【作者】 胡淑敏；

【导师】 陈誉华；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2007， 博士

【摘要】 目的神经管畸形(neural tube defects，NTD)是人类常见先天畸形，是影响人口素质的最常见、最严重的出生缺陷性疾病之一。它是由于在胚胎发育过程中，神经管闭合不全所引起的一组缺陷，包括无脑畸形、脑膨出和脊柱裂等。它们在病因学上是多因素的，是遗传学因素和环境因素共同作用结果。胚胎脑发育的早期阶段主要是神经板隆起、弯曲、闭合形成神经管，神经管的头部从前向后依次发育形成脑的基本组织形态，即端脑、间脑、中脑、后脑，在神经管闭合过程中出现异常就会导致NTD。研究表明一氧化氮(nitric oxide，NO)与脑内许多生理功能相关，尤其在神经系统分化过程中扮演重要角色。另有文献报道在鸡胚NO通过细胞凋亡调节细胞数目影响神经管的闭合，从而影响神经管发育。由于NO极不稳定，半衰期短，目前尚无法直接定位组织中NO的分布，主要通过对一氧化氮合酶(nitric oxide synthase，NOS)的定位来反映NO的分布。NOS是催化合成内源性NO的唯一酶，在很大程度上决定着NO的生物学效应。NOS分为3型，分别为神经元型一氧化氮合酶(neuronal NOS，nNOS)；诱导型一氧化氮合酶(inducible NOS，iNOS)；内皮型一氧化氮合酶(endothelial NOS，eNOS)。研究资料表明NO增高可以促使细胞发生凋亡，从而影响神经管的发育，但在三种类型NOS中，究竟是来源于哪一种类型NOS的NO在神经管发育过程中起主导作用目前尚不清楚。因此，本研究采用乙烯硫脲(Ethylenethiourea，ETU)致畸的大鼠神经管畸形动物模型观察nNOS、iNOS、eNOS在胎鼠大脑皮质中表达情况，从血管内皮细胞与神经细胞相互作用影响神经管发育角度，探讨NOS与脑发育异常的关系，希望为脑发育异常机制提供理论根据。材料和方法一、神经管畸形大鼠模型制备与动物分组Wister大鼠80只(中国医科大学第一临床学院实验动物中心提供)体重250～300克。雌雄(比例3：1)交配后，清晨阴道涂片发现精子定为孕0天，在孕10天时，随机选取50只孕鼠为ETU致畸组，经胃管注入1％ETU，剂量为125mg/kg，制作神经管畸形大鼠模型。10只为对照组，注入等量生理盐水。分别在大鼠孕14d、20d时，剖宫取出胎鼠，一部分胎鼠用0.9％的生理盐水10毫升及4％多聚甲醛20毫升分别进行左心室灌注固定，然后在显微镜下取出胎鼠的大脑组织，置于4％多聚甲醛后固定3小时，转入30％蔗糖过夜，将固定好的标本连续冠状冰冻切片或石蜡包埋；另一部分胎鼠在显微镜下分离新鲜未经固定大脑组织，液氮速冻后于-70℃保存。实验分四组：对照组、给药无畸形组、脊柱裂组、脑膨出组。二、神经管畸形胎鼠大脑皮质的病理改变观测1、对E20胎鼠大脑皮质进行HE染色，以明确脑膨出胎鼠大脑皮质的病理结构变化。2、应用细胞凋亡原位检测法检测不同组E20胎鼠大脑皮质神经元凋亡情况，并计算凋亡指数，进行X~2检验。3、应用免疫荧光技术检测bcl-2在E20胎鼠大脑皮质神经元细胞中的分布和表达，应用Western blot法检测胎鼠大脑皮质中caspase-3、bcl-2的蛋白表达半定量分析。三、神经管畸形胎鼠大脑皮质中NOS的差异表达检测1、应用免疫荧光技术检测nNOS、iNOS、eNOS在E20胎鼠大脑皮质神经元和脑血管内皮细胞中的分布和表达，应用免疫荧光技术检测V8脑血管内皮细胞中的分布和表达；应用Western blot法检测胎鼠大脑皮质中nNOS、iNOS、eNOS的蛋白表达半定量分析。2、应用实时定量PCR对nNOSmRNA、eNOSmRNA、iNOS mRNA的表达作相对定量分析。结果一、神经管畸形模型成功制备在大鼠孕10d给予ETU处理后，孕20d剖腹取出胎鼠发现可出现脑膨出、脊柱裂、无尾、肛门闭锁等多种畸形。对对照组、给药无畸形组、脊柱裂组、脑膨出组E20胎鼠大脑皮质进行HE染色，发现给药无畸形组、脊柱裂组与对照组的胎鼠大脑皮质无明显异常改变，而脑膨出组胎鼠大脑皮质明显变薄，细胞数量减少，部分细胞变性(细胞核淡染，肿胀)。二、应用细胞凋亡原位检测法检测不同组E20胎鼠大脑皮质中细胞凋亡情况E20胎鼠大脑皮质中Tunel阳性细胞数目在正常组、给药无畸形组及脊柱裂组细胞凋亡数无明显改变，而在脑膨出组胎鼠大脑皮质细胞凋亡数明显多于对照组，计算凋亡细胞百分率，然后用X~2检验进行分析，脑膨出组和对照组的细胞凋亡指数差异有统计学意义(P<0.05)。三、免疫荧光技术检测E20胎鼠大脑皮质中bcl-2的表达差异E20胎鼠大脑皮质中bcl-2阳性神经元表达量在对照组、给药无畸形组、脊柱裂组无明显改变，而脑膨出组bcl-2阳性神经元表达量明显减少。四、Western-blot检测E14、E20胎鼠大脑皮质中caspase-3、bcl-2蛋白的表达1、用Western-blot方法检测不同组E20胎鼠大脑皮质中caspase-3、bcl-2蛋白表达水平，结果表明对照组、给药无畸形组、脊柱裂组胎鼠大脑皮质中caspase-3、bcl-2蛋白表达水平无明显改变，而脑膨出组胎鼠大脑皮质中caspase-3蛋白活化片段表达水平明显增加，bcl-2蛋白表达水平明显减少。2、用Western-blot方法检测脑膨出组、对照组E14、E20胎鼠大脑皮质中caspase-3蛋白表达水平，结果表明E14、E20脑膨出组大脑皮质中caspase-3活化片段表达明显高于对照组(P<0.01)。五、胎鼠大脑皮质中nNOS、eNOS和iNOS蛋白的差异表达1、利用免疫荧光技术检测到给药无畸形组、脊柱裂组和对照组E20胎鼠大脑皮质中nNOS阳性神经元数量无明显改变，而脑膨出组胎鼠大脑皮质中nNOS阳性神经元数量明显减少，计算平均光密度值发现与其他组相比，脑膨出组胎鼠大脑皮质中nNOS表达量明显减少；给药无畸形组、脊柱裂组和对照组E20胎鼠大脑皮质中eNOS阳性血管数量无明显改变，而脑膨出组胎鼠大脑皮质中eNOS阳性血管数量增多，计算平均荧光密度值发现各组胎鼠大脑皮质中eNOS的表达无明显改变，同样计算V8染色血管数量及平均荧光密度值亦得到同样结果；在各组未检测到iNOS阳性染色。