發(fā)布時間：2018-06-17 18:09

  本文選題：乙烯硫脲 + 神經(jīng)管畸形��； 參考：《中國醫(yī)科大學(xué)》2007年博士論文

【摘要】： 目的 神經(jīng)管畸形(neural tube defects，NTD)是人類常見先天畸形，是影響人口素質(zhì)的最常見、最嚴重的出生缺陷性疾病之一。它是由于在胚胎發(fā)育過程中，神經(jīng)管閉合不全所引起的一組缺陷，包括無腦畸形、腦膨出和脊柱裂等。它們在病因?qū)W上是多因素的，是遺傳學(xué)因素和環(huán)境因素共同作用結(jié)果。胚胎腦發(fā)育的早期階段主要是神經(jīng)板隆起、彎曲、閉合形成神經(jīng)管，神經(jīng)管的頭部從前向后依次發(fā)育形成腦的基本組織形態(tài)，即端腦、間腦、中腦、后腦，在神經(jīng)管閉合過程中出現(xiàn)異常就會導(dǎo)致NTD。 研究表明一氧化氮(nitric oxide，NO)與腦內(nèi)許多生理功能相關(guān)，尤其在神經(jīng)系統(tǒng)分化過程中扮演重要角色。另有文獻報道在雞胚NO通過細胞凋亡調(diào)節(jié)細胞數(shù)目影響神經(jīng)管的閉合，從而影響神經(jīng)管發(fā)育。由于NO極不穩(wěn)定，半衰期短，目前尚無法直接定位組織中NO的分布，主要通過對一氧化氮合酶(nitric oxide synthase，NOS)的定位來反映NO的分布。NOS是催化合成內(nèi)源性NO的唯一酶，在很大程度上決定著NO的生物學(xué)效應(yīng)。NOS分為3型，分別為神經(jīng)元型一氧化氮合酶(neuronal NOS，nNOS)；誘導(dǎo)型一氧化氮合酶(inducible NOS，iNOS)；內(nèi)皮型一氧化氮合酶(endothelial NOS，eNOS)。 研究資料表明NO增高可以促使細胞發(fā)生凋亡，從而影響神經(jīng)管的發(fā)育，但在三種類型NOS中，究竟是來源于哪一種類型NOS的NO在神經(jīng)管發(fā)育過程中起主導(dǎo)作用目前尚不清楚。因此，本研究采用乙烯硫脲(Ethylenethiourea，ETU)致畸的大鼠神經(jīng)管畸形動物模型觀察nNOS、iNOS、eNOS在胎鼠大腦皮質(zhì)中表達情況，從血管內(nèi)皮細胞與神經(jīng)細胞相互作用影響神經(jīng)管發(fā)育角度，探討NOS與腦發(fā)育異常的關(guān)系，希望為腦發(fā)育異常機制提供理論根據(jù)。 材料和方法 一、神經(jīng)管畸形大鼠模型制備與動物分組 Wister大鼠80只(中國醫(yī)科大學(xué)第一臨床學(xué)院實驗動物中心提供)體重250～300克。雌雄(比例3：1)交配后，清晨陰道涂片發(fā)現(xiàn)精子定為孕0天，在孕10天時，隨機選取50只孕鼠為ETU致畸組，經(jīng)胃管注入1％ETU，劑量為125mg/kg，制作神經(jīng)管畸形大鼠模型。10只為對照組，注入等量生理鹽水。分別在大鼠孕14d、20d時，剖宮取出胎鼠，一部分胎鼠用0.9％的生理鹽水10毫升及4％多聚甲醛20毫升分別進行左心室灌注固定，然后在顯微鏡下取出胎鼠的大腦組織，置于4％多聚甲醛后固定3小時，轉(zhuǎn)入30％蔗糖過夜，將固定好的標(biāo)本連續(xù)冠狀冰凍切片或石蠟包埋；另一部分胎鼠在顯微鏡下分離新鮮未經(jīng)固定大腦組織，液氮速凍后于-70℃保存。