免疫荧光双标法应用于小鼠表皮干细胞分子标记的筛选

发布时间：2018-02-25 16:31

  本文关键词： 表皮干细胞 标记滞留细胞 免疫荧光双标 Brdu 整合素β1 P63 CD34　出处：《山东大学》2011年硕士论文　论文类型：学位论文

【摘要】：研究背景： 皮肤是人体最大的组织器官,它有很强的自我更新能力和损伤后修复能力,其关键就在于表皮干细胞(epidermal stem cells, ESCs)的存在。此外,已有研究证明表皮干细胞对肿瘤的发生起始和基因治疗有着靶向作用,并可能成为肿瘤治疗的关键。因此,表皮干细胞一直是近年来研究的热点。而如何从皮肤组织中将表皮干细胞进行分离、纯化和培养,是进行表皮干细胞研究的最基本条件。目前学术界仍没有公认的表皮干细胞特异性标记物,因此其表面分子标记物的确定是一个必要而且关键的研究环节。标记滞留细胞(label retaining cell, LRCs)技术是近年来发展起来的一种鉴定成体干细胞及其定位的方法：其原理就是选取5-溴,2-脱氧尿嘧啶(5-bromodeoxyuridine, BrdU)作为示踪剂,利用其可与内源性胸腺嘧啶核苷竞争掺入S期(DNA合成期)单链DNA核苷酸序列中的特点,通过皮下或腹腔注射的方法,引入外源性特异抗原BrdU,在组织增殖期将BrdU标记到细胞中。由于成体干细胞有慢细胞周期、半保留复制以及不对称分裂等特点,通过利用特异性的抗BrdU抗体,检测到的BrdU阳性细胞就是标记滞留细胞。有研究已经证实,这些标记滞留细胞就是表皮干细胞。 目的： 用标记滞留细胞(LRCs)以及免疫荧光双标记方法进行小鼠表皮干细胞(ESCs)可靠分子标记的筛选,为进一步分离和研究表皮干细胞提供线索和基础。 方法： 1、选择出生5d的昆明小乳鼠进行腹腔注射5-溴,2-脱氧尿嘧啶(5-bromodeoxyuridine,BrdU),隔日注射,连续7d。 2、于注射后7d、30d、60d、90d杀死小鼠并褪毛,取下老鼠背部皮肤,保存于液氮罐中。 3、将标本制成冰冻切片及石蜡切片。 4、在注射后7d、30d、60d、90d的石蜡切片上进行BrdU染色。 5、选取注射后第90天的冰冻切片,分别进行BrdU与整合素β1、P63和CD34的双重免疫荧光标记,观察这些标记物与BrdU的符合度。 6、在荧光显微镜下(200×)随机选取视野,观察每组标本中的随机10个视野并计数其中的阳性细胞数,最后计算出每组标本平均阳性细胞数目及平均阳性细胞百分比。 结果： 1、随着注射BrdU时间的延长,石蜡切片中BrdU阳性细胞数逐渐减少,但减少到90天后就维持在一个相对稳定的数量不再减少。90天后的石蜡切片免疫组化结果显示：有少数阳性细胞存在于小鼠皮肤上皮细胞中,这些阳性细胞主要位于毛囊的隆突部位,皮肤基底层中也有少数阳性细胞散落。 2、免疫荧光双标的结果显示：Brdu与整合素β1双标阳性的细胞占所有整合素β1阳性细胞的81%,占所有Brdu阳性细胞的94%; Brdu与P63双标阳性的细胞占所有P63阳性细胞的65%,占所有Brdu阳性细胞的86%; Brdu与CD34双标阳性的细胞占所有CD34阳性细胞的30%,占所有Brdu阳性细胞的62%。 结论： 整合素β1与BrdU的符合率最高,P63其次,CD34最低。因此整合素β1和P63是这些分子标记物中筛选干细胞较好的指标。可以通过整合素β1和P63双重标记实现对表皮干细胞的初步筛选。
[Abstract]:Research background:
The skin is the largest organ of human body, it has strong self-renewal ability and ability to repair after injury, the key lies in the epidermal stem cells (epidermal stem cells, ESCs) exist. In addition, it has been shown that the initiation of epidermal stem cells and gene therapy with targeted against cancer, and may become the key in the treatment of cancer. Therefore, epidermal stem cells has been a research hotspot in recent years. And how to be isolated from the skin tissue in the epidermal stem cells, purified and cultured, is the most basic conditions of epidermal stem cell research. The current academic circles there is no recognized epidermal stem cell specific markers, so the the surface of molecular markers is a necessary and key research areas. The label retaining cells (label retaining cell LRCs) technology developed in recent years is a kind of identification of adult stem cells and its location Method: the principle is to select 5- bromide, 2- deoxyuridine (5-bromodeoxyuridine, BrdU) as a tracer, which can compete with endogenous thymidine incorporation in S phase (DNA synthesis) characteristics of single stranded DNA nucleotide sequence, by subcutaneous or intraperitoneal injection, introducing exogenous antigen BrdU in tissue proliferation the BrdU labeled cells. The adult stem cells have slow cell cycle, semiconservative replication and asymmetric division characteristics, through the use of specific anti BrdU antibody, BrdU positive cells detected is labeled stranded cells. Previous studies have shown that these label retaining cells is the epidermal stem cells.
Objective:
Marker linked cell (LRCs) and immunofluorescence double labeling methods were used to screen reliable molecular markers of mouse epidermal stem cells (ESCs), providing clues and basis for further separation and research of epidermal stem cells.
Method锛，

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