2、在Western blot分析中，发现E20胎鼠大脑皮质中nNOS、eNOS蛋白表达在给药无畸形组、脊柱裂组和对照组间无明显改变，而脑膨出组E20胎鼠大脑皮质中nNOS蛋白表达明显减少，eNOS蛋白表达增加；在各组未检测到iNOS蛋白表达。3、在Western blot分析中，发现与对照组比较，E14脑膨出组胎鼠大脑皮质中nNOS蛋白表达明显减少，eNOS蛋白表达水平明显增加，与E20结果相一致；在E14胎鼠大脑皮质中检测到iNOS蛋白微量表达，但脑膨出组与对照组比较无明显差异，E20胎鼠大脑皮质中未检测到iNOS表达。六、不同组E20胎鼠大脑皮质中nNOS mRNA、eNOS mRNA和iNOS mRNA的表达1、实时定量PCR检测发现对照组、给药无畸形组、脊柱裂组和脑膨出组E20胎鼠大脑皮质中nNOS mRNA表达无明显改变；2、给药无畸形组、脊柱裂组和对照组胎鼠大脑皮质中eNOSmRNA表达无明显改变，而脑膨出组胎鼠大脑皮质中eNOS mRNA表达明显增多；3、在各组未检测到iNOSmRNA表达。结论1、在用ETU成功制备神经管畸形大鼠模型过程中，发现孕10天是获得神经管畸形大鼠模型的最佳给药时间。2、神经管畸形中脑膨出胎鼠大脑组织中eNOS表达升高，引起NO释放增加，可能是通过caspase-3、bcl-2途径促发神经元凋亡，导致神经管闭合缺陷引起脑发育异常，说明eNOS在神经管畸形发育过程中起主导作用。3、脑血管与神经元发育比例失衡，可能也是脑发育异常的一个重要因素。更多还原

【Abstract】 ObjectiveNeural tube defects(NTD) are a common congenital malformation in humans, which have affected on the human quality and are the most severe desease of birth defects, which comprises a series of defects such as anencephalia, encephalocoele, myeloschisis and so on because of no and delay closure of the neuropore in the progress of developing embryo.NTD are the multi-factors in the etiology, are caused by both genetic factors and environmental factors. Neural tube was formed by the apophysis,bend and close of neural crest. The top of neural tube forms basic encenphalic shape,namely endbrain, interbrain, midbrain and afterbrain. The abnormal closure of neural tube will result in neural tube detects. Encephalocoele is a common congenital malformation whose mechanism was no known. NO participates in the physiologic process of brain. In the process of development,NO can promote the differentiation of nervous system by cell apoptosis and promote both the growth and contact of nerve cell. In ECs,NO is a product of the conversion of L-arginine by endothelial NO synthase,which determines the biologic domino offect of NO. NOS isoforms-neuronal (nNOS), endothelial (eNOS),and induciable(iNOS) were found in the brain.NO induce apoptosis in the early NT development. However,it is yet unclear to which types of NOS play a dominant role in NT development. The present study was undertaken in an attempt to determine if morphogenesis malformation of the NTD could be induced in wister rat by administration of ETU.The morphogenesis of encephalocoele in rats given ETU was investigated by light microscopy. From mutually affect on endothelial cell and the neuron, We explored the molecule bases of changes in encephalocoele through studied expression of eNOS, nNOS, iNOS protein and mRNA. And illuminated