一氧化氮合酶与神经管畸形关系的研究

发布时间：2018-06-17 18:09

本文选题：乙烯硫脲 + 神经管畸形　；参考：《中国医科大学》2007年博士论文

【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(P0.05)。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(P0.01)。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。
[Abstract]:Purpose

Neural tube defects ( NTD ) , one of the most common and most serious birth defects affecting population quality , is one of the most common and most serious birth defects affecting population quality .

Nitric oxide ( NO ) plays an important role in the process of nervous system differentiation , especially in the course of nervous system differentiation .
inducible nitric oxide synthase ( iNOS ) ;
Endothelial nitric oxide synthase ( eNOS ) .

In this study , the expression of nitric oxide synthase ( iNOS ) and eNOS ( eNOS ) in the cerebral cortex of fetal mice was investigated by using ethynylthiourea ( ETU ) as an animal model for neural tube development .

Materials and Methods

1 . Preparation of Rat Model of Neurovascular malformation and Animal Grouping

In the control group , 50 pregnant rats were randomly selected to be ETU group . At the end of pregnancy , 50 pregnant rats were randomly selected as ETU group . Then , the brain tissues of fetal mice were taken from the control group and fixed for 3 hours with 0.9 % normal saline and 20 ml of 4 % polyformaldehyde respectively . After the rats were placed in 4 % polyformaldehyde for 3 hours , the rats were transferred to 30 % sucrose overnight , and the fixed specimen was frozen in coronal ice sections or paraffin embedded ;
The experiment was divided into four groups : the control group , the drug - free group , the bifida group and the brain swelling group .

Observation on pathological changes of cerebral cortex in fetal rat with neural tube malformation

1 . HE staining was performed on the cerebral cortex of E20 fetal rat , so as to clarify the pathological changes of cerebral cortex .

2 . The apoptosis of cerebral cortex neurons in different groups of E20 fetuses was detected by in situ detection method , and the apoptotic index was calculated and X ~ 2 test was performed .

3 . Using immunofluorescence technique to detect the distribution and expression of bcl - 2 in cerebral cortex of E20 fetal rat . Western blot was used to detect the semi - quantitative analysis of caspase - 3 and bcl - 2 protein expression in fetal rat cerebral cortex .

Differential expression of NOS in cerebral cortex of fetal rat with neural tube malformation

1 . The distribution and expression of NOS , iNOS and eNOS in cerebral cortex neurons and cerebral vascular endothelial cells of E20 fetal mice were detected by immunofluorescence technique , and the distribution and expression of eNOS , iNOS and eNOS in cerebral vascular endothelial cells were detected by immunofluorescence technique .
Western blot was used to detect the semi - quantitative analysis of the expression of nitric oxide synthase , iNOS and eNOS in rat cerebral cortex .

2 . The expression of nNOSmRNA , eNOSmRNA and iNOS mRNA was analyzed quantitatively by real - time quantitative PCR .

Results

I . Successful preparation of neural tube deformity model

In the control group , no abnormality was found in the cerebral cortex of the fetuses in the control group , the bifida group and the control group , and the cerebral cortex of the brain expanded group was significantly thinner , the number of cells decreased , some cell degeneration ( nuclear staining and swelling ) were observed .

2 . Apoptosis of the cerebral cortex of E20 fetuses in different groups was detected by in situ cell apoptosis assay .

There was no significant change in the number of Tunel - positive cells in the cerebral cortex of E20 fetuses than in the control group , and the percentage of apoptotic cells was calculated . The percentage of apoptotic cells was calculated by X - 2 test , and the difference of apoptosis index between the brain - expanded group and the control group was statistically significant ( P0.05 ) .

Detection of bcl - 2 expression in E20 fetal rat cerebral cortex by immunofluorescence technique

In the control group , the expression of bcl - 2 positive neurons in the cerebral cortex of E20 fetuses was significantly lower than that in the control group .

Expression of caspase - 3 and bcl - 2 protein in E14 and E20 fetal rat cerebral cortex by Western - blot

1 . The expression level of caspase - 3 and bcl - 2 protein was detected by Western - blot . The results showed that the expression level of caspase - 3 and bcl - 2 protein in the cerebral cortex of rats with no deformity was significantly higher than that in control group .

2 . The expression level of caspase - 3 protein in the cerebral cortex of E14 and E20 rats was detected by Western - blot . The results showed that the expression of caspase - 3 in the cerebral cortex of E14 and E20 was significantly higher than that of the control group ( P0.01 ) .

Differential expression of nitric oxide synthase , eNOS and iNOS protein in rat cerebral cortex

1 . There was no significant change in the number of NOS - positive neurons in the cerebral cortex of E20 fetal mice , but the number of NOS - positive neurons in the cerebral cortex of the brain expanded group was significantly decreased , and the average optical density value was found to be significantly decreased compared with other groups .
There was no significant change in eNOS positive blood vessels in the cerebral cortex of E20 fetal mice , but the number of eNOS positive vessels in the cerebral cortex of the brain expanded group was increased , and the mean fluorescence density value was calculated to show that the expression of eNOS in the cerebral cortex of each group did not change significantly , and the same result was also obtained by calculating the number of V8 stained vessels and the average fluorescence density value .
iNOS positive staining was not detected in each group .

2 . In the Western blot analysis , it was found that in E20 fetal rat cerebral cortex , the expression of eNOS protein in the cerebral cortex of E20 fetal rat was not changed obviously , but the expression of the expression of eNOS protein in the cerebral cortex of E20 fetal rat was significantly decreased , and the expression of eNOS protein was increased .
iNOS protein expression was not detected in each group .

3 . In the Western blot analysis , the expression of eNOS protein in the cerebral cortex of E14 brain was significantly decreased compared with the control group , and the expression level of eNOS protein increased significantly , which was consistent with the results of E20 .
The expression of iNOS was detected in the cerebral cortex of E14 fetal rat , but no significant difference was found between the brain swelling group and the control group , and no iNOS expression was detected in E20 fetal rat cerebral cortex .

Expression of eNOS mRNA , eNOS mRNA and iNOS mRNA in cerebral cortex of E20 fetal mice in different groups

1 . Real - time quantitative PCR assay showed that there was no significant change in the mRNA expression in the cerebral cortex of E20 fetal mice in the control group , the drug - free group , the spinal bifida group and the brain expansion group .

2 . There was no significant change in eNOSmRNA expression in the cerebral cortex of rats with no deformity group , bifida group and control group , but the expression of eNOS mRNA in cerebral cortex of brain expanded group was significantly increased .

3 . iNOSmRNA expression was not detected in each group .

Conclusion

1 . In the process of successfully preparing the rat model of the neural tube with ETU , it was found that 10 days pregnant was the best time to administer the rat model of the neural tube .

2 . The expression of eNOS in the brain tissue of the brain tissue of the neural tube was increased , which caused the increase of NO release , which could lead to the apoptosis of the neurons through the caspase - 3 and bcl - 2 pathway , which led to abnormal brain development caused by the closure defect of the neural tube , suggesting that the eNOS plays a leading role in the development of the neural tube .

3 . The imbalance of cerebral vascular and neuronal development may also be an important factor in brain development .
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

【引证文献】