本文選題：喉癌 + miR-133a�。� 參考：《吉林大學(xué)》2015年博士論文

【摘要】：研究背景及目的喉癌是我國(guó)北方地區(qū)發(fā)病率較高的腫瘤之一,發(fā)病率約占全身腫瘤的1~5%。盡管目前在喉癌的診斷和治療方面取得了很大進(jìn)展,但部分喉癌患者預(yù)后差,生存率低。對(duì)于晚期不能手術(shù)和術(shù)后復(fù)發(fā)的患者,常采用化學(xué)治療方法,但化療效果不佳,推測(cè)與喉癌細(xì)胞對(duì)化療藥物耐藥有關(guān)。腫瘤細(xì)胞的多藥耐藥是化療失敗的主要原因之一,喉癌的化療藥物的耐藥常表現(xiàn)為多藥耐藥。逆轉(zhuǎn)腫瘤細(xì)胞的耐藥一直是腫瘤研究的一個(gè)重點(diǎn),近年來(lái)通過(guò)mi RNAs逆轉(zhuǎn)腫瘤耐藥的研究取得了進(jìn)展,但應(yīng)用mi RNAs針對(duì)喉癌多藥耐藥的干預(yù)研究較少。本論文采用微陣列芯片技術(shù)研究喉鱗癌及癌旁正常黏膜中mi RNAs表達(dá)譜差異,篩選參與喉癌發(fā)生的特異mi RNAs。通過(guò)比較特異mi RNA在親本細(xì)胞Hep-2細(xì)胞與VCR耐藥細(xì)胞株Hep-2/v之間的表達(dá)差異驗(yàn)證與喉癌多藥耐藥有關(guān)的mi RNAs。材料和方法分離8例喉癌組織及其對(duì)應(yīng)癌旁正常黏膜中的mi RNA,采用熒光染料Cy3標(biāo)記,并與制備好的哺乳動(dòng)物mi RNA芯片V3.0雜交,用Lux Scan 10K/A掃描儀掃描熒光信號(hào),采用Lux Scan3.0圖像分析軟件對(duì)芯片圖像進(jìn)行分析,用SAM和Cluster3.0軟件進(jìn)行差異基因篩選和聚類分析,對(duì)部分差異表達(dá)mi RNA采用RNA加尾和引物延伸實(shí)時(shí)定量RT-PCR法對(duì)芯片檢測(cè)結(jié)果進(jìn)行驗(yàn)證。通過(guò)給予50u M、100u M和200u M的順鉑作用Hep-2/v細(xì)胞。應(yīng)用細(xì)胞增殖實(shí)驗(yàn)、Tunel細(xì)胞凋亡確定Hep-2和Hep-2/v對(duì)順鉑耐藥的差別,確認(rèn)Hep-2/v耐藥株存在多藥耐藥性。采用Poly加尾PCR的方法檢測(cè)正常咽上皮細(xì)胞NP69,Hep-2和Hep-2/v細(xì)胞mi R-133a的水平;采用免疫熒光染色和PCR、Western Blotting方法同時(shí)檢測(cè)兩種細(xì)胞在加入順鉑后ATP7B的變化。采用慢病毒重組載體p EZX-mi R-133a轉(zhuǎn)染Hep-2細(xì)胞和Hep-2/v耐藥細(xì)胞株,CCK8細(xì)胞活力實(shí)驗(yàn)觀察不同濃度順鉑對(duì)細(xì)胞存活的影響;通過(guò)免疫熒光染色和實(shí)時(shí)定量PCR方法檢測(cè)p EZX-mi R-133a轉(zhuǎn)染細(xì)胞ATP7B的轉(zhuǎn)錄和蛋白表達(dá)變化。結(jié)果1.通過(guò)mi RNA表達(dá)譜分析共篩選出47個(gè)喉癌相關(guān)的差異表達(dá)mi RNAs基因,包括24個(gè)在喉癌組織中表達(dá)下調(diào)的mi RNAs和23個(gè)在喉癌組織中表達(dá)上調(diào)的mi RNAs。其中在喉癌組織中下調(diào)5倍以上的有mi R-1、mi R-486-5p、mi R-206、mi R-487a、mi R-375、mi R-422a、mi R-144、hsa-mi R-384、mi R-378和mi R-133a共10個(gè)。在喉癌組織中表達(dá)上調(diào)3倍以上的mi RNA有3個(gè),分別是mi R-93、mi R-31和mi R-20b。在喉癌差異表達(dá)的mi RNA中有13個(gè)成5簇共表達(dá),分別位于8、13、14、18和X染色體上。熒光定量RT PCR結(jié)果與mi RNAs芯片的結(jié)果較符合。2.50u M和100u M的順鉑作用24h后,Hep-2細(xì)胞存活為71%和73%,而對(duì)Hep-2/v的細(xì)胞的存活沒(méi)有影響統(tǒng)計(jì)學(xué)分析有顯著差異(p0.01);Hep-2/v的生存在100u M時(shí)為85%明顯高于同劑量的Hep-2細(xì)胞(p0.05);50u M的順鉑作用Hep-2細(xì)胞24h,細(xì)胞凋亡指數(shù)為31.12±4.75,Hep-2/v細(xì)胞的凋亡指數(shù)僅為3.5±1.23,卡方檢驗(yàn)p0.01;Hep-2/v細(xì)胞的ATP7B蛋白表達(dá)明顯高于Hep-2細(xì)胞。3.順鉑作用后Hep-2和Hep-2/v表達(dá)ATP7B蛋白與對(duì)照組相比增強(qiáng)。實(shí)時(shí)定量PCR檢測(cè)順鉑作用后不同時(shí)間ATP7A和ATP7B基因轉(zhuǎn)錄變化,結(jié)果發(fā)現(xiàn)50u M順鉑能夠誘導(dǎo)Hep-2和Hep-2/v表達(dá)ATP7B,Hep-2/v細(xì)胞在作用后3h表達(dá)增強(qiáng),6h達(dá)到峰值,持續(xù)表達(dá)24h無(wú)明顯變化。Hep-2細(xì)胞和Hep-2/v表達(dá)ATP7B的熒光值明顯高于NP-69細(xì)胞。4.Hep-2細(xì)胞mi R-133a的表達(dá)量與NP-69細(xì)胞相比下降4.76±0.54倍,Hep-2/v細(xì)胞的mi R-133a的表達(dá)量與NP-69相比下降5.55±0.14倍。重組慢病毒介導(dǎo)mi R-133a轉(zhuǎn)染Hep-2細(xì)胞和Hep-2/v細(xì)胞48h后CCK8檢測(cè)細(xì)胞存活。結(jié)果顯示順鉑50u M和100u M作用組,轉(zhuǎn)染p EZX-Control的Hep-2/v細(xì)胞存活明顯高于轉(zhuǎn)染p EZX-mi R-133a的Hep-2/v細(xì)胞;mi R-133a能夠降低Hep-2/v細(xì)胞ATP7B的轉(zhuǎn)錄和表達(dá)。結(jié)論mi R-133a在喉癌組織中表達(dá)下調(diào),喉癌組織中差異表達(dá)的mi R-133a可能是參與喉癌的發(fā)生的因素之一。喉癌耐藥細(xì)胞Hep-2/v的ATP7B蛋白表達(dá)明顯高于Hep-2細(xì)胞,ATP7B蛋白表達(dá)增高為喉癌細(xì)胞對(duì)順鉑耐藥的研究提供了一個(gè)新思路。實(shí)驗(yàn)中發(fā)現(xiàn)了mi R-133a能重新提高喉癌耐藥細(xì)胞對(duì)順鉑的化療敏感性這一現(xiàn)象,推測(cè)其機(jī)制可能與mi R-133a下調(diào)ATP7B蛋白表達(dá)有關(guān)。
