本文選題：神經(jīng)管畸形 + 神經(jīng)干細胞��； 參考：《山西醫(yī)科大學》2009年碩士論文

【摘要】： 神經(jīng)系統(tǒng)是機體最重要和最復(fù)雜的系統(tǒng),起源于神經(jīng)管(neuarl tube)和神經(jīng)峭,神經(jīng)管形成中樞神經(jīng)系統(tǒng)(central nevrous system, CNS)(腦和脊髓),神經(jīng)峭形成周圍神經(jīng)系統(tǒng)的神經(jīng)節(jié)等。CNS正常發(fā)生的關(guān)鍵是神經(jīng)板內(nèi)外相關(guān)組織細胞行為的準確進行和協(xié)同,神經(jīng)管的形成是該過程結(jié)束的標志。神經(jīng)管發(fā)生是一個重要的涉及到建立中樞神經(jīng)系統(tǒng)原基的胚胎學事件,是指從神經(jīng)板出現(xiàn)到神經(jīng)管關(guān)閉的發(fā)育過程。神經(jīng)管管壁最初是由一層較厚的假復(fù)層上皮組成,稱為神經(jīng)上皮(neuroepithelium)。神經(jīng)上皮不斷增殖的同時細胞也逐漸開始進行遷移和分化,逐漸形成三層結(jié)構(gòu)的管壁,由內(nèi)向外依次為室管膜層,套層和邊緣層。在此過程中,神經(jīng)上皮細胞處于活躍的細胞增殖周期中,為具有多種分化潛能的神經(jīng)干細胞(neutal stem cell, NSC),伴隨著分化演變。神經(jīng)上皮中神經(jīng)干細胞的增殖遷移以及分化是神經(jīng)系統(tǒng)發(fā)育的關(guān)鍵環(huán)節(jié)。研究發(fā)現(xiàn)神經(jīng)管形成時期極易受多種內(nèi)外因素的干擾而致畸,神經(jīng)管缺陷(neural tube defects, NTDs)就是其中發(fā)病率最高的一種,表現(xiàn)為各種腦和脊髓的發(fā)育畸形。多年來的研究提示NTDs的發(fā)生與神經(jīng)上皮的異常發(fā)育密切相關(guān)。從基因水平而言,神經(jīng)上皮的發(fā)育過程是一系列基因按照高度特異的時空模式表達并相互作用的結(jié)果,但迄今對此復(fù)雜過程的基因表達與調(diào)控的了解還很少。 在各種調(diào)控機制中,Notch信號通路(Notch signaling pathway)與神經(jīng)管的發(fā)育有著密切關(guān)系,并且由它介導(dǎo)的“旁側(cè)抑制(lateral inhibition)”機制被認為是決定NSC分化命運的一個關(guān)鍵環(huán)節(jié)。反式維甲酸(all-trans retinoic acid, ATRA)是維生素A在體內(nèi)的正常代謝產(chǎn)物,它是一種作用很強的誘導(dǎo)分化劑,在神經(jīng)管的形成過程中,它具有很重要的作用。由維甲酸誘導(dǎo)形成的NTDs模型在科學研究中已經(jīng)得到了廣泛的應(yīng)用,但其具體的致畸作用機制尚未見文獻報道。鑒于NSC增殖與分化的機制在神經(jīng)管形成時期具有重要意義,所以本課題利用Western blot、Real-time PCR、雙熒光素酶報告基因系統(tǒng)、神經(jīng)干細胞體外培養(yǎng)、質(zhì)粒轉(zhuǎn)染及免疫熒光染色等技術(shù),初步研究了ATRA對NSC分化過程中Notch信號通路的具體調(diào)節(jié)途徑,為進一步探討Notch信號通路在胚胎神經(jīng)系統(tǒng)發(fā)育中的作用機制,尤其是對神經(jīng)上皮細胞的增殖分化的影響以及在神經(jīng)管發(fā)育和NTDs中可能分子機制奠定了理論基礎(chǔ)和實驗依據(jù)。 第一章神經(jīng)干細胞分化過程中Notch信號通路相關(guān)基因的表達變化 目的 探討神經(jīng)干細胞體外分離培養(yǎng)的方法、增殖特點和多向分化的特性。使用ATRA作用于處于分化狀態(tài)的NSCs,檢測其對Notch信號通路相關(guān)基因(Notch 1, Musashi 1, Numb, Presenilin 1, Rbpj, Hes 1, Sox 1, Mash 1和Neurogenin 2)表達變化的影響。 方法 取15天C57BL/6胎鼠的大腦皮質(zhì)組織,采用機械分離的方法獲得神經(jīng)干細胞,并應(yīng)用無血清神經(jīng)干細胞培養(yǎng)基進行原代和傳代培養(yǎng)。對培養(yǎng)的神經(jīng)干細胞進行Nestin(巢蛋白)免疫細胞化學鑒定;將獲得的神經(jīng)干細胞進行體外分化,對分化結(jié)果進行NF(Neurofilament,神經(jīng)絲蛋白,為神經(jīng)元標志抗原)、GFAP(Glial Fibrillary Acidic protein,膠質(zhì)原纖維酸性蛋白,為星形膠質(zhì)細胞標志抗原)和GALC(Galactocerebroside,半乳糖腦苷脂,為少突膠質(zhì)細胞標志抗原)免疫細胞化學鑒定。并在神經(jīng)干細胞增殖和分化過程中進行細胞形態(tài)學觀察。 將第5代經(jīng)鑒定過的神經(jīng)干細胞分為兩組:對照組使用分化培養(yǎng)基進行培養(yǎng),實驗組就是在對照組的基礎(chǔ)上再同時加入1μmol/L ATRA。每組分別取NSC(分化0d),分化1,3,5,7d共5個時間點進行實驗,每個時間點分別重復(fù)三次。在各個時間點通過實時熒光定量PCR反應(yīng)檢測Notch通路相關(guān)基因在mRNA表達水平的相對變化情況,通過Western Blot測定Notch1蛋白胞內(nèi)段NICD的相對表達變化。 結(jié)果 采用機械分離和無血清培養(yǎng)的方法可以獲得大量的神經(jīng)干細胞,對其進行Nestin免疫細胞化學鑒定為陽性,證明為神經(jīng)干細胞;神經(jīng)干細胞體外分化可獲得各種神經(jīng)終末細胞,NF、GFAP和GALC鑒定結(jié)果均為陽性,證明可分化為神經(jīng)元、星形膠質(zhì)細胞和少突膠質(zhì)細胞。 使用ATRA后,Notch 1表達降低,其胞內(nèi)段NICD的生成量也降低,Musashi 1表達增高,Numb表達先增高后降低,Presenilin 1表達增高,Rbpj表達先降低后增高,Hes 1表達降低,Sox 1表達降低,Mash 1表達降低,Neurogenin 2表達降低。 