實驗分四組：對照組、給藥無畸形組、脊柱裂組、腦膨出組。 二、神經(jīng)管畸形胎鼠大腦皮質(zhì)的病理改變觀測 1、對E20胎鼠大腦皮質(zhì)進行HE染色，以明確腦膨出胎鼠大腦皮質(zhì)的病理結(jié)構(gòu)變化。 2、應(yīng)用細胞凋亡原位檢測法檢測不同組E20胎鼠大腦皮質(zhì)神經(jīng)元凋亡情況，并計算凋亡指數(shù)，，進行X~2檢驗。 3、應(yīng)用免疫熒光技術(shù)檢測bcl-2在E20胎鼠大腦皮質(zhì)神經(jīng)元細胞中的分布和表達，應(yīng)用Western blot法檢測胎鼠大腦皮質(zhì)中caspase-3、bcl-2的蛋白表達半定量分析。 三、神經(jīng)管畸形胎鼠大腦皮質(zhì)中NOS的差異表達檢測 1、應(yīng)用免疫熒光技術(shù)檢測nNOS、iNOS、eNOS在E20胎鼠大腦皮質(zhì)神經(jīng)元和腦血管內(nèi)皮細胞中的分布和表達，應(yīng)用免疫熒光技術(shù)檢測V8腦血管內(nèi)皮細胞中的分布和表達；應(yīng)用Western blot法檢測胎鼠大腦皮質(zhì)中nNOS、iNOS、eNOS的蛋白表達半定量分析。 2、應(yīng)用實時定量PCR對nNOSmRNA、eNOSmRNA、iNOS mRNA的表達作相對定量分析。 結(jié)果 一、神經(jīng)管畸形模型成功制備 在大鼠孕10d給予ETU處理后，孕20d剖腹取出胎鼠發(fā)現(xiàn)可出現(xiàn)腦膨出、脊柱裂、無尾、肛門閉鎖等多種畸形。對對照組、給藥無畸形組、脊柱裂組、腦膨出組E20胎鼠大腦皮質(zhì)進行HE染色，發(fā)現(xiàn)給藥無畸形組、脊柱裂組與對照組的胎鼠大腦皮質(zhì)無明顯異常改變，而腦膨出組胎鼠大腦皮質(zhì)明顯變薄，細胞數(shù)量減少，部分細胞變性(細胞核淡染，腫脹)。 二、應(yīng)用細胞凋亡原位檢測法檢測不同組E20胎鼠大腦皮質(zhì)中細胞凋亡情況 E20胎鼠大腦皮質(zhì)中Tunel陽性細胞數(shù)目在正常組、給藥無畸形組及脊柱裂組細胞凋亡數(shù)無明顯改變，而在腦膨出組胎鼠大腦皮質(zhì)細胞凋亡數(shù)明顯多于對照組，計算凋亡細胞百分率，然后用X~2檢驗進行分析，腦膨出組和對照組的細胞凋亡指數(shù)差異有統(tǒng)計學(xué)意義(P0.05)。 三、免疫熒光技術(shù)檢測E20胎鼠大腦皮質(zhì)中bcl-2的表達差異 E20胎鼠大腦皮質(zhì)中bcl-2陽性神經(jīng)元表達量在對照組、給藥無畸形組、脊柱裂組無明顯改變，而腦膨出組bcl-2陽性神經(jīng)元表達量明顯減少。 四、Western-blot檢測E14、E20胎鼠大腦皮質(zhì)中caspase-3、bcl-2蛋白的表達 1、用Western-blot方法檢測不同組E20胎鼠大腦皮質(zhì)中caspase-3、bcl-2蛋白表達水平，結(jié)果表明對照組、給藥無畸形組、脊柱裂組胎鼠大腦皮質(zhì)中caspase-3、bcl-2蛋白表達水平無明顯改變，而腦膨出組胎鼠大腦皮質(zhì)中caspase-3蛋白活化片段表達水平明顯增加，bcl-2蛋白表達水平明顯減少。 2、用Western-blot方法檢測腦膨出組、對照組E14、E20胎鼠大腦皮質(zhì)中caspase-3蛋白表達水平，結(jié)果表明E14、E20腦膨出組大腦皮質(zhì)中caspase-3活化片段表達明顯高于對照組(P0.01)。 