本文選題：Cajal間質(zhì)細(xì)胞 + 神經(jīng)上皮干細(xì)胞�。� 參考：《山東大學(xué)》2013年博士論文

【摘要】：先天性巨結(jié)腸病(Hirschsprung's disease, HD)是一種常見的、嚴(yán)重危害嬰幼兒健康的先天性畸形,根據(jù)有關(guān)統(tǒng)計,其發(fā)病率高達(dá)1/3000-1/5000。其主要病理變化是病變腸壁內(nèi)神經(jīng)節(jié)細(xì)胞缺如和Cajal間質(zhì)細(xì)胞減少,病變腸段持續(xù)性痙攣,腸腔狹窄,其近端腸腔擴(kuò)張,內(nèi)容物潴留；主要臨床表現(xiàn)是腹脹,頑固性便秘,不完全性腸梗阻。目前認(rèn)為,HD是由遺傳因素和環(huán)境因素共同作用而導(dǎo)致的一種多因子、多基因遺傳病。調(diào)控腸壁內(nèi)神經(jīng)節(jié)細(xì)胞發(fā)育的基因如RET,EDNRB、EDN3、GDNF等的異常可能是HD發(fā)病的內(nèi)因,同時缺血、缺氧、毒素、炎癥等可能是HD發(fā)病的環(huán)境因素。研究證明,腸神經(jīng)系統(tǒng)(Enteric Nervous System, ENS)能調(diào)節(jié)消化管的運動功能,并且它是一個相對獨立的神經(jīng)調(diào)節(jié)系統(tǒng)。腸神經(jīng)系統(tǒng)位于腸壁內(nèi),主要由神經(jīng)節(jié)細(xì)胞和神經(jīng)纖維構(gòu)成。神經(jīng)節(jié)中包含神經(jīng)細(xì)胞和神經(jīng)膠質(zhì)細(xì)胞,這些細(xì)胞均由原始的神經(jīng)嵴細(xì)胞遷移、分化而來。在人胚發(fā)育的過程中,如果由于某種原因造成神經(jīng)嵴細(xì)胞沒能遷入原始消化管,或者遷入的神經(jīng)嵴細(xì)胞未能正常分化為神經(jīng)節(jié)細(xì)胞,就會引起像HD這樣的胃腸道神經(jīng)運動障礙性疾病。Cajal間質(zhì)細(xì)胞(Interstitial cell of Cajal, ICC)位于神經(jīng)叢周圍及靠近環(huán)形肌處,是一種來自間充質(zhì)的細(xì)胞。有研究顯示,這種細(xì)胞具有產(chǎn)生電慢波,調(diào)控胃腸平滑肌自主節(jié)律性運動及調(diào)節(jié)腸神經(jīng)系統(tǒng)中興奮性和抑制性神經(jīng)遞質(zhì)釋放等功能。ICC的缺如或減少可影響腸道起搏活動,阻礙神經(jīng)遞質(zhì)傳遞,從而導(dǎo)致腸道自主運動功能出現(xiàn)紊亂,腸道不能正常蠕動,從而引起HD的發(fā)病。由此可見,腸神經(jīng)節(jié)細(xì)胞和腸壁內(nèi)Cajal間質(zhì)細(xì)胞的缺如、減少或功能異常是HD發(fā)病的直接原因�；谏厦嫠�,我們設(shè)計了該項研究課題,研究了大鼠的Cajal間質(zhì)細(xì)胞和神經(jīng)上皮干細(xì)胞分離、培養(yǎng)的方法。同時還將兩種細(xì)胞進(jìn)行了體外的聯(lián)合培養(yǎng),以探討兩種細(xì)胞的相互作用,并為進(jìn)一步的細(xì)胞聯(lián)合移植治療無神經(jīng)節(jié)細(xì)胞巨結(jié)腸病的可行性,提供理論依據(jù)。實驗分三部分進(jìn)行：第一部分大鼠結(jié)腸Cajal間質(zhì)細(xì)胞的分離和體外培養(yǎng)通過體外細(xì)胞培養(yǎng),我們才能深入的研究細(xì)胞功能以及細(xì)胞信號的轉(zhuǎn)導(dǎo)機(jī)制。為了更好地認(rèn)識Cajal間質(zhì)細(xì)胞,我們對新生大鼠結(jié)腸的Cajal間質(zhì)細(xì)胞進(jìn)行了分離和體外培養(yǎng)。我們采用酶解法和密度梯度離心法,成功分離出了Cajal間質(zhì)細(xì)胞。然后,在體外環(huán)境下,用含干細(xì)胞因子(SCF)的有血清M199培養(yǎng)基進(jìn)行進(jìn)行培養(yǎng)。24小時后,我們通過光學(xué)顯微鏡可以看到ICCs貼壁生長；培養(yǎng)72小時后,可以觀察到細(xì)胞生長良好,胞體呈現(xiàn)類梭形。5天后,由ICCs發(fā)出的突起逐漸伸長,并與周圍相鄰細(xì)胞的突起互相靠近。7天后,顯微鏡下可以觀察到,相鄰的ICCs之間突起相互交織,形成網(wǎng)絡(luò)結(jié)構(gòu)。7天后進(jìn)行免疫細(xì)胞染色,熒光顯微鏡下可以觀察到Cajal間質(zhì)細(xì)胞呈c-kit陽性表達(dá)。第二部分大鼠神經(jīng)上皮干細(xì)胞的分離和培養(yǎng)神經(jīng)上皮干細(xì)胞(neuroepithelial stem cells, NESCs)的分離和培養(yǎng)是我們認(rèn)識干細(xì)胞的基礎(chǔ)和前提。經(jīng)過多次試驗,我們摸索出了神經(jīng)上皮干細(xì)胞分離培養(yǎng)的最佳方案。將從孕11.5天的大鼠胚胎中分離出的神經(jīng)管進(jìn)行機(jī)械吹打得到單細(xì)胞懸液,接種于含B27的無血清培養(yǎng)基DMEM/F12中進(jìn)行培養(yǎng)。24小時后,神經(jīng)上皮干細(xì)胞出現(xiàn)核分裂相。6天后,出現(xiàn)幾十甚至幾百個細(xì)胞聚集形成的集落,即神經(jīng)球,Nesting染色呈陽性。將培養(yǎng)基更換為含10%FBS的DMEM/F12培養(yǎng)7天后,進(jìn)行膠原纖維酸性蛋白(glial fibrillary acidic protein, GFAP)和微管相關(guān)蛋白2 (microtubule associated protein 2, MAP2)細(xì)胞染色,結(jié)果顯示,NESCs分化為GFAP陽性的膠質(zhì)細(xì)胞和MAP2陽性的神經(jīng)細(xì)胞。第三部分Cajal間質(zhì)細(xì)胞和神經(jīng)上皮干細(xì)胞的聯(lián)合培養(yǎng)將兩種細(xì)胞進(jìn)行聯(lián)合培養(yǎng),可以更好的觀察細(xì)胞之間的相互作用。