (2.5mL/kg) olive oil and continued to be fed. The two groups were subjected to caesarean section under E21 abdominal anesthesia. The specimens of the colorectal electron microscope were made, the pre section location, ultrathin section, transmission electron microscope observation and recording analysis were made.
Result
1, immunohistochemistry:
(1) E16, the brown PGP9.5 positive cells scattered scattered in the rectal wall were seen in the rectal wall, and the SYP positive cells were not seen in the colon wall of the upper rectum. From E16-18, the positive cells appeared in the serous side of the PGP9.5 ring muscle. The positive cells were scattered in a single appearance and the submucosal positive cells were rare. The positive expression of the SYP brown yellow streak like expression in the rectal wall was visible. It is distributed in the subserous layer of the intestinal wall.E20, PGP9.5 positive cells are yellow brown clusters, the structure of the ring muscle and the longitudinal muscle layer is clear and orderly, and the positive cells.SYP in the mucous membrane are enhanced in the positive expression of E20.
(2) the experimental group: the E16 rectum intermuscular tissue began to differentiate and the structure was neat. PGP9.5 could display the scattered positive cells in the rectum. There was no obvious change in the rectum. Most of the rectum had no SYP positive cells.E18. The PGP9.5 positive expression was found in the proximal rectum, and the positive expression of the brown yellow streak like SYP was found in the rectal wall, but it was found in the rectal wall. Compared with normal group, the.E18-E20 positive cells were less developed than those in normal group.
2, ultrastructural observation of transmission electron microscope:
(1) the control group: the nerve plexus is mainly distributed between the ring muscle and the longitudinal muscle. The intermuscular structure is arranged neatly. Microtubules, microfilaments, mitochondria and dense ribosomes are seen in the interstitial cells. There are non myelinated nerve fibers and synapse connections at the end of the axon.
(2) the experimental group: muscular intermuscular structure disorder, axon degeneration or occasional untypical synaptic structure, no typical unmyelinated non myelinated nerve fibers, microtubules, microfilament display indistinct, various organelle structures blurred, mitochondria swollen. The distribution of immature neuroglia cells, nuclear condensation, chromatin aggregation and relative body body relative to the glial cells Smaller and less cytoplasm.
conclusion
1, ATRA can induce the abnormal development of ENS in rats, and the rate of teratogenicity is high.
2, the development of ENS is a gradual maturation process, and there is no obvious difference in the morphology between the.E16 experimental group and the control group. With the increase of fetal age, the development of ENS in the experimental group is abnormal and gradually significant.
3, the ultrastructure of E21 experimental group is different from that of normal tissue during embryonic development.

【學位授予單位】：寧夏醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R-332

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