[Abstract]:Background and objective laryngeal cancer is one of the higher incidence of cancer in northern China. The incidence of the disease is about 1~5%. of the whole body tumor. Although great progress has been made in the diagnosis and treatment of larynx cancer, some patients with larynx cancer have poor prognosis and low survival rate. But the effect of chemotherapy is not good. It is presumed that the drug resistance of the laryngeal cancer cells is related to the drug resistance. The multidrug resistance of the tumor cells is one of the main reasons for the failure of chemotherapy. The drug resistance of the larynx cancer is often characterized by multidrug resistance. The reversal of the drug resistance of the tumor cells has been a key point in the cancer research. In recent years, the reversal of the tumor resistance through mi RNAs Progress has been made in the study, but the application of MI RNAs for multidrug resistance of larynx is less. This paper uses microarray technology to study the MI RNAs expression profiles in laryngeal squamous cell carcinoma and normal mucosa adjacent to cancer, and to screen specific mi RNAs. involved in the occurrence of larynx cancer by comparing the specific mi RNA in the parent cell Hep-2 cells and VCR resistant cell He. The expression difference between p-2/v and MI RNAs. related to multidrug resistance of larynx was used to separate 8 cases of larynx tissues and the MI RNA corresponding to the normal mucosa adjacent to the cancer, using a fluorescent dye Cy3 marker, and a V3.0 hybridization with a prepared mammalian mi RNA chip. The fluorescent signal was scanned with Lux Scan 10K/A scanner, and the fluorescent signal was scanned by Lux Scan 10K/A scanner. Analysis software was used to analyze the chip image. The differential gene screening and cluster analysis were carried out with SAM and Cluster3.0 software. A partial differential expression of MI RNA was verified by RNA plus tail and primer extension real-time quantitative RT-PCR method. By giving 50U M, 100u M and 200u M with cisplatin, the cell proliferation was applied. Tunel cell apoptosis determined the difference between Hep-2 and Hep-2/v on cisplatin resistance, and confirmed the multidrug resistance of Hep-2/v resistant strains. Poly plus tail PCR was used to detect the level of MI R-133a in normal pharyngeal epithelial cells, NP69, Hep-2 and Hep-2/v cells, and two cells were simultaneously detected by immunofluorescence staining and PCR. Changes in ATP7B after cisplatin. Transfection of