本文關(guān)鍵詞： 喉癌 多藥耐藥 Hep-2/v細(xì)胞系 miRNA 基因芯片 基因表達(dá)譜 粉防己堿 川芎嗪　出處：《吉林大學(xué)》2010年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 喉癌是頭頸部較為常見(jiàn)的惡性腫瘤之一,但在其綜合治療中,化療的療效欠佳,其主要原因是由于多藥耐藥性的產(chǎn)生。因此研究喉癌多藥耐藥相關(guān)基因的表達(dá)和調(diào)控機(jī)制以及篩選具有高效、低毒及多靶點(diǎn)性的中藥逆轉(zhuǎn)劑,對(duì)于提高喉癌的療效,尤其是遠(yuǎn)期療效,有重要的意義。 本文以長(zhǎng)春新堿為誘導(dǎo)劑,采用藥物濃度梯度遞增法體外誘導(dǎo)建立了喉癌多藥耐藥Hep-2/v細(xì)胞模型。在此基礎(chǔ)上應(yīng)用基因芯片技術(shù)研究喉癌耐藥細(xì)胞株Hep-2/v與其親本細(xì)胞Hep-2之間的多藥耐藥相關(guān)miRNA及mRNA表達(dá)譜,篩選出差異表達(dá)的miRNA及mRNA,并利用Target數(shù)據(jù)庫(kù)初步預(yù)測(cè)了miRNA的靶基因。MTT、流式細(xì)胞術(shù)及RT-PCR、Western Blot等技術(shù)研究中藥對(duì)喉癌細(xì)胞的逆轉(zhuǎn)特性及多藥耐藥相關(guān)基因mRNA的表達(dá)變化。統(tǒng)計(jì)學(xué)方法對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行了分析。 實(shí)驗(yàn)結(jié)果如下: (1)以藥物濃度梯度遞增法成功構(gòu)建了喉癌多藥耐藥細(xì)胞模型。 (2)AFFX miRNA芯片篩查出差異表達(dá)的多藥耐藥相關(guān)miRNA共7個(gè),其中表達(dá)上調(diào)2個(gè),表達(dá)下調(diào)5個(gè)。 (3)全基因組寡核酸微陣列芯片篩查出差異表達(dá)的多藥耐藥相關(guān)基因共有605個(gè),其中表達(dá)上調(diào)的基因270個(gè),表達(dá)水平下調(diào)的基因335個(gè)。 (4)結(jié)合基因芯片篩查結(jié)果,初步預(yù)測(cè)miRNA-210的靶基因?yàn)镠TRA1及NUPR1;miRNA-923的靶基因?yàn)镹UPR1;miRNA-93的靶基因?yàn)镽GS10。 (5)粉防己堿及川芎嗪對(duì)Hep-2/v細(xì)胞的逆轉(zhuǎn)倍數(shù)分別為2.22倍和1.88倍。RT-PCR及Western Blot檢測(cè)顯示粉防己堿能夠逆轉(zhuǎn)Hep-2/v細(xì)胞內(nèi)HTRA1、RGS10及MDR1基因的表達(dá),但對(duì)NUPR1基因的表達(dá)無(wú)逆轉(zhuǎn)作用。 本實(shí)驗(yàn)對(duì)喉癌多藥耐藥相關(guān)基因的表達(dá)及調(diào)控機(jī)制進(jìn)行了研究,表明miRNA可通過(guò)調(diào)控其靶基因的表達(dá)參與喉癌的多藥耐藥性。同時(shí)對(duì)中藥的逆轉(zhuǎn)機(jī)制進(jìn)行了研究,并篩選出對(duì)喉癌耐藥細(xì)胞有明顯逆轉(zhuǎn)作用的中藥逆轉(zhuǎn)劑,為進(jìn)一步研究奠定了基礎(chǔ)。
[Abstract]:Laryngeal carcinoma is one of the most common malignant tumors in head and neck. The main reason is the production of multidrug resistance. Therefore, to study the mechanism of expression and regulation of multidrug resistance-related genes in laryngeal cancer and to screen Chinese medicine reversal agents with high efficiency, low toxicity and multiple targets can improve the curative effect of laryngeal cancer. Especially the long-term effect, has the important significance. In this paper, vincristine was used as inducer. The multidrug resistant (Hep-2/v) cell model of laryngeal carcinoma was established by the method of drug concentration gradient increasing in vitro. The multidrug resistance-related miRNA and mRNA expression profiles between the drug resistant laryngeal cancer cell line Hep-2/v and their parent cell Hep-2 were studied by gene chip technique. The differentially expressed miRNA and mRNAs were screened, and the target gene. MTT, flow cytometry and RT-PCR- Western Blot of miRNA were preliminarily predicted by Target database to study the reverse characteristics of Chinese herbal medicine on laryngeal cancer cells and the expression of multidrug resistance-associated gene mRNA. The experimental results were analyzed by statistical method. The results are as follows:. 1) A multidrug resistant cell model of laryngeal carcinoma was successfully constructed by increasing drug concentration gradient. A total of 7 differentially expressed multidrug resistance-related miRNA were screened by AFFX miRNA microarray, of which 2 were up-regulated and 5 down-regulated. A total of 605 differentially expressed multidrug resistance-related genes were screened by genomic oligonucleic acid microarray, among which 270 genes were up-regulated and 335 genes were down-regulated. Combined with the results of gene microarray screening, it was preliminarily predicted that the target of miRNA-210 was HTRA1 and NUPR1 miRNA-923 because of NUPR1 miRNA-93. The reverse times of Tetrandrine and tetramethylpyrazine on Hep-2/v cells were 2.22 and 1.88 times respectively. RT-PCR and Western Blot analysis showed that Tetrandrine could reverse the expression of RGS10 and MDR1 genes in Hep-2/v cells, but had no reverse effect on NUPR1 gene expression. In this study, the expression and regulatory mechanism of multidrug resistance-associated genes in laryngeal carcinoma were studied. The results showed that miRNA could participate in the multidrug resistance of laryngeal carcinoma by regulating the expression of its target genes, and the reversal mechanism of traditional Chinese medicine was also studied. The Chinese medicine reversal agents which can reverse the drug resistance of laryngeal cancer cells were screened, which laid a foundation for further study.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R739.65;R285.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 郭娟娟,潘祥林,馮長(zhǎng)偉,鄒俊暉;冬凌草甲素誘導(dǎo)多藥耐藥細(xì)胞系K562/A02凋亡、逆轉(zhuǎn)耐藥性的研究[J];安徽中醫(yī)學(xué)院學(xué)報(bào);2000年03期

