【摘要】：背景腫瘤細(xì)胞耐藥是導(dǎo)致腫瘤化學(xué)治療失敗的重要原因,長(zhǎng)春新堿(vincristine, VCR)是一類(lèi)廣泛用于腫瘤治療的化學(xué)藥物。但是,長(zhǎng)期使用可使腫瘤細(xì)胞對(duì)VCR產(chǎn)生耐藥性,腫瘤耐藥機(jī)制仍不清楚。本研究探討與結(jié)腸癌耐藥的微小RNA(microRNA, miRNA),旨在揭示腫瘤耐藥機(jī)制及為腫瘤細(xì)胞耐藥逆轉(zhuǎn)提供基礎(chǔ)。目的建立長(zhǎng)春新堿耐藥結(jié)腸癌細(xì)胞模型,篩選與結(jié)腸癌細(xì)胞VCR耐藥相關(guān)的miRNA。方法1.培養(yǎng)結(jié)腸癌HCT-8細(xì)胞,采用VCR梯度誘導(dǎo),建立HCT-8/VCR耐藥細(xì)胞株。2.采用全基因組篩選的研究策略,運(yùn)用HiSeq 2500測(cè)序技術(shù)和生物信息學(xué)的方法,檢測(cè)和分析耐藥HCT-8/VCR細(xì)胞的和非耐藥的HCT-8細(xì)胞差異性表達(dá)的miRNAs,構(gòu)建其miRNA的差異表達(dá)譜。3.針對(duì)鑒定的差異miRNA,通過(guò)靶基因預(yù)測(cè)軟件進(jìn)行靶位點(diǎn)確認(rèn),通過(guò)靶基因所在基因的GO進(jìn)行功能注釋。結(jié)果1.利用濃度梯度的VCR建立了VCR耐藥的結(jié)腸癌細(xì)胞HCT-8/VCR, HCT-8/VCR細(xì)胞在2×103ng/mLVCR中生長(zhǎng)良好。2.共進(jìn)行了1651種miRNA測(cè)序,其中有差異的有48種miRNA,與非耐藥的結(jié)腸癌HCT-8細(xì)胞相比,24種miRNA差異具有統(tǒng)計(jì)學(xué)意義(P0.05),其中有17種miRNA表達(dá)上調(diào)(P0.05)。hsa-miR-675-3p表達(dá)量上調(diào)最高,高達(dá)7.497681倍,其次是hsa-miR-100-3p, hsa-miR-125b-1-3p, hsa-miR-582-5p, hsa-miR-3141, chr15_10055, hsa-miR-582-5p, hsa-miR-582-3p, hsa-miR-3687。7種miRNA表達(dá)下調(diào)(P0.05),下調(diào)最高的是hsa-miR-146a-5p,下調(diào)7.75199倍,其次是hsa-miR-1247-3p, hsa-miR-181 a-3p, hsa-miR-1247-5p。結(jié)論]miRNA的異常表達(dá)與結(jié)腸癌細(xì)胞VCR耐藥相關(guān)。miRNA表達(dá)量的差異可能在結(jié)腸癌細(xì)胞VCR耐藥中起關(guān)鍵作用。
[Abstract]:Background Drug resistance of tumor cells is an important factor leading to the failure of tumor chemotherapy. Vincristine (vincristine, VCR) is a kind of chemotherapeutic drugs widely used in tumor therapy. However, long-term use can result in drug resistance of tumor cells to VCR, and the mechanism of drug-resistance remains unclear. The purpose of this study was to explore the mechanism of drug resistance and to provide a basis for reversal of drug resistance in cancer cells. Objective to establish vincristine resistant colon cancer cell model and screen miRNA. associated with VCR resistance of colon cancer cells. Method 1. Colon cancer HCT-8 cells were cultured and induced by VCR gradient to establish HCT-8/VCR resistant cell line. 2. By using HiSeq 2500 sequencing technique and bioinformatics method, the differentially expressed miRNAs, of drug-resistant HCT-8/VCR cells and non-drug-resistant HCT-8 cells were detected and analyzed by using the strategy of genome screening. The differential expression profile of miRNA was constructed. The target site was identified by target gene prediction software and the functional annotation was performed by GO of the gene in which the target gene was located. Result 1. The VCR resistant colon cancer cell HCT-8/VCR, HCT-8/VCR cells were established by using concentration gradient VCR. The cells grew well in 2 脳 103ng/mLVCR. A total of 1651 miRNA sequences were performed. Among them, 48 miRNA, were significantly different from non-drug-resistant colon cancer HCT-8 cells in 24 miRNA (P0.05), 17 of which were miRNA up-regulated (P0.05), the highest (7.497681 times) in hsa-miR-675-3p expression, and the highest in Hsa-miR-675-3p expression (P0.05). The miRNA expression of hsa-miR-100-3p, hsa-miR-125b-1-3p, hsa-miR-582-5p, hsa-miR-3141, chr15_10055, hsa-miR-582-5p, hsa-miR-582-3p, hsa-miR-3687.7 species was down-regulated (P0.05), and the highest down-regulation of hsa-miR-146a-5p, was 7.75199 times, followed by hsa-miR-1247-3p, hsa-miR-181 a-3p, hsa-miR-1247-5p.. Conclusion: the difference of the expression of miRNA and VCR related to drug resistance in colon cancer cells may play a key role in the drug resistance of colon cancer cell line VCR.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.35

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