【摘要】： 目的 神經(jīng)嵴干細(xì)胞(neural crest stem cells, NCSC)起源于胚胎期的神經(jīng)管背側(cè),部分NCSC定向分化成腸神經(jīng)系統(tǒng)(enteric nervous system, ENS)的神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞,被稱(chēng)為腸神經(jīng)嵴干細(xì)胞(gut neural crest stem cells, GNCSC)。GNCSC的發(fā)育異常會(huì)導(dǎo)致腸神經(jīng)系統(tǒng)疾病,其數(shù)量、功能及分化細(xì)胞的類(lèi)型在ENS發(fā)育過(guò)程中起關(guān)鍵作用。已有研究證實(shí),肛門(mén)直腸畸形(Anorectal Malformations, ARMs)患者術(shù)后排便功能不良的部分原因是由末端直腸的腸神經(jīng)系統(tǒng)的發(fā)育異常引起,先天性巨結(jié)腸(Hirschsprung's Disease, HD)為小兒外科的常見(jiàn)疾病,其病理基礎(chǔ)也是先天性腸神經(jīng)系統(tǒng)發(fā)育異常的結(jié)果。GNCSC的發(fā)現(xiàn)和體外培養(yǎng)的成功,為我們利用GNCSC進(jìn)行干細(xì)胞移植,從而徹底治愈先天性巨結(jié)腸及解決肛門(mén)直腸畸形患兒術(shù)后便失禁等問(wèn)題提供了一個(gè)很好的前景。 雖然近些年來(lái)關(guān)于GNCSC體外培養(yǎng)的文章不在少數(shù),但是在GNCSC的分離提取及培養(yǎng)的關(guān)鍵步驟上說(shuō)法模糊,對(duì)于材料的選擇也沒(méi)有統(tǒng)一的標(biāo)準(zhǔn)。本實(shí)驗(yàn)旨在完善GNCSC體外的培養(yǎng)方法,比較從不同胎齡大鼠胚胎及生后新生大鼠提取的GNCSC體外培養(yǎng)形成的神經(jīng)球樣結(jié)構(gòu)(neurosphere-like bodies, NLB)生長(zhǎng)情況,探討最佳的取材時(shí)機(jī)及培養(yǎng)方法。 近年的文獻(xiàn)證實(shí)Wnt蛋白通過(guò)不同信號(hào)分子間的相互作用,觸發(fā)了調(diào)節(jié)細(xì)胞生長(zhǎng)、遷移、分化及發(fā)育等多方面的復(fù)雜信號(hào)級(jí)聯(lián)反應(yīng),其中對(duì)Wnt5a的研究正在逐漸增多,Wnt5a在調(diào)節(jié)中樞神經(jīng)系統(tǒng)神經(jīng)干細(xì)胞的增殖分化中起重要作用,本文通過(guò)免疫熒光染色技術(shù)研究Wnt5a在GNCSC增殖及分化過(guò)程中的表達(dá)情況,研究Wnt5a在ENS形成及發(fā)育過(guò)程的可能調(diào)節(jié)作用。 1、不同胎齡大鼠GNCSC的分離培養(yǎng)、鑒定及比較 分別在成熟健康Wistar孕鼠妊娠第17天(E17)、第19天(E19),第21天(E21)剖宮取出胎鼠,和生后2天(P2)大鼠,利用顯微外科手術(shù)器械取其腸管做GNCSC原代培養(yǎng)。待NLB出現(xiàn)后進(jìn)行傳代培養(yǎng),并利用傳代5-7天后的GNCSC做免疫熒光鑒定。比較不同胎齡胎鼠GNCSC體外培養(yǎng)形成的NLB的生長(zhǎng)情況。 2、Wnt5a在GNCSC增殖及分化過(guò)程中的表達(dá) 提取E19天大鼠胚胎GNCSC,將傳代培養(yǎng)后6天的GNCSC分為5組。第1組直接爬片進(jìn)行免疫熒光染色,另外5組利用含血清的培養(yǎng)基誘導(dǎo)分化,并于誘導(dǎo)分化開(kāi)始后第1天、3天、5天、7天進(jìn)行Wnt5a的免疫熒光染色。 結(jié)果 1、比較不同胎齡及生后提取的GNCSC形成神經(jīng)球的時(shí)間發(fā)現(xiàn),E17、E19提取的GNCSC于原代后第3天形成神經(jīng)球,E21,P2提取的GNCSC于提取后第5天形成。 2、在GNCSC中及GNCSC分化后第1天均發(fā)現(xiàn)Wnt5a有較強(qiáng)表達(dá),第3天表達(dá)較弱,第5-7天幾乎無(wú)表達(dá)。 結(jié)論 1、本實(shí)驗(yàn)中,GNCSC體外培養(yǎng)取材時(shí)機(jī),選擇E19大鼠胚胎為宜。 2、Wnt5a在GNCSC及早期分化過(guò)程中起著重要的作用。
[Abstract]:Purpose