Hep-2 and Hep-2/v resistant cells with lentivirus recombinant vector p EZX-mi R-133a. The effect of cisplatin on cell viability was observed at different concentrations in CCK8 cell viability test. The transcriptional and protein expression changes of P EZX-mi R-133a transfected cell ATP7B were detected by immunofluorescence staining and real-time quantitative PCR method. Results 1. the differential expression of MI RNAs gene related to 47 laryngomas, including 24 down regulated mi RNAs in Laryngic carcinoma and 23 mi RNAs. in Laryngic cancer tissues, was screened by Mi RNA expression analysis. R-144, hsa-mi R-384, MI R-378 and MI R-133a are 10. There are 3 mi RNA expressed in the larynx cancer tissues, which are mi R-93. 13 of 5 clusters are co expressed in the differential expression of the larynx cancer. After 24h of.50u M and 100u M, Hep-2 cells survived 71% and 73%, while the survival of Hep-2/v cells had no significant difference (P0.01), and the survival of Hep-2/v was 85% higher than the same dose of Hep-2 cells at 100u M; the apoptosis index was 31.12 + 4.75. The apoptotic index of V cells was only 3.5 + 1.23, chi square test was P0.01, and the expression of ATP7B protein in Hep-2/v cells was significantly higher than that of.3. cisplatin in Hep-2 cells. Hep-2 and Hep-2/v expressed ATP7B protein compared with those of the control group. Hep-2 and Hep-2/v expressed ATP7B, and the expression of 3H was enhanced after the action of Hep-2/v cells, and the 6h reached its peak value. There was no obvious change in the expression of 24h. The fluorescent value of.Hep-2 cells and Hep-2/v expressed ATP7B was significantly higher than that of NP-69 cell.4.Hep-2 cells. P-69 decreased by 5.55 + 0.14 times. Recombinant lentivirus mediated mi R-133a transfection of Hep-2 cells and Hep-2/v cells for 48h to detect cell survival. The results showed that cisplatin 50U M and 100u M cells were significantly higher than those transfected with Hep-2 cells. Conclusion the expression of MI R-133a in larynx tissues is downregulated. The differential expression of MI R-133a in larynx cancer tissues may be one of the factors involved in the occurrence of larynx cancer. The expression of ATP7B protein in the Hep-2/v of larynx cancer cells is obviously higher than that of Hep-2 cells. The increase of ATP7B protein expression provides a new thought for the study of the larynx cell cell for the study of cisplatin resistance. In the experiment, it was found that MI R-133a could improve the chemosensitivity of cisplatin in larynx resistant cells. It is presumed that the mechanism may be related to the down regulation of ATP7B protein expression by Mi R-133a.
【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】：2015
【分類號(hào)】：R739.65

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