結(jié)論 應(yīng)用機械分離和無血清神經(jīng)干細胞培養(yǎng)基培養(yǎng)的方法可獲得大量的神經(jīng)干細胞,這些神經(jīng)干細胞具有多向分化的潛能,可分化為神經(jīng)元等多種神經(jīng)終末細胞。 Notch 1、Hes 1表達的降低和NICD生成量的降低說明ATRA可能直接或間接地通過抑制Notch 1的表達從而促進了神經(jīng)干細胞的分化,Musashi 1表達增高以及Numb在后期表達相應(yīng)的降低可能與Notch 1胞內(nèi)段NICD減少從而產(chǎn)生一種反饋性調(diào)節(jié)機制有關(guān),Presenilin 1表達增高以及Rbpj表達在后期的增高也可能屬于這種反饋調(diào)節(jié)機制。Sox 1表達的降低可能并不是因為ATRA的作用引起的,而是因為Hes 1表達降低產(chǎn)生反饋調(diào)節(jié)引起的。Mash 1和Neurogenin 2表達降低說明,使用ATRA后NSC向神經(jīng)元方向的分化減少了。 第二章γ-分泌酶在神經(jīng)干細胞分化過程中的作用 目的 建立基于Gal4-VP16/UAS系統(tǒng)和雙熒光素酶報告基因系統(tǒng)檢測神經(jīng)干細胞分化過程中γ分泌酶活性的方法。對神經(jīng)干細胞分化過程中γ分泌酶的活性、酶解產(chǎn)物NICD和活性中心—早老素1(Presenilin 1, PS1)進行檢測,為后期深入研究神經(jīng)干細胞分化過程的調(diào)節(jié)機制提供一個重要的實驗基礎(chǔ)。檢測神經(jīng)干細胞分化過程中ATRA對γ分泌酶活性的影響。 方法 將編碼攜帶有轉(zhuǎn)錄激活因子Gal4-VP16的小鼠Notch1跨膜與胞內(nèi)段的質(zhì)粒Notch1△E-GVP,編碼螢火蟲熒光素酶基因和上游激活序列(UAS)的質(zhì)粒MH100,以及海腎熒光素酶質(zhì)粒pRL-CMV,用脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)入處于分化狀態(tài)的神經(jīng)干細胞,利用Gal4-VP16/UAS系統(tǒng)和雙熒光素酶報告基因系統(tǒng)測定神經(jīng)干細胞分化過程中γ分泌酶的活性;通過Western blot技術(shù)檢測γ分泌酶酶解產(chǎn)物NICD的生成量;采用實時熒光定量PCR測定γ分泌酶活性中心PS1的表達。 結(jié)果 在γ分泌酶抑制劑DAPT作用下,γ分泌酶活性呈劑量依賴性降低,NICD的生成量也同步減少,而其催化中心組份PS1的表達則呈反饋性的同步增高。使用ATRA作用于處于分化狀態(tài)的NSCs后,γ分泌酶的活性增高了。 結(jié)論 利用Gal4-VP16/UAS系統(tǒng)和雙熒光素酶報告基因系統(tǒng)來檢測神經(jīng)干細胞分化過程中γ分泌酶的活性,為研究神經(jīng)干細胞分化過程的調(diào)節(jié)提供了一個重要的實驗方法。在神經(jīng)干細胞分化過程中,從γ分泌酶對Notch通路的這個調(diào)控范圍來說,ATRA抑制Notch通路的作用在于抑制Notch 1的表達,而不在于直接抑制γ分泌酶的活性及其催化組份早老素1的表達。
[Abstract]:The nervous system is the most important and complex system of the body, which originates from the Neuarl tube and the nerve, and the nerve canal forms the central nevrous system (CNS) (brain and spinal cord). The key to the normal hair growth of the ganglion, such as the ganglion that forms the peripheral nervous system, is the accuracy of the behavior of the cells inside and outside the nerve plate. The formation of neural tube is the sign of the end of the process. Neural tube formation is an important embryological event involving the establishment of the central nervous system. It refers to the development process from the nerve plate to the closure of the nerve canal. The nerve tube wall was initially composed of a thicker layer of false lamina, called the neuroepithelium (neuro Epithelium). At the same time, the cells gradually proliferate and the cells gradually begin to migrate and differentiate, and gradually form the three layer structure of the tube wall, from the inside to the outer layer, the interlayer and the marginal layer. In this process, the neuroepithelial cells are in the active cell proliferation cycle, and are the neural stem cells with multiple differentiation potential (neutal Stem cell, NSC), with the differentiation and evolution. The proliferation, migration