【摘要】：基因表達(dá)檢測(cè)與分析是研究細(xì)胞功能的重要手段之一,但傳統(tǒng)的分析方法通常是以細(xì)胞群體為研究對(duì)象,得到的是細(xì)胞群體mRNA的整體或平均表達(dá)水平,缺少針對(duì)單細(xì)胞中mRNA定位和定量分析的有效手段。鎖式探針滾環(huán)擴(kuò)增(Padlock rolling-circle amplification, Padlock-RCA)原位檢測(cè)技術(shù)是基于鎖式探針與單鏈DNA靶點(diǎn)的特異性結(jié)合,連接轉(zhuǎn)換為環(huán)形分子后滾環(huán)擴(kuò)增原位檢測(cè)單鏈DNA靶點(diǎn)。該技術(shù)具有高特異性和高敏感性,能定位和定量分析單細(xì)胞內(nèi)DNA靶點(diǎn),因此與PCR和傳統(tǒng)原位雜交技術(shù)相比有明顯優(yōu)勢(shì)。Padlock-RCA是一系列反應(yīng)的整合,首先,利用特異的鎖核酸(Locked nucleic acid, LNA)與mRNA結(jié)合,可在細(xì)胞內(nèi)原位將mRNA反轉(zhuǎn)錄成cDNA,通過(guò)RNase H將mRNA降解的同時(shí),鎖式探針(Padlock probe)與新合成的cDNA中靶序列結(jié)合,并通過(guò)連接酶的作用,鎖式探針自連環(huán)化,形成單鏈環(huán)化模板。隨后在Phi29聚合酶的作用下,對(duì)環(huán)化產(chǎn)物進(jìn)行滾環(huán)擴(kuò)增(RCA),最后熒光探針與擴(kuò)增產(chǎn)物上的靶序列特異性結(jié)合,從而實(shí)現(xiàn)對(duì)細(xì)胞內(nèi)mRNA的表達(dá)水平的檢測(cè)。Padlock-RCA對(duì)組織細(xì)胞中含量極低的mRNA有極高的敏感性,能夠在單細(xì)胞水平檢測(cè)mRNA的表達(dá),因此可以實(shí)現(xiàn)在細(xì)胞群體中發(fā)現(xiàn)某些基因特異表達(dá)的罕見(jiàn)細(xì)胞和細(xì)胞亞群。 本研究的目的就是優(yōu)化并利用鎖式探針滾環(huán)擴(kuò)增方法對(duì)小鼠畸胎瘤細(xì)胞F9單個(gè)細(xì)胞中的基因表達(dá)進(jìn)行原位檢測(cè)。研究主要包括四個(gè)部分,首先建立滾環(huán)復(fù)制擴(kuò)增的方法,通過(guò)對(duì)隨機(jī)引物,Phi29聚合酶,擴(kuò)增時(shí)間等方面的條件摸索,建立了穩(wěn)定、高效的滾環(huán)復(fù)制擴(kuò)增體系；其次,優(yōu)化鎖式探針滾環(huán)擴(kuò)增方法并檢測(cè)小鼠畸胎瘤細(xì)胞F9單細(xì)胞中Nanog基因及Perl基因的表達(dá)；而后利用維甲酸(RA)誘導(dǎo)小鼠畸胎瘤細(xì)胞F9分化模型,對(duì)細(xì)胞分化前后Nanog基因mRNA表達(dá)水平的改變進(jìn)行分析；最后將F9細(xì)胞通過(guò)皮下注射,接種到129SV小鼠體內(nèi),待成瘤后,將畸胎瘤組織制備冰凍切片,利用Padlock-RCA原位檢測(cè)小鼠畸胎瘤組織中Nanog基因的表達(dá)。本研究成功的應(yīng)用鎖式探針滾環(huán)擴(kuò)增方法檢測(cè)了Nanog基因和Perl基因在部分F9單細(xì)胞中的表達(dá)；并發(fā)現(xiàn)Nanog基因在F9細(xì)胞分化前后表達(dá)水平的差異；同時(shí)在小鼠畸胎瘤冰凍組織切片中發(fā)現(xiàn)大量Nanog基因表達(dá)陽(yáng)性的細(xì)胞。為進(jìn)一步研究單細(xì)胞基因表達(dá)及細(xì)胞亞群功能分析開(kāi)辟了一條新途徑。
[Abstract]:The detection and analysis of gene expression is one of the important methods to study cell function, but the traditional analysis method usually takes the cell population as the research object, and obtains the whole or average expression level of mRNA in the cell population. Lack of effective means for localization and quantitative analysis of mRNA in single cell. The technique of lock-type probe rolling amplification (Padlock rolling-circle amplification, Padlock-RCA) in situ detection is based on the specific binding of the lock-type probe to the single-stranded DNA target, and the ligation is transformed into the ring molecular roller-loop amplification in situ detection of single-stranded DNA target. This technique has high specificity and sensitivity, and can locate and analyze the target of DNA in single cell, so it has obvious advantages over PCR and traditional in situ hybridization. Padlock-RCA is the integration of a series of reactions. By using specific lock-nucleic acid (Locked nucleic acid, LNA) binding to mRNA, mRNA could be inverted into cDNA, in situ to degrade mRNA by RNase H, and the lock-probe (Padlock probe) could bind to the target sequence of the newly synthesized cDNA. The single chain cyclization template was formed by self-linking of the lock-type probe by ligase. Then under the action of Phi29 polymerase, the cyclized product was rolled to amplify the (RCA), and the final fluorescent probe was specifically bound to the target sequence of the amplified product. Padlock-RCA is highly sensitive to mRNA, which is very low in tissue cells, and can detect the expression of mRNA at single cell level. Therefore, rare cell and cell subsets with specific expression of some genes can be found in cell populations. The aim of this study was to optimize and detect in situ gene expression in single mouse teratoma cell line F9 by locking probe rolling amplification method. The research mainly includes four parts. Firstly, the method of rolling loop replication and amplification was established. By exploring the conditions of random primer, Phi29 polymerase, amplification time and so on, a stable and efficient rolling loop replication amplification system was established. Secondly, the Nanog gene and Perl gene expression in mouse teratoma cell F9 single cell were detected by optimizing the method of loop amplification with locking probe. Then the F9 differentiation model of mouse teratoma cells induced by retinoic acid (RA) was used to analyze the changes of mRNA expression of Nanog gene before and after differentiation. Finally, F9 cells were injected subcutaneously into 129SV mice. After tumorigenesis, frozen sections of teratoma tissue were prepared and the expression of Nanog gene in mouse teratoma tissue was detected by Padlock-RCA in situ. In this study, the expression of Nanog gene and Perl gene in F9 single cell was detected by the method of locking probe rolling amplification, and the difference of Nanog gene expression before and after F9 cell differentiation was found. At the same time, a large number of Nanog gene positive cells were found in frozen tissue sections of mouse teratoma. It opens a new way for further study of single cell gene expression and functional analysis of cell subsets.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R346

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本文編號(hào)：2402468