the effect of eNOS, nNOS, iNOS on brain in neural tube defects. Materials and methodsMature Wister rats, aged frome 2 to 3 months and weighing approximately 300g,were used in this experiment. sixty female(Animal Laboratory.china medical university)were time mated with a male rat overnight.The day on which sperm was found on vaginal smear was designated as day 0 of gestation.The pregnant rats were divided randomly into control (10rats) and experimental group (50 rats).After pregnant, experimental rats received 1% ETU(125mg/kg) by garage on gestation day 10.Control rats received only the vehicle at the same volume. All dams received cesarean section on gestation day 14,20.Fetuses aged E20 brains were perfused via the left ventricle,first with heparin (15,000IU/I)in 0.1 M potassium phosphate-buffered saline(KPBS)for half a minute,and then with 4% paraformaldehyde and immersed in 30% sucrose-KPBS overnight at 4℃,and processed for paraffin sectioning or cut into 10-um cryostat sections.Some brain were stored fluid nitrogen and then stored at-70℃refrigerator.Sections were stained with H￡E for light microscopic examination.1. H￡E staining was carried out in cerebral cortex of rat embryo to determine the pathological changes of rat cerebral cortex in animal model with neural tube defects(NTD).2. In situ Cell Apoptosis Detection (Tunel)Sections are examined by Tunel in order to observe the apoptosis cells in E20 cerebral cortex of all groups.3. The immune-fluorescence method was used to test the distribution and expression of bcl-2 in cerebral cortex of rat embryos; Western blot analysis was performed to determine the semiquantitative of caspase-3 and bcl-2 protern expression.4. The immune-fluorescence method was used to test the distribution and expression of NOS in cerebral cortex of rat embryos;Western blot analysis was performed to determine the semiquantitative of nNOS、eNOS and iNOS protern expression.5. Brain RNA was extracted in cerebral cortex of rat embryo by guanidine isothiocyanate-phenol-chloroform method.Tagman quantitative real time PCR method was used to analysis the quantitative of NOS mRNA in animal model with neural tube defects. Results1. NTD animal model:NTD can be produced by intragastric administration of ETU to timed-pregnant rats:about 80% offspring have NTD. The volume and cells of cerebral cortex were thinner in encephalocoele embryos than in other ETU-injected and in control rat embryos.And there were no evidence changes in histopathologic analysis of brain tissues in other ETU-injected and in control rat