The neural crest stem cells ( NCSC ) are derived from the dorsal side of the neural tube in the embryonic stage . Some NCSC is directed to the neurons and glial cells of the nervous system ( ENS ) , known as gut neural crest stem cells ( GNCSC ) . The abnormal development of GNCSC has played a key role in the development of the intestinal nervous system . It has been proved that the postoperative defecating function of the patients with Anorectal Malocclusion ( ARMs ) is caused by the abnormal development of the enteric nervous system of the terminal rectum . The pathological basis is also the result of the abnormal development of the congenital intestinal nervous system . The findings of GNCSC and the success of in vitro culture provide us a good prospect for the complete cure of congenital megacons and the resolution of the incontinence of anorectal malformation .



Although there are few articles about the in vitro culture of GNCSC in recent years , it is suggested that the selection of GNCSC is not uniform . The aim of this experiment is to improve the culture method of GNCSC in vitro , to compare the growth of neurosphere - like bodies ( NLB ) formed by GNCSC in vitro and to explore the best time and method of culture .



Recent studies have shown that Wnt protein plays an important role in regulating cell growth , migration , differentiation and development . Wnt5a plays an important role in regulating the proliferation and differentiation of neural stem cells in the central nervous system . Wnt5a plays an important role in regulating the proliferation and differentiation of neural stem cells in the central nervous system .



Isolation , Culture , Identification and Comparison of GNCSC



The rats were taken out from the uterus of mature healthy Wistar rats ( E17 ) , 19 ( E19 ) , 21 ( E21 ) , and 2 days after birth ( P2 ) rats . The intestinal canals were cultured in the primary culture of GNCSC . After the appearance of NLB , the GNCSC was used as the immunofluorescence assay .



2 . Expression of Wnt5a during proliferation and differentiation of GNCSC



GNCSC was extracted from E19 , and GNCSC for 6 days after subculture was divided into 5 groups . Group 1 direct climbing was used for immunofluorescence staining . In addition , 5 groups were induced to differentiate with serum - containing medium , and Wnt5a immunofluorescence staining was performed on day 1 , 3 days , 5 days and 7 days after induction differentiation .



Results



1 . It was found that GNCSC extracted from E17 and E19 was formed on the 3rd day after primary generation . GNCSC extracted from E17 and E19 was formed on the 5th day after extraction .



2 . Wnt5a was found to be strongly expressed on day 1 after GNCSC and GNCSC . The expression of Wnt5a was weak on Day 3 and almost no expression on Day 5 - 7 .



Conclusion



1 . In this experiment , GNCSC is suitable for selecting E19 rat embryos in vitro .



2 . Wnt5a plays an important role in GNCSC and early differentiation .
【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R329

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相關(guān)期刊論文 前2條

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本文編號(hào)：2119994