大鼠肠神经嵴干细胞的分离培养材料的选择及Wnt5a在大鼠肠神经嵴干细胞及其分化过程中的表达
发布时间：2018-07-13 16:09
【摘要】： 目的 神经嵴干细胞(neural crest stem cells, NCSC)起源于胚胎期的神经管背侧,部分NCSC定向分化成肠神经系统(enteric nervous system, ENS)的神经元和神经胶质细胞,被称为肠神经嵴干细胞(gut neural crest stem cells, GNCSC)。GNCSC的发育异常会导致肠神经系统疾病,其数量、功能及分化细胞的类型在ENS发育过程中起关键作用。已有研究证实,肛门直肠畸形(Anorectal Malformations, ARMs)患者术后排便功能不良的部分原因是由末端直肠的肠神经系统的发育异常引起,先天性巨结肠(Hirschsprung's Disease, HD)为小儿外科的常见疾病,其病理基础也是先天性肠神经系统发育异常的结果。GNCSC的发现和体外培养的成功,为我们利用GNCSC进行干细胞移植,从而彻底治愈先天性巨结肠及解决肛门直肠畸形患儿术后便失禁等问题提供了一个很好的前景。 虽然近些年来关于GNCSC体外培养的文章不在少数,但是在GNCSC的分离提取及培养的关键步骤上说法模糊,对于材料的选择也没有统一的标准。本实验旨在完善GNCSC体外的培养方法,比较从不同胎龄大鼠胚胎及生后新生大鼠提取的GNCSC体外培养形成的神经球样结构(neurosphere-like bodies, NLB)生长情况,探讨最佳的取材时机及培养方法。 近年的文献证实Wnt蛋白通过不同信号分子间的相互作用,触发了调节细胞生长、迁移、分化及发育等多方面的复杂信号级联反应,其中对Wnt5a的研究正在逐渐增多,Wnt5a在调节中枢神经系统神经干细胞的增殖分化中起重要作用,本文通过免疫荧光染色技术研究Wnt5a在GNCSC增殖及分化过程中的表达情况,研究Wnt5a在ENS形成及发育过程的可能调节作用。 1、不同胎龄大鼠GNCSC的分离培养、鉴定及比较 分别在成熟健康Wistar孕鼠妊娠第17天(E17)、第19天(E19),第21天(E21)剖宫取出胎鼠,和生后2天(P2)大鼠,利用显微外科手术器械取其肠管做GNCSC原代培养。待NLB出现后进行传代培养,并利用传代5-7天后的GNCSC做免疫荧光鉴定。比较不同胎龄胎鼠GNCSC体外培养形成的NLB的生长情况。 2、Wnt5a在GNCSC增殖及分化过程中的表达 提取E19天大鼠胚胎GNCSC,将传代培养后6天的GNCSC分为5组。第1组直接爬片进行免疫荧光染色,另外5组利用含血清的培养基诱导分化,并于诱导分化开始后第1天、3天、5天、7天进行Wnt5a的免疫荧光染色。 结果 1、比较不同胎龄及生后提取的GNCSC形成神经球的时间发现,E17、E19提取的GNCSC于原代后第3天形成神经球,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天均发现Wnt5a有较强表达,第3天表达较弱,第5-7天几乎无表达。 结论 1、本实验中,GNCSC体外培养取材时机,选择E19大鼠胚胎为宜。 2、Wnt5a在GNCSC及早期分化过程中起着重要的作用。
[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 .
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2119994
[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 .
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前2条
1 周青,张世忠,徐如祥,徐凯;神经干细胞移植及术后处理:附70例报告[J];第一军医大学学报;2004年10期
2 朱巍,毛颖,周良辅,汪洋,江永,朱剑虹;转基因神经干细胞移植在缓解脑缺血性神经血管损害中的作用[J];中华实验外科杂志;2005年02期
，本文编号：2119994
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