【作者】 骆晓峰；

【导师】 陈志哲； 刘庭波；

【作者基本信息】 福建医科大学 ， 内科学血液病学， 2008， 硕士

【摘要】 研究背景:c-myc基因是调控细胞增殖与分化的癌基因,在急性白血病细胞株及急性白血病、恶性淋巴瘤患者的细胞中均出现高表达,是影响这些恶性肿瘤发生、发展的重要基因之一。RNA干扰(RNA interference)是许多生物体内的一种保守机制,是指一种双链RNA(double-stranded RNA,dsRNA)分子在mRNA水平关闭相应序列基因的表达使其沉默的过程,是一种序列特异性的转录后基因沉默(post-transcriptional gene silencing,PTGS)。其基本原理是dsRNA通过RNaseⅢ内切酶Dicer的作用产生21～23 nt有活性的小干扰RNA(small interfering RNA,siRNA),然后在细胞内形成RNA诱导沉默复合体(RNA induced silence complex,RISC),RISC根据碱基互补以siRNA为模板特异地识别其同源基因mRNA,并对其进行递进式剪切,诱导序列特异性mRNA的降解,从而抑制基因表达。RNAi是新近发展起来的一种基因功能研究的新方法[1,2],因而得到迅速发展,目前已广泛应用于功能基因组和基因治疗的研究[3,4]。本研究选择c-myc表达较高且c-myc在细胞增殖、凋亡中起重要作用的Jurkat细胞[5,6]作为研究对象,观察c-myc siRNA对其增殖、凋亡的影响。目的:探讨c-myc小干扰RNA对急性淋巴细胞白血病细胞系Jurkat细胞的增殖、凋亡的影响及对c-myc基因、蛋白表达的影响。对白血病的基因治疗提供新方法和靶点。方法:针对c-myc mRNA的第1545-1565靶位点设计siRNA,采取化学合成法合成。合成的c-myc siRNA,经转染剂转染入Jurkat细胞,观察细胞形态学变化,应用四唑氮化合物(MTS)法绘制细胞生长曲线,细胞集落培养观察c-myc siRNA对Jurkat细胞增殖的影响,应用流式细胞术和TdT酶介导的末端缺失原位标记(TUNEL)法分析细胞的凋亡,RT-PCR及Western blot检测c-myc siRNA作用前后c-myc、hTERT基因的mRNA及蛋白表达水平的变化。结果:(1) c-myc siRNA能明显抑制Jurkat细胞的增殖,作用48小时的半数抑制浓度(IC50)约为75nM,MTS法检测c-myc siRNA对Jurkat细胞的生长有抑制作用,对克隆形成也有明显的抑制作用。(2) c-myc siRNA可引起Jurkat细胞凋亡,经流式细胞仪、TdT酶介导的末端缺失原位标记(TUNEL)法均检测出细胞凋亡,且随着作用时间的延长,凋亡率也逐渐上升。(3) c-myc siRNA可引起Jurkat细胞的c-myc、hTERT mRNA表达水平的降低。(4) c-myc siRNA可引起Jurkat细胞的c-myc、hTERT蛋白表达水平的降低。结论:化学合成法合成的c-myc siRNA能明显抑制Jurkat细胞的增殖与克隆形成,并可显著诱导Jurkat细胞发生凋亡。c-myc siRNA明显降低Jurkat细胞c-myc、hTERT基因的mRNA及蛋白表达水平,有望成为白血病靶向基因治疗的新工具。更多还原

【Abstract】 Backgrounds: c-myc gene is an oncogene regulating proliferation and differentiation of cell. It has been found to be overexpressed in acute leukemia cell lines and cells of acute leukemia and malignant lymphoma patients. It’s one of the important genes influencing growth and progression of these malignancies. RNA interference(RNAi) is the sequence-specific gene silencing induced by double-stranded RNA(dsRNA), is the process of sequence-specific post-transcriptional gene silencing(PTGS) in animals and plants, initiated by double-stranded RNA that is homologous in sequence to the silenced gene. This phenomenon is conserved in a variety of organisms. RNAi is mediated by small interfering RNAs(siRNAs) that are produced from long dsRNAs of exogenous or endogenous origin by an endonuclease-Ⅲtype, called Dicer. The resulting siRNAs are about 21-23 nucleotides(nt) long and are then incorporated into a nuclease complex, the RNA-inducing silencing complex (RISC), which then targets and cleaves mRNA containing a sequence identical to that of the siRNA. Recently RNAi has became the new tool used to study the functions of genes and was used widly in the fields of genemic function research and gene therapy. In this study, Jurkat cell line with rather high expression level of c-myc which plays an important role in its proliferation and apoptosis was chosen to be the object which was treated with the anti c-myc siRNA.Objective: The aim of this study is to investigate the effects of anti c-myc siRNA on apoptosis and proliferation and on c-myc protein and mRNA expression in human lymphoblastic leukemia cell line Jurkat cells and is to provide a target for treatment and new gene therapy method of leukemia.Methods: siRNA targeting the site 1545-1565 of c-myc mRNA was designed and chemically synthesized. In vitro cultured Jurkat cells were transfected with transfection agent. Growth inhibition was detected by MTS assay and colony formation assay, and cell apoptosis by flow cytometry and detection of TdT mediated dUTP nick end labeling(TUNEL). The expression of c-myc and hTERT was detected by RT-PCR and Western blot.Result: (1) c-myc siRNA remarkably inhibited the cell proliferation, and the inhibitory concentration 50%(IC50) after 48 hour of treatment was about 75nM. By MTS assay, c-myc siRNA had inhibition effects on the growth of Jurkat cells and inhibited clone growth significantly. (2) c-myc siRNA induced apoptosis in Jurkat cells that could be detected by flow cytometry and detection of TdT mediated dUTP nick end labeling(TUNEL), These also showed that c-myc siRNA induced apoptosis in Jurkat cells in time-dependent manner. (3) c-myc siRNA can decrease the expression levels of c-myc and hTERT mRNA expression in Jurkat cells. (4) c-myc siRNA can decrease the expression levels of c-myc and hTERT proteins in Jurkat cells.Conclusion: c-myc siRNA chemically synthesized could inhibit significently Jurkat cells proliferation and induce apoptosis. c-myc siRNA decreased c-myc and hTERT mRNA and proteins expression in Jurkat cells, it may become one of the new tools of gene therapy to acute leukemia.更多还原