全反式维甲酸诱导大鼠肠神经系统发育异常的实验研究

发布时间：2018-04-19 19:54

本文选题：全反式维甲酸 + 肠神经系统　；参考：《宁夏医科大学》2012年硕士论文

【摘要】：目的利用全反式维甲酸（all-trans retinoic acid, ATRA）制作大鼠肠神经系统（Enteric nervous system）发育异常的动物模型，应用蛋白基因产物9.5（protein geneproduct9.5, PGP9.5）及突触素(synaptophysin, SYP)动态观察ENS在胚胎发育过程的阳性表达及变化并进行透射电镜超微结构研究，以期了解ENS发育及ATRA是否诱导ENS异常发育，为临床防治ENS发育异常诱发的疾病提供实验基础。方法 1.实验一 40只健康成熟未孕的SD雌鼠，体重250～300g，由宁夏医科大学实验动物中心提供。雌雄大鼠按1：1比例于午夜合笼，上午8:10-9:10时检测有阴道栓排出的雌鼠，即认为已交配，此日标为妊娠第0天（Embryonic day0，E0），正常饲养。将已孕雌鼠30只随机分为2组，每组15只。实验组于E10将ATRA按100mg/kg一次性灌胃；对照组于E10给予体积(2.5mL/kg)橄榄油，继续饲养。两组分别于E14-20腹腔麻醉下行剖宫术。检查判定所有胚胎存活数量及畸形并记录。制作结直肠段标本，行HE常规染色及PGP9.5和SYP免疫组化。已知阳性切片作阳性对照，磷酸盐缓冲液代替一抗作空白对照。图像分析、采图、数据记录分析。 2.实验二已孕母鼠8只随机分为2组，每组4只。实验组于E10将ATRA按100mg/kg灌胃；对照组于E10给予体积(2.5mL/kg)橄榄油，继续饲养。两组于E21腹腔麻醉下行剖宫术。制作结直肠电镜标本，预切片定位，超薄切片，透射电镜观察，拍片记录分析。结果 1、免疫组织化学：（1）对照组: E16，在直肠肠壁内可见散在稀疏分布的棕色PGP9.5阳性细胞，直肠上端结肠壁内未能见SYP阳性细胞。从E16-18，PGP9.5环肌浆膜侧出现阳性细胞，成散在单个出现，粘膜下阳性细胞少见。直肠壁内可见SYP棕黄色成条索状的阳性表达，主要分布在肠壁浆膜下层。E20， PGP9.5阳性细胞呈黄褐色团簇状，环肌层和纵肌层结构清晰，排列整齐，粘膜下可见阳性细胞。SYP在E20阳性表达增强。（2）实验组: E16直肠肌间组织开始分化，结构整齐，PGP9.5在直肠中可显示散在分布的阳性细胞，与正常相比无明显改变，直肠大部分未见SYP阳性细胞。E18开始，近端直肠可见PGP9.5阳性表达，直肠壁内可见棕黄色成条索状的SYP阳性表达，但是与正常相比，，发育滞后，体积较小，分布稀疏。E18-E20阳性细胞较正常组密度减低。 2、透射电镜超微结构观察：（1）对照组：神经丛主要分布在环形肌和纵行肌之间，肌间结构排列整齐，细胞间质内可见微管、微丝、线粒体和密集的核糖体，有无髓神经纤维分布，轴突终末都有突触联系。（2）实验组：肌层肌间结构紊乱，可见轴突变性或偶见不典型突触结构，未见典型无髓神经纤维，微管、微丝显示不清，各种细胞器结构模糊，线粒体肿胀。不成熟神经胶质细胞分布，胶质细胞核膜有不规则突起，核固缩、染色质聚集，胞体相对较小，胞浆少。结论 1、ATRA可诱导大鼠ENS发育异常，致畸率高。 2、ENS的发育是一个逐渐成熟的过程。E16实验组和对照组形态无明显差异，随着胎龄的增加，实验组的ENS发育出现异常，且逐渐显著。 3、E21实验组胚胎发育过程中其超微结构与正常组织结构不同。
[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.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332

【参考文献】