and differentiation of neural stem cells in the neural epithelium are the key link in the development of neural system. It is found that the period of neural tube formation is easily teratogenic by the interference of various internal and external factors. Neural tube defects (neural tube defects, NTDs) is one of the highest incidence of the neural tube (NTDs). Various brain and spinal deformities. Years of research have suggested that the occurrence of NTDs is closely related to the abnormal development of the neuroepithelium. From the level of gene, the development of the neuroepithelium is the result of a series of genes expressed and interacting in a highly specific spatio-temporal pattern, but the gene expression and regulation of this complex process have been present to date. There is little understanding.
In various regulatory mechanisms, the Notch signaling pathway (Notch signaling pathway) is closely related to the development of neural tubes, and the mechanism of "side inhibition (lateral inhibition)" mediated by it is considered to be a key link in determining the fate of NSC differentiation. Trans retinoic acid (all-trans retinoic acid, ATRA) is in the body of vitamin A. Normal metabolites, which are a strong inducing differentiation agent, play an important role in the formation of neural tubes. The NTDs model induced by retinoic acid has been widely used in scientific research, but its specific mechanism for teratogenicity has not been reported in the literature. In view of the mechanism of NSC proliferation and differentiation The period of neural tube formation is of great significance, so this subject uses Western blot, Real-time PCR, double luciferase reporter gene system, neural stem cells in vitro culture, plasmid transfection and immunofluorescence staining and other techniques, preliminary study the specific regulation pathway of ATRA on NSC signaling pathway in the process of NSC differentiation, in order to further explore Notch letter. The mechanism of the signaling pathway in the development of embryonic neural system, especially the effect on the proliferation and differentiation of neuroepithelial cells, and the theoretical basis and experimental basis for the development of neural tube and possible molecular mechanism in NTDs.
Chapter one expression changes of Notch signaling pathway related genes during neural stem cell differentiation
objective
To investigate the methods of isolation and culture of neural stem cells in vitro, the characteristics of proliferation and multidirectional differentiation. The effects of ATRA on the differentiated NSCs were used to detect the changes in the expression of Notch signaling pathway related genes (Notch 1, Musashi 1, Numb, Presenilin 1, Rbpj, Hes 1, Sox 1, Mash 1 and Neurogenin 2).