embryos.2. Results of In situ cell apoptosis detectionIn E20 cerebral cortex, more Tunel(+) cells were found in encephalocoele group than control group,and the results were significant.3. Expression of Bcl-2 in cerebral neronsIn encephalocoele groups,the bcl-2 immunoreative signal showed trace staining in E20 cerebral cortex compared with other groups.4. Expression of Caspase-3 and bcl-2 proteinIn E20 cerebral cortex, the expression of cleaved Caspase-3 in the encephalocoele group are higher than that in control group, and the expression of bcl-2 in the encephalocoele group are lower than that in control group. These findings were consistent with the results of the western blot analysis in E14 Brain.Comparison of gray values both Caspase-3、bcl-2 and GAPDH was done through Band Scan analysis soft, and the results were significant.5. Expression of nNOS、eNOS and iNOS protein:In encephalocoele groups,the nNOS immunoreative signal showed trace staining in E20 cerebral cortex compared with other groups. The eNOS immunoreative signal showed no change in E20 cerebral cortex in all groups,But the more numbers of eNOS (+) vessel were found in encephalocoele groups than that in other groups. iNOS wasnot detected in all groups. These findings were consistent with the results of the western blot analysis.Western blot analysis confirmed that lower expression levels of nNOS and higher expression of eNOS in E14 brain tissue in the encephalocoele group compared with the control group. These findings were consistent with the results of E20 cerebral cortex. iNOS was detected in E14 brain and There were no evidence changes of expression of iNOS in the encephalocoele group compared with the control group.6. Expression of nNOS mRNA, eNOS mRNA and iNOS mRNA in the E 20 cerebral cortex:There were no obvious changes of expression of nNOS mRNA in ETU-injected and control groups. Expression of eNOS mRNA increased significantly in cerebral cortex of the encephalocoele group, And there were no obvious changes of expression of eNOS mRNA in cerebral cortex of the control group and the other ETU-injection groups. iNOS mRNA weren’t dectected in all groups.Conclusion1. Pregnancy rats treatment with ETU on gestation 10 successily induced NTD.2. The neurons apoptosis induced by eNOS-secreted NO may be involved in caspase-3, bcl-2 singaling pathway in the encephalocoele group, which leads to neural tube closure defects. It suggested that eNOS played a dominant role in the process of NTD.3. The unbalanced proportion between vessels and neurons could resposible for NTD.更多还原