將培養(yǎng)第三代的NESCs接種至生長良好的ICCs上,培養(yǎng)液為含10%FBS的DMEM/F12,每3天換液。對照組為用含10%FBS的DMEM/F12培養(yǎng)的NESCs.將經(jīng)過聯(lián)合培養(yǎng)7天的NESCs和單獨培養(yǎng)的NESCs進(jìn)行蛋白基因產(chǎn)物9.5 (protein gene product 9.5, PGP9.5)免疫熒光染色,結(jié)果發(fā)現(xiàn)聯(lián)合培養(yǎng)組的陽性細(xì)胞數(shù)明顯高于單獨培養(yǎng)的NESCs 。將聯(lián)合培養(yǎng)組的NESCs進(jìn)行神經(jīng)型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)免疫熒光染色,可以見到NESCs分化成nNOS陽性的神經(jīng)元。將經(jīng)過聯(lián)合培養(yǎng)7天的ICCs和NESCs進(jìn)行c-kit和MAP2免疫熒光雙染,可見兩種細(xì)胞相互交聯(lián)成網(wǎng)絡(luò)狀。結(jié)論本項研究成功地摸索出了大鼠結(jié)腸Cajal間質(zhì)細(xì)胞以及胚胎神經(jīng)上皮干細(xì)胞分離和體外培養(yǎng)的有效方法。并在此基礎(chǔ)上進(jìn)行了兩種細(xì)胞的聯(lián)合培養(yǎng),證明了ICCs能促進(jìn)NESCs向神經(jīng)元和神經(jīng)細(xì)胞的分化,并在形態(tài)上與分化的神經(jīng)細(xì)胞相互連接成網(wǎng)絡(luò)狀,進(jìn)而可能參與神經(jīng)信號的傳導(dǎo)。這一研究發(fā)現(xiàn)為我們將兩種細(xì)胞聯(lián)合移植來治療腸神經(jīng)系統(tǒng)疾病提供了理論依據(jù)。
[Abstract]:Hirschsprung's disease (HD) is a common congenital malformation which seriously endangering infant health. According to the relevant statistics, the incidence of the disease is up to 1/3000-1/5000.. The main pathological changes are the absence of ganglion cells in the intestinal wall and the decrease of interstitial cells in the Cajal, the persistent spasm of the intestinal segment, the narrowing of the intestinal cavity, and the narrowing of the intestinal cavity. The main clinical manifestations are abdominal distention, intractable constipation, and incomplete intestinal obstruction. At present, HD is a multi factor, multi gene genetic disease caused by the combination of genetic factors and environmental factors. The abnormalities of the genes regulating the development of ganglion cells in the intestinal wall, such as RET, EDNRB, EDN3, and GDNF, may be HD The internal cause of the disease, while ischemia, hypoxia, toxin, and inflammation may be the environmental factors of HD. Studies have shown that the Enteric Nervous System (ENS) can regulate the motor function of the digestive tube, and it is a relatively independent nervous system. The intestinal nervous system is located in the wall of the intestines, mainly by ganglion cells and nerve fibers. The ganglia consists of nerve cells and glial cells, which are migrated and differentiated from the original neural crest cells. In the process of human embryo development, the neural crest cells fail to move into the original digestive tube for some reason or the migratory neural crest cells fail to differentiate into ganglion cells. The.Cajal interstitial cells (Interstitial cell of Cajal, ICC), such as HD, are located around the nerve plexus and near the ring muscles. It is a cell from mesenchyme. Studies have shown that this cell has electrical slow