Method
The neural stem cells were obtained by mechanical separation in the cerebral cortex of C57BL/6 fetal rat for 15 days. The cultured neural stem cell culture medium was used for primary and subculture. The cultured neural stem cells were identified by Nestin (nestin) immunocytochemical identification, and the obtained neural stem cells were differentiated in vitro, and the differentiation results were obtained. NF (Neurofilament, neurofilament protein, neuron marker antigen), GFAP (Glial Fibrillary Acidic protein, glial fibrillary acidic protein, astrocyte marker antigen) and GALC (Galactocerebroside, galactose brain glycoside, oligodendrocyte marker antigen) immunocytochemical identification, and proliferation and proliferation in neural stem cells. Cell morphology was observed during the differentiation process.
The fifth generation of identified neural stem cells were divided into two groups: the control group was cultured with the differentiation medium. The experimental group was added to the control group and added 1 mu mol/L ATRA. at the same time to take NSC (differentiation 0d), and the 1,3,5,7d was divided into three times at each time point. The relative changes in the expression level of the Notch pathway related genes were detected by the fluorescence quantitative PCR reaction, and the relative expression of the NICD in the intracellular segment of the Notch1 protein was measured by Western Blot.
Result
A large number of neural stem cells can be obtained by mechanical separation and serum-free culture. Nestin immunocytochemical identification of them is positive. It is proved to be neural stem cells. The differentiation of neural stem cells in vitro can obtain all kinds of nerve terminal cells. NF, GFAP and GALC identify the positive results, which prove that it can be differentiated into neurons and astrocytes. Cells and oligodendrocytes.
After the use of ATRA, the expression of Notch 1 decreased, the production of NICD in the intracellular segment decreased, the expression of Musashi 1 increased, the expression of Numb increased first and then decreased, the expression of Presenilin 1 increased, the expression of Rbpj decreased first, the expression of Hes 1 decreased, the expression of Sox 1 decreased, the expression of Mash 1 decreased, Neurogenin 2 was reduced.
conclusion
A large number of neural stem cells can be obtained by means of mechanical separation and culture medium culture of serum-free neural stem cells. These neural stem cells have the potential of multidifferentiation and can differentiate into a variety of nerve terminal cells, such as neurons.
The decrease of expression of Notch 1, Hes 1 and the decrease of NICD production indicate that ATRA may directly or indirectly inhibit the differentiation of neural stem cells by inhibiting the expression of Notch 1, the increased expression of Musashi 1 and the corresponding decrease in the expression of Numb in the later period of Numb may be related to the mechanism of feedback regulation, Pr, to produce a feedback regulation mechanism, Pr. The increased expression of esenilin 1 and the increase of Rbpj expression at the later stage may also belong to the feedback regulation mechanism of.Sox 1, which may not be caused by the effect of ATRA, but because of the decrease in the expression of.Mash 1 and Neurogenin 2 expressed by the Hes 1 expression. The reduction has been reduced.
The second chapter is the role of gamma secretase in the differentiation of neural stem cells.
objective
A method based on Gal4-VP16/UAS system and double luciferase reporter gene system was established to detect the activity of gamma secretase during the differentiation of neural stem cells. The activity of gamma secretase in the differentiation process of neural stem cells, the enzyme hydrolysate NICD and the active center - Presenilin 1 (Presenilin 1, PS1) were detected for the later study of neural stem cells. The regulation mechanism of the chemical process provides an important experimental basis to detect the effect of ATRA on the activity of gamma secretase during the differentiation of neural stem cells.
Method
The Notch1 transmembrane and plasmid Notch1 Delta E-GVP of the mice were encoded with the transcription activator Gal4-VP16, encoding the luciferase luciferase gene and the plasmid MH100 of the upstream activation sequence (UAS), and the luciferase plasmid pRL-CMV of the sea kidney, and transfected into the differentiated neural stem cells by liposome transfection, and using Gal4-VP16/UAS. The system and the double luciferase reporter gene system were used to determine the activity of gamma secretase during the differentiation of neural stem cells; the production of NICD was detected by Western blot technique and the expression of PS1 in the activity center of gamma secretase was measured by real-time fluorescence quantitative PCR.
Result
Under the action of gamma secretase inhibitor DAPT, the activity of gamma secretase decreased in a dose-dependent manner and the production of NICD decreased synchronously, while the expression of PS1 in the catalytic central component increased synchronously. After the use of ATRA in the differentiated NSCs, the activity of gamma secretase increased.
conclusion
Using the Gal4-VP16/UAS system and the double luciferase reporter gene system to detect the activity of gamma secretase during the differentiation of neural stem cells, it provides an important experimental method for the study of the regulation of neural stem cells differentiation process. In the process of neural stem cell differentiation, from this regulation of the Notch pathway, ATRA The inhibitory effect of Notch pathway is on the inhibition of Notch 1 expression, not on the direct inhibition of the activity of gamma secretase and the expression of the catalytic component of the precursor protein 1.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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