【摘要】：耐藥性的出現(xiàn)是目前癌癥治療中存在的一大難題。為了分析癌細(xì)胞的耐藥機(jī)制及篩選耐藥相關(guān)基因,一種最常用的實(shí)驗(yàn)策略是通過(guò)持續(xù)增加藥物濃度處理親本細(xì)胞系來(lái)產(chǎn)生耐藥細(xì)胞株,并檢測(cè)相應(yīng)的基因表達(dá)譜數(shù)據(jù)。大量的研究通過(guò)篩選藥物誘導(dǎo)的耐藥細(xì)胞系與親本細(xì)胞系間的差異表達(dá)的基因(Differentially expressed genes,DEGs)來(lái)識(shí)別耐藥相關(guān)基因及生物學(xué)通路。但是基于耐藥細(xì)胞系模型篩選的耐藥相關(guān)基因常難以應(yīng)用于指導(dǎo)臨床用藥。藥物誘導(dǎo)的耐藥細(xì)胞系與親本細(xì)胞系間的差異表達(dá)的基因可能主要反映細(xì)胞系對(duì)藥物處理產(chǎn)生的應(yīng)答變化,而不一定與細(xì)胞系耐藥相關(guān)。因此,有必要通過(guò)臨床組織樣本來(lái)評(píng)價(jià)基于細(xì)胞系耐藥模型篩選的耐藥基因標(biāo)志的臨床相關(guān)性。此外,由于同一種細(xì)胞系無(wú)生物學(xué)差異,因此研究者通常只檢測(cè)親本與耐藥細(xì)胞系各少數(shù)幾個(gè)(如2-3個(gè))技術(shù)重復(fù)樣本。對(duì)此類小樣本數(shù)據(jù),研究者普遍按基因在兩類細(xì)胞中平均表達(dá)值之間的倍數(shù)變化(Fold Change,FC)排秩篩選差異表達(dá)基因。顯然,在對(duì)照組中高表達(dá)的基因難以有較大的倍數(shù)增加,因此難以被FC排秩方法判斷為差異高表達(dá);而在對(duì)照組中表達(dá)量低的基因則容易受檢測(cè)偏差的影響而隨機(jī)產(chǎn)生較大的倍數(shù)變化,導(dǎo)致出現(xiàn)較多的假陽(yáng)性結(jié)果。針對(duì)FC方法存在的偏倚問(wèn)題,已有研究工作者提出了根據(jù)基因在兩類樣本中的平均表達(dá)差值(Average Difference,AD)大小排秩及加權(quán)平均差異排秩的方法,以識(shí)別那些在兩類樣本間表達(dá)水平差值較大而改變倍數(shù)不大的基因。因此,有必要評(píng)價(jià)AD方法用于識(shí)別小樣本細(xì)胞系實(shí)驗(yàn)研究的意義。本文將接受基于氟尿嘧啶(5-FU)與奧沙利鉑(L-OHP)聯(lián)合化療的臨床非響應(yīng)-響應(yīng)結(jié)腸癌患者的化療前取樣的組織樣本間的差異基因定義為臨床耐藥相關(guān)基因(clinically relevant drug resistance genes,記為CRG5-FU/L-OHP),并以CRG5-FU/L-OHP基因?yàn)閰⒄?用于評(píng)價(jià)細(xì)胞耐藥基因的臨床相關(guān)性。我們利用結(jié)腸癌細(xì)胞系HCT116與其經(jīng)氟尿嘧啶、奧沙利鉑分別誘導(dǎo)的耐藥細(xì)胞系的基因表達(dá)譜數(shù)據(jù),證實(shí):(1)耐藥細(xì)胞系與親本細(xì)胞系之間的差異表達(dá)基因主要反映藥物處理親本細(xì)胞系后引起的變化,其大多數(shù)可能并不與耐藥相關(guān);(2)反映親本及耐藥細(xì)胞系對(duì)藥物短暫處理(如24h)誘導(dǎo)的差異表達(dá)基因與CRG5-FU/L-OHP基因顯著吻合,并且與化療后取樣的臨床非響應(yīng)-響應(yīng)組織樣本間的差異基因顯著吻合;(3)不同于FC排秩方法,采用AD排秩方法可以識(shí)別出在兩類細(xì)胞中表達(dá)豐度高的差異基因,它們與CRG5-FU/L-OHP基因顯著一致。本文提出了新的基于耐藥細(xì)胞系模型識(shí)別腫瘤耐藥標(biāo)志的實(shí)驗(yàn)設(shè)計(jì)與數(shù)據(jù)分析策略。
[Abstract]:The emergence of drug resistance is a major problem in cancer treatment. In order to analyze the mechanism of drug resistance of cancer cells and to screen drug-resistance related genes, one of the most commonly used experimental strategies is to produce drug-resistant cell lines by continuously increasing drug concentration to produce drug-resistant cell lines, and to detect the corresponding gene expression profile data. A large number of studies have been conducted to identify drug-resistance-related genes and biological pathways by screening differentially expressed genes (Differentially expressed genes,DEGs between drug-induced drug resistant cell lines and parental cell lines. However, drug resistance related genes based on drug resistance cell line model are difficult to be used to guide clinical drug use. The differentially expressed genes between drug-resistant cell lines and parent cell lines may reflect the response of drug resistant cell lines to drug treatment, but may not be related to drug resistance of cell lines. Therefore, it is necessary to evaluate the clinical correlation of drug resistance gene markers based on cell line resistance model through clinical tissue samples. In addition, because there is no biological difference in the same cell line, researchers usually detect only a few (e.g. 2-3) technical repeat samples of each parent and drug-resistant cell line. For such small sample data, the researchers generally ranked the multiple variation (Fold Change,FC) between the average expression values of genes in the two types of cells to screen differentially expressed genes. It is obvious that the high expression genes in the control group are difficult to increase by a large multiple, so it is difficult to be judged as differential high expression by the FC rank method. However, the genes with low expression in the control group were susceptible to the influence of the detection deviation, resulting in a large number of random changes, resulting in more false positive results. In view of the bias problem of FC method, some researchers have proposed a method to rank the genes according to the average expression difference (Average Difference,AD) size and weighted average difference in the two kinds of samples. In order to identify those between the two types of samples between the level of expression difference and the change is not multiple genes. Therefore, it is necessary to evaluate the significance of AD method in identifying small cell lines. In this study, the difference gene between tissue samples taken before chemotherapy in patients with nonresponsive colon cancer was defined as clinical drug-resistance correlation based on combination chemotherapy of fluorouracil (5-FU) and oxaliplatin (L-OHP). Gene (clinically relevant drug resistance genes, To evaluate the clinical association of cell resistance genes with CRG5-FU/L-OHP as reference. We used the gene expression profile data of colon cancer cell line HCT116 and its drug-resistant cell lines induced by fluorouracil and oxaliplatin respectively. The results showed that: (1) the differentially expressed genes between drug-resistant cell lines and parental cell lines mainly reflect the changes caused by drug treatment of parent cell lines, and most of them may not be related to drug resistance; (2) the differentially expressed genes induced by transient treatment (such as 24 h) by their parents and drug resistant cell lines were significantly consistent with those of CRG5-FU/L-OHP gene. And the difference gene between the clinical non-response-response tissue samples sampled after chemotherapy was significantly consistent. (3) different from the FC method, AD rank method can identify the differentially expressed genes in the two types of cells, which are significantly consistent with the CRG5-FU/L-OHP gene. In this paper, a new experimental design and data analysis strategy for the identification of tumor resistance markers based on drug resistant cell line model is proposed.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R730.5

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