慢病毒介导RNA干扰抑制RRS1基因表达对胆管癌RBE细胞增殖的影响

发布时间：2018-06-15 13:19

本文选题：RRS1 + 胆管癌　；参考：《南华大学》2015年硕士论文

【摘要】：目的：本研究将探讨运用慢病毒介导RNAi干扰技术抑制RRS1基因在胆管癌RBE细胞表达后,能否在体外实验中对胆管癌RBE细胞增殖产生影响。材料与方法：1.第一步确定在胆管癌中RRS1基因是否异常表达,运用RT-PCR技术手段检测湖南省人民医院肝脏外二科2013年5月至2013年11月间10例手术切除的胆管癌与癌旁胆管组织标本RRS1mRNA基因水平的表达。2.第二步拟通过慢病毒载体介导RNA干扰技术,从转录水平沉默RRS1基因表达,RT-PCR技术检测抑制RRS 1后在胆管癌mRNA基因水平的表达；体外实验观察抑制RRS1基因后对胆管癌RBE细胞增殖的影响,初步探讨抑制RRS1基因表达后对胆管癌细胞增殖的影响。结果：1.在10例临床样本中,RT-PCR检测结果发现在胆管癌组织中的RRS1 mRNA基因表达水平较癌旁胆管组织明显升高,差异具有统计学意义(P0.05)。提示在胆管癌中RRS1基因表达上调。2.成功通过慢病毒介导RNA干扰技术阻断了转录后水平RRS1基因的表达,RT-PCR技术手段检测实验组(转染RRS1-shRNA组)与阴性对照组(转染shRNA慢病毒载体组)RRS1mRNA基因表达水平,结果表明抑制了RRS1表达的实验组其mRNA的相对表达量受到明显抑制,差异具有统计学意义(P0.05)。评估其基因敲减效率为80.10%,提示RNA干扰RRS1基因效果显著。3.在体外实验中运用cellomics检测实验组和阴性对照组两组细胞生长计数并绘制其生长曲线,结果表明RNA干扰抑制RRS1基因在胆管癌RBE细胞中表达后,胆管癌RBE细胞的增殖能力受到了明显下降。结论：1.通过实时荧光定量PCR检测临床样本,其数据表明在胆管癌组织中的RRS1 mRNA表达水平较癌旁胆管组织显著增高,提示RRS1在胆管癌组织中表达上调。2.慢病毒RNAi干扰抑制RRS1基因在胆管癌RBE细胞中的表达后,胆管癌RBE细胞生长与增殖能力有明显下降,提示RRS1可能会促进胆管癌细胞的增殖能力。
[Abstract]:Aim: to investigate the effect of lentivirus-mediated RNAi interference on the proliferation of RBE cells of cholangiocarcinoma after inhibiting the expression of RRS1 gene in RBE cells in vitro. Materials and methods: 1. The first step is to determine the abnormal expression of RRS1 gene in cholangiocarcinoma. RT-PCR technique was used to detect the expression of RRS1 mRNA gene in 10 cases of cholangiocarcinoma and adjacent bile duct tissues surgically resected from May 2013 to November 2013 in the second Department of external liver of Hunan Provincial people's Hospital. In the second step, RNA interference mediated by lentivirus vector was used to detect the mRNA expression of RRS1 in cholangiocarcinoma after inhibition of RRS1 by reverse transcriptional silencing of RRS1 gene. To observe the effect of inhibiting RRS1 gene on the proliferation of cholangiocarcinoma RBE cells in vitro and to explore the effect of inhibition of RRS1 gene expression on the proliferation of cholangiocarcinoma cells in vitro. The result is 1: 1. The results of RT-PCR in 10 clinical samples showed that the expression level of RRS1 mRNA in cholangiocarcinoma tissues was significantly higher than that in adjacent bile duct tissues, and the difference was statistically significant (P 0.05). These results suggest that RRS1 gene expression is up-regulated in cholangiocarcinoma. The expression of RRS1 gene at post-transcriptional level was blocked by lentivirus mediated RNA interference. RT-PCR was used to detect the expression of RRS1 mRNA in experimental group (transfected with RRS1-shRNA) and negative control group (transfected shRNA lentivirus vector group). The results showed that the relative expression of RRS1 mRNA in the experimental group was significantly inhibited, and the difference was statistically significant (P 0.05). The gene knockout efficiency of RRS1 gene was evaluated to be 80.10%, indicating that RNA interference with RRS1 gene had a significant effect on RRS1 gene. In vitro, cellomics was used to detect the cell growth of the experimental group and the negative control group. The results showed that RNAi inhibited the expression of RRS1 gene in bile duct carcinoma cells. The proliferative ability of RBE cells in cholangiocarcinoma was significantly decreased. Conclusion 1. Clinical samples were detected by real-time fluorescence quantitative PCR. The results showed that the expression of RRS1 mRNA in cholangiocarcinoma tissues was significantly higher than that in adjacent bile duct tissues, suggesting that RRS1 mRNA expression was up-regulated in cholangiocarcinoma tissues. Lentivirus RNAi interference inhibited the expression of RRS1 gene in bile duct carcinoma RBE cells, and the growth and proliferation ability of bile duct carcinoma RBE cells decreased significantly, suggesting that RRS1 might promote the proliferation of cholangiocarcinoma cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.8

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