waves, regulates autonomic rhythmic movement of the gastrointestinal smooth muscle and regulates the intestinal nervous system. The absence or reduction of the function of.ICC, such as stimulating and inhibiting neurotransmitter release, may affect the intestinal pacing activities and obstruct the transmission of neurotransmitters, which leads to the disorder of the autonomic movement of the intestines and the unnormal peristalsis of the intestines, thus causing the onset of HD. Thus, the absence of the interstitial cells between the intestinal God ganglion cells and the intestinal wall of the intestinal wall is reduced. Less or less functional is a direct cause of the onset of HD. Based on the above, we have designed the subject to study the isolation and culture of Cajal mesenchymal cells and neuroepithelial stem cells in rats. At the same time, the two cells were co cultured in vitro to explore the interaction of the two cells and for further cell union. The feasibility of transplantation in the treatment of anthetic cytomegaloenteropathy is feasible and provides theoretical basis. The experiment is divided into three parts: the first part of the rat colon Cajal mesenchymal cells isolation and in vitro culture through in vitro cell culture, we can deeply study cell function and cell signal transduction mechanism. In order to better understand the Cajal stroma Cells, we isolated and cultured the Cajal stromal cells from the colon of newborn rats. We successfully isolated Cajal stromal cells by enzymolysis and density gradient centrifugation. Then, in vitro, we used the M199 medium containing stem cell factor (SCF) to culture for.24 hours, we passed the optical microscopy. The growth of ICCs adherent wall can be seen by the mirror. After 72 hours of culture, it is observed that the cell growth is good, the cell body appears shuttle like.5 days, the protuberances from ICCs are elongated gradually, and the protuberances of adjacent cells are close to each other for.7 days. Under the microscope, the protuberances of adjacent ICCs are interwoven with each other to form a network structure after.7 days. Immunocyte staining was carried out and c-kit positive expression of interstitial cells of Cajal could be observed under the fluorescence microscope. Separation and culture of neuroepithelial stem cells (neuroepithelial stem cells, NESCs) in second parts of rats were the basis and prerequisite for our understanding of stem cells. The optimal scheme for isolation and culture of neuroepithelial stem cells was obtained. The neural tube separated from the rat embryo of 11.5 days of pregnancy was mechanically blown to get single cell suspension and inoculated in the serum-free medium DMEM/F12 