【摘要】：背景腫瘤細胞耐藥是導致腫瘤化學治療失敗的重要原因,長春新堿(vincristine, VCR)是一類廣泛用于腫瘤治療的化學藥物。但是,長期使用可使腫瘤細胞對VCR產生耐藥性,腫瘤耐藥機制仍不清楚。本研究探討與結腸癌耐藥的微小RNA(microRNA, miRNA),旨在揭示腫瘤耐藥機制及為腫瘤細胞耐藥逆轉提供基礎。目的建立長春新堿耐藥結腸癌細胞模型,篩選與結腸癌細胞VCR耐藥相關的miRNA。方法1.培養(yǎng)結腸癌HCT-8細胞,采用VCR梯度誘導,建立HCT-8/VCR耐藥細胞株。2.采用全基因組篩選的研究策略,運用HiSeq 2500測序技術和生物信息學的方法,檢測和分析耐藥HCT-8/VCR細胞的和非耐藥的HCT-8細胞差異性表達的miRNAs,構建其miRNA的差異表達譜。3.針對鑒定的差異miRNA,通過靶基因預測軟件進行靶位點確認,通過靶基因所在基因的GO進行功能注釋。結果1.利用濃度梯度的VCR建立了VCR耐藥的結腸癌細胞HCT-8/VCR, HCT-8/VCR細胞在2×103ng/mLVCR中生長良好。2.共進行了1651種miRNA測序,其中有差異的有48種miRNA,與非耐藥的結腸癌HCT-8細胞相比,24種miRNA差異具有統(tǒng)計學意義(P0.05),其中有17種miRNA表達上調(P0.05)。hsa-miR-675-3p表達量上調最高,高達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表達下調(P0.05),下調最高的是hsa-miR-146a-5p,下調7.75199倍,其次是hsa-miR-1247-3p, hsa-miR-181 a-3p, hsa-miR-1247-5p。結論]miRNA的異常表達與結腸癌細胞VCR耐藥相關。miRNA表達量的差異可能在結腸癌細胞VCR耐藥中起關鍵作用。
[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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.35

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