本文關(guān)鍵詞： 表皮干細(xì)胞 標(biāo)記滯留細(xì)胞 免疫熒光雙標(biāo) Brdu 整合素β1 P63 CD34　出處：《山東大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景： 皮膚是人體最大的組織器官,它有很強(qiáng)的自我更新能力和損傷后修復(fù)能力,其關(guān)鍵就在于表皮干細(xì)胞(epidermal stem cells, ESCs)的存在。此外,已有研究證明表皮干細(xì)胞對(duì)腫瘤的發(fā)生起始和基因治療有著靶向作用,并可能成為腫瘤治療的關(guān)鍵。因此,表皮干細(xì)胞一直是近年來(lái)研究的熱點(diǎn)。而如何從皮膚組織中將表皮干細(xì)胞進(jìn)行分離、純化和培養(yǎng),是進(jìn)行表皮干細(xì)胞研究的最基本條件。目前學(xué)術(shù)界仍沒(méi)有公認(rèn)的表皮干細(xì)胞特異性標(biāo)記物,因此其表面分子標(biāo)記物的確定是一個(gè)必要而且關(guān)鍵的研究環(huán)節(jié)。標(biāo)記滯留細(xì)胞(label retaining cell, LRCs)技術(shù)是近年來(lái)發(fā)展起來(lái)的一種鑒定成體干細(xì)胞及其定位的方法：其原理就是選取5-溴,2-脫氧尿嘧啶(5-bromodeoxyuridine, BrdU)作為示蹤劑,利用其可與內(nèi)源性胸腺嘧啶核苷競(jìng)爭(zhēng)摻入S期(DNA合成期)單鏈DNA核苷酸序列中的特點(diǎn),通過(guò)皮下或腹腔注射的方法,引入外源性特異抗原BrdU,在組織增殖期將BrdU標(biāo)記到細(xì)胞中。由于成體干細(xì)胞有慢細(xì)胞周期、半保留復(fù)制以及不對(duì)稱分裂等特點(diǎn),通過(guò)利用特異性的抗BrdU抗體,檢測(cè)到的BrdU陽(yáng)性細(xì)胞就是標(biāo)記滯留細(xì)胞。有研究已經(jīng)證實(shí),這些標(biāo)記滯留細(xì)胞就是表皮干細(xì)胞。 目的： 用標(biāo)記滯留細(xì)胞(LRCs)以及免疫熒光雙標(biāo)記方法進(jìn)行小鼠表皮干細(xì)胞(ESCs)可靠分子標(biāo)記的篩選,為進(jìn)一步分離和研究表皮干細(xì)胞提供線索和基礎(chǔ)。 方法： 1、選擇出生5d的昆明小乳鼠進(jìn)行腹腔注射5-溴,2-脫氧尿嘧啶(5-bromodeoxyuridine,BrdU),隔日注射,連續(xù)7d。 2、于注射后7d、30d、60d、90d殺死小鼠并褪毛,取下老鼠背部皮膚,保存于液氮罐中。 3、將標(biāo)本制成冰凍切片及石蠟切片。 4、在注射后7d、30d、60d、90d的石蠟切片上進(jìn)行BrdU染色。 5、選取注射后第90天的冰凍切片,分別進(jìn)行BrdU與整合素β1、P63和CD34的雙重免疫熒光標(biāo)記,觀察這些標(biāo)記物與BrdU的符合度。 6、在熒光顯微鏡下(200×)隨機(jī)選取視野,觀察每組標(biāo)本中的隨機(jī)10個(gè)視野并計(jì)數(shù)其中的陽(yáng)性細(xì)胞數(shù),最后計(jì)算出每組標(biāo)本平均陽(yáng)性細(xì)胞數(shù)目及平均陽(yáng)性細(xì)胞百分比。 結(jié)果： 1、隨著注射BrdU時(shí)間的延長(zhǎng),石蠟切片中BrdU陽(yáng)性細(xì)胞數(shù)逐漸減少,但減少到90天后就維持在一個(gè)相對(duì)穩(wěn)定的數(shù)量不再減少。90天后的石蠟切片免疫組化結(jié)果顯示：有少數(shù)陽(yáng)性細(xì)胞存在于小鼠皮膚上皮細(xì)胞中,這些陽(yáng)性細(xì)胞主要位于毛囊的隆突部位,皮膚基底層中也有少數(shù)陽(yáng)性細(xì)胞散落。 2、免疫熒光雙標(biāo)的結(jié)果顯示：Brdu與整合素β1雙標(biāo)陽(yáng)性的細(xì)胞占所有整合素β1陽(yáng)性細(xì)胞的81%,占所有Brdu陽(yáng)性細(xì)胞的94%; Brdu與P63雙標(biāo)陽(yáng)性的細(xì)胞占所有P63陽(yáng)性細(xì)胞的65%,占所有Brdu陽(yáng)性細(xì)胞的86%; Brdu與CD34雙標(biāo)陽(yáng)性的細(xì)胞占所有CD34陽(yáng)性細(xì)胞的30%,占所有Brdu陽(yáng)性細(xì)胞的62%。 結(jié)論： 整合素β1與BrdU的符合率最高,P63其次,CD34最低。因此整合素β1和P63是這些分子標(biāo)記物中篩選干細(xì)胞較好的指標(biāo)。可以通過(guò)整合素β1和P63雙重標(biāo)記實(shí)現(xiàn)對(duì)表皮干細(xì)胞的初步篩選。
[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錛，

本文編號(hào)：1534316