五、胎鼠大腦皮質(zhì)中nNOS、eNOS和iNOS蛋白的差異表達 1、利用免疫熒光技術(shù)檢測到給藥無畸形組、脊柱裂組和對照組E20胎鼠大腦皮質(zhì)中nNOS陽性神經(jīng)元數(shù)量無明顯改變，而腦膨出組胎鼠大腦皮質(zhì)中nNOS陽性神經(jīng)元數(shù)量明顯減少，計算平均光密度值發(fā)現(xiàn)與其他組相比，腦膨出組胎鼠大腦皮質(zhì)中nNOS表達量明顯減少；給藥無畸形組、脊柱裂組和對照組E20胎鼠大腦皮質(zhì)中eNOS陽性血管數(shù)量無明顯改變，而腦膨出組胎鼠大腦皮質(zhì)中eNOS陽性血管數(shù)量增多，計算平均熒光密度值發(fā)現(xiàn)各組胎鼠大腦皮質(zhì)中eNOS的表達無明顯改變，同樣計算V8染色血管數(shù)量及平均熒光密度值亦得到同樣結(jié)果；在各組未檢測到iNOS陽性染色。 2、在Western blot分析中，發(fā)現(xiàn)E20胎鼠大腦皮質(zhì)中nNOS、eNOS蛋白表達在給藥無畸形組、脊柱裂組和對照組間無明顯改變，而腦膨出組E20胎鼠大腦皮質(zhì)中nNOS蛋白表達明顯減少，eNOS蛋白表達增加；在各組未檢測到iNOS蛋白表達。 3、在Western blot分析中，發(fā)現(xiàn)與對照組比較，E14腦膨出組胎鼠大腦皮質(zhì)中nNOS蛋白表達明顯減少，eNOS蛋白表達水平明顯增加，與E20結(jié)果相一致；在E14胎鼠大腦皮質(zhì)中檢測到iNOS蛋白微量表達，但腦膨出組與對照組比較無明顯差異，E20胎鼠大腦皮質(zhì)中未檢測到iNOS表達。 六、不同組E20胎鼠大腦皮質(zhì)中nNOS mRNA、eNOS mRNA和iNOS mRNA的表達 1、實時定量PCR檢測發(fā)現(xiàn)對照組、給藥無畸形組、脊柱裂組和腦膨出組E20胎鼠大腦皮質(zhì)中nNOS mRNA表達無明顯改變； 2、給藥無畸形組、脊柱裂組和對照組胎鼠大腦皮質(zhì)中eNOSmRNA表達無明顯改變，而腦膨出組胎鼠大腦皮質(zhì)中eNOS mRNA表達明顯增多； 3、在各組未檢測到iNOSmRNA表達。 結(jié)論 1、在用ETU成功制備神經(jīng)管畸形大鼠模型過程中，發(fā)現(xiàn)孕10天是獲得神經(jīng)管畸形大鼠模型的最佳給藥時間。 2、神經(jīng)管畸形中腦膨出胎鼠大腦組織中eNOS表達升高，引起NO釋放增加，可能是通過caspase-3、bcl-2途徑促發(fā)神經(jīng)元凋亡，導(dǎo)致神經(jīng)管閉合缺陷引起腦發(fā)育異常，說明eNOS在神經(jīng)管畸形發(fā)育過程中起主導(dǎo)作用。 3、腦血管與神經(jīng)元發(fā)育比例失衡，可能也是腦發(fā)育異常的一個重要因素。
[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 .
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R363

【引證文獻】

相關(guān)博士學(xué)位論文 前1條

1 韓莉妲;基于GC/MS技術(shù)的脂肪酸代謝輪廓譜方法和應(yīng)用研究[D];華東理工大學(xué);2011年

本文編號：2031975