2 姜潤(rùn)德,張立新,岳文,朱運(yùn)峰,路浩軍,劉昕,岑信棠,關(guān)新元,李春海;人鼻咽癌順鉑耐藥細(xì)胞系(CNE2/DDP)的建立及耐藥相關(guān)基因的篩選[J];癌癥;2003年04期

3 姜潤(rùn)德,胡亮,關(guān)新元,張立新,岳文,岑信棠,李春海;人鼻咽癌順鉑耐藥細(xì)胞系(CNE2/DDP)及其親代細(xì)胞(CNE2)的比較基因組雜交研究[J];癌癥;2004年04期

4 趙殿鳳;劉子玲;馬寧;張永峰;屈蓓蓓;王志虹;李志毅;;漢防己甲素逆轉(zhuǎn)白血病細(xì)胞株K562/A02耐藥的機(jī)制[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2009年01期

5 馬海英;趙瑾瑤;金偉;孔力;;川芎嗪對(duì)轉(zhuǎn)基因多藥耐藥細(xì)胞K562/MDR耐藥性的逆轉(zhuǎn)作用[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2009年04期

6 曲正,侯培珍,曲偉,王穎慧;川芎嗪逆轉(zhuǎn)人胃癌耐藥細(xì)胞SGC7901/ADM多藥耐藥的實(shí)驗(yàn)研究[J];包頭醫(yī)學(xué)院學(xué)報(bào);2005年03期

7 史曦凱,張翼軍,趙春竟;人參皂甙單體Rb1對(duì)多藥耐藥細(xì)胞系K562/HHT細(xì)胞內(nèi)柔紅霉素濃度的影響[J];重慶醫(yī)學(xué);2001年06期

8 徐建業(yè),周琦,湯偉;漢防已甲素、羅通定及川芎嗪對(duì)腫瘤細(xì)胞株KBV200多藥耐藥性逆轉(zhuǎn)作用的研究[J];重慶醫(yī)學(xué);2005年09期

9 丁世凱;游海波;李起;劉作金;龔建平;;中藥復(fù)方“肝癌-1號(hào)”治療晚期肝癌的初步臨床研究[J];重慶醫(yī)學(xué);2008年15期

10 葉琳;王馳;謝明均;陳鴻雁;;粉防己堿對(duì)喉癌耐藥細(xì)胞株Hep-2/ADM多藥耐藥逆轉(zhuǎn)作用研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2009年12期

，

本文編號(hào)：1530840