肝癌5-氟尿嘧啶耐药细胞株的构建及其耐药机制的初步研究

发布时间：2018-04-06 04:01

本文选题：肝细胞癌　切入点：5-氟尿嘧啶　出处：《西北大学》2017年硕士论文

【摘要】：肝细胞癌(hepatocellular carcinoma,HCC)是一种世界性的恶性肿瘤,其发病率随着环境和生活方式的改变而逐年增高。针对肝癌晚期患者,化学疗法是一种有效的治疗手段。而5-氟尿嘧啶(5-Fluorouracil,5-FU)作为首选的一线化疗药物之一,常在临床上被用于治疗肝癌晚期患者。但是,在长期使用的过程中,患者易对5-氟尿嘧啶等常规化疗药物产生耐药性,从而导致治疗效果减弱甚至失效。现阶段对于5-氟尿嘧啶在肝癌中的耐药机制仍不完全清楚。本实验旨在筛选出与5-氟尿嘧啶的耐药机制密切相关的基因和信号通路。我们使用浓度梯度递增的方法药物诱导人肝癌HepG2细胞,再通过有限稀释法得到了由单细胞衍生而来的两种细胞株,分别为HepG2敏感细胞株和HepG2/5-FU耐药细胞株。利用转录组测序(Transcriptome Sequencing,RNA-Seq)技术,我们得到了这两个细胞株样品的转录组信息(总共检测了 21409个基因),随后分析了这两个样本中的基因表达量,并筛选出641个差异表达基因(differentially expressed genes,DEGs),包含350个上调基因和291个下调基因。在完成对基因的功能注释和富集分析之后,我们发现,共有13个差异表达基因被富集到了 Wnt信号通路,其中三个基因具有上下游关系且其表达差异符合预期,它们分别为 Wnt 通路抑制因子 1(dickkopf WNT signaling pathway inhibitor 1,DKK1)、卷曲受体 10(frizzled class receptor 10,FZD10)、和人散乱蛋白 1(dishevelled segment polarity protein 1,DVL1)。随后,采用实时荧光定量PCR(q-PCR)验证了测序结果的可靠性以及三个Wnt信号(DKK1、FZD10和DVL1)的表达差异。最后,利用siRNA干扰技术验证了这三个Wnt信号与HepG2细胞5-FU耐药之间的关联。在本研究中,我们筛选出三个新的生物标志物(DKK1、FZD10和DVL1)(位于Wnt信号通路中),其表达调控与肝癌5-FU耐药性密切相关。研究表明,Wnt信号通路在肝癌5-FU耐药性的形成过程中可能起到了重要的作用,这为肝癌耐药患者的个体化治疗提供了一个潜在靶点。
[Abstract]:Hepatocellular carcinoma (HCC) is a worldwide malignant tumor. The incidence of HCC increases year by year with the change of environment and lifestyle.Chemotherapy is an effective treatment for advanced liver cancer patients.5-Fluorouraciline 5-FU (5-Fluorouraciline 5-FU), as one of the first-line chemotherapeutic drugs, is often used in the treatment of advanced liver cancer patients.However, in the long-term use, patients tend to develop drug resistance to 5-fluorouracil and other conventional chemotherapeutic drugs, resulting in weakened or even ineffective treatment.At present, the mechanism of 5-fluorouracil resistance in hepatocellular carcinoma is not fully understood.The aim of this study was to screen genes and signaling pathways closely related to the resistance mechanism of 5-fluorouracil.We used the method of increasing concentration gradient to induce human hepatoma HepG2 cells. Then we obtained two kinds of cell lines derived from single cell by finite dilution method: HepG2 sensitive cell line and HepG2/5-FU drug resistant cell line.Using transcriptome sequencing RNA-Seq-based technique, we obtained transcriptome sequencing RNA-Seq-based information for the two cell lines (21409 gene loci were detected, and then the amount of gene expression in the two samples was analyzed.A total of 641 differentially expressed genes were screened, including 350 up-regulated genes and 291 down-regulated genes.After functional annotation and enrichment analysis, we found that 13 differentially expressed genes were enriched into the Wnt signaling pathway.They are 1(dickkopf WNT signaling pathway inhibitor 1 (DKK1), 10(frizzled class receptor 10 (FZD10), and 1(dishevelled segment polarity protein 1 (DVL1), respectively.The reliability of the sequencing results and the differences in the expression of three Wnt signals DKK1FZD10 and DVL1) were verified by real-time fluorescence quantitative PCRQ-PCR.Finally, siRNA interference technique was used to verify the correlation between the three Wnt signals and 5-FU resistance in HepG2 cells.In this study, we screened out three new biomarkers, DKK1FZD10 and DVL1 (located in the Wnt signaling pathway), whose expression regulation is closely related to 5-FU resistance in HCC.It is suggested that the Wnt signaling pathway may play an important role in the formation of 5-FU resistance in hepatocellular carcinoma, which provides a potential target for individualized treatment of drug-resistant HCC patients.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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