containing B27 for.24 hours. After.6 days of the nucleus mitotic phase of the neural epithelial stem cells appeared, dozens or even hundreds of them appeared. The colony formed by the aggregation of the cells, that is, the nerve bulb, and the Nesting staining was positive. The culture medium was replaced by a 10%FBS containing DMEM/F12 for 7 days, and the collagen fibrillary acidic protein (glial fibrillary acidic protein, GFAP) and microtubule related protein 2 (microtubule associated protein 2, MAP2) cells were stained. Positive glial cells and MAP2 positive nerve cells. The combined culture of third parts of Cajal stromal cells and neuroepithelial stem cells combined culture of two cells to better observe the interaction between cells. The culture of third generation of NESCs was inoculated to a good growing ICCs, and the culture medium was 10%FBS containing DMEM/F12, every 3 days. The control group was immunofluorescent staining of protein gene product 9.5 (protein gene product 9.5, PGP9.5) after 7 days of combined culture of NESCs and NESCs cultured with 10%FBS containing DMEM/F12. The results showed that the number of positive cells in the combined culture group was significantly higher than that of the single culture NESCs. The NESCs of the joint culture group was NESCs. The neuronal nitric oxide synthase (neuronal nitric oxide synthase, nNOS) immunofluorescence staining, NESCs can be seen to differentiate into nNOS positive neurons. Through the combined culture of ICCs and NESCs for 7 days, c-kit and MAP2 immunofluorescence double staining, can be seen that two kinds of cells interlinked into the network. Conclusion this study successfully found out The effective methods for the isolation and culture of Cajal mesenchymal cells and embryonic neuroepithelial stem cells in the rat colon were used. On this basis, two kinds of cells were co cultured. It was proved that ICCs could promote the differentiation of NESCs into neurons and nerve cells, and form a network form with the differentiated nerve cells. This discovery provides a theoretical basis for the combined transplantation of two kinds of cells in the treatment of enteric nervous system diseases.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R725.7

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前3條

1 楊平;雞腸道Cajal間質(zhì)細(xì)胞與腸神經(jīng)系統(tǒng)的特性研究[D];南京農(nóng)業(yè)大學(xué);2012年

2 丁雄輝;RET/CXCR4通路介導(dǎo)神經(jīng)嵴衍生細(xì)胞遷移及其在先天性腸無神經(jīng)節(jié)細(xì)胞癥發(fā)生中的作用[D];重慶醫(yī)科大學(xué);2013年

3 普佳睿;神經(jīng)調(diào)節(jié)素1在斑馬魚腸神經(jīng)系統(tǒng)發(fā)育中的作用[D];華中科技大學(xué);2013年

相關(guān)碩士學(xué)位論文 前1條

1 劉揚;先天性巨結(jié)腸癥及其類緣病的腹腔鏡診療策略[D];河北醫(yī)科大學(xué);2013年

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本文編號：2088816