【作者】 马常慧；

【导师】 陈海滨；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 目的:肿瘤坏死因子受体1(tumor necrosis factor receptor 1,TNFR1)是肿瘤坏死因子(tumor necrosis factor,TNF)的膜受体之一。TNFR1在人体内广泛分布于正常细胞膜表面,也存在于多种肿瘤细胞表面。TNFR1与配体TNF-α或TNF-β结合后,启动相关的信号通路,功能涉及到细胞的活化、增殖、凋亡及细胞毒活性等方面,但在不同的细胞,可以出现相反的结果,其作用机制目前尚不完全清楚。RNA干扰(RNA interference,RNAi)技术是由与靶基因序列同源的双链RNA(double-stranded RNA,dsRNA)引发的普遍存在于生物体中的序列特异性基因转录后沉默过程,越来越广泛地应用于基础实验研究。本研究主要应用RNAi的实验方法,沉默食管癌细胞系EC109中TNFR1的表达,随后探讨对照组与实验组细胞增殖、凋亡等生物学行为的变化。材料和方法:1. EC109细胞用DMEM+F12培养基常规培养传代,RNAiso Reagent提取细胞总RNA,RT-PCR检测细胞中TNFR1在基因水平的表达。EC109细胞用DMEM+F12培养基常规培养传代,胰酶消化后收集贴壁生长的细胞,PBS清洗、离心、裂解,试剂盒分级提取可溶性亚细胞蛋白组分,所得膜蛋白进行Western Blot分析,检测EC109中TNFR1在蛋白水平的表达。2. EC109细胞用DMEM+F12培养基在六孔板内培养,在对转染条件进行优化后,对细胞进行转染,细胞分三组:空白对照组(未加转染试剂及siRNA),阴性对照组(加入转染试剂及阴性对照siRNA),实验组(加入转染试剂及TNFR1-siRNA)。RNAiso Reagent消化细胞后提取各组细胞总RNA,RT-PCR检验基因水平转染效果。试剂盒分级提取可溶性亚细胞蛋白组分,Western Blot技术检验蛋白水平转染效果。3.转染24小时后倒置相差显微镜观察细胞形态。4.将EC109细胞种于96孔板,对转染条件进行优化,细胞转染后用四甲基偶氮唑蓝(MTT)检测转染前后细胞增殖能力的改变,绘制细胞生长曲线。5.收集转染后六孔板中各组细胞,应用流式细胞术检测细胞凋亡的变化情况。结果:1.由RT-PCR以及Western Blot等手段验证TNFR1在食管癌细胞系EC109中高表达。2.将TNFR1-siRNA转染进EC109细胞中,成功封闭了TNFR1的表达。3. RNAi处理后,三组细胞在形态上没有显示差别;RNAi处理后MTT检测结果显示,空白对照组和阴性对照组的细胞32小时内的细胞增殖没有差异(P>0.05),实验组的细胞增殖加快(P<0.05);RNAi处理后空白对照组和阴性对照组的细胞凋亡情况无差异(P>0.05),实验组的细胞凋亡率降低(P<0.05)。结论:1. TNFR1在食管癌细胞系EC109中高表达。2.阻断TNFR1的表达使食管癌细胞的增殖、凋亡均发生变化,提示TNFR1在食管癌发生发展中发挥十分重要的作用。3.在食管癌细胞系EC109中,TNFR1主要发挥促进细胞凋亡的作用。更多还原

【Abstract】 Objective:Tumor necrosis factor receptor 1(TNFR1) is one of the membrane receptors of tumor necrosis factor (TNF). TNFR1 distributes on the surface of human normal cell membranes generally, as well as many kinds of tumor cell membranes. After binding with its’specific ligand TNF-αor TNF-β, TNFR1 plays an important part in many aspects, such as cell activation, multiplication, apoptosis and toxicity; but the effect may be opposite in different cells. At present, the concrete mechanism is not clear. RNA interference (RNAi), a new experimental technique in recent years, is a specific post-transcriptional gene silencing progress exsisting in the organisms widely, triggered by the double-stranded RNA which is homologous with the target gene sequence. The technology is used in the experimental research more and more extensively. Applying the RNAi, we silenced the expression of TNFR1 in esophageal carcinoma cell line, and then showed the biological behavior changes between experimental group and control group.Materials and methods:1. EC109 cell line was cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Total RNA was extracted from cells by RNAiso Reagent, the genetic level of TNFR1 was detected by RT-PCR. EC109 cell line was cultivated in DMEM supplemented with 10% fetal bovine serum, then the adherent cells were collected by trypsinization, soluble subcellular protein components were extracted fractionally by ProteoExtract Subcellular Proteome Extraction Kit after PBS washing, centrifugation and spallation, membrane protein component was analysed by Western Blot detecting the protein level of TNFR1 in EC109 cell line.2. EC109 cell line was cultivated in DMEM supplemented with 10% fetal bovine serum in six pore plate preparing for transfection, after optimization of cell transfection conditions, cells were divided into three groups : blank control group(without transfection regent and siRNA), negative control group (added transfection regent and negative control-siRNA),experimental group(added transfection regent and TNFR1-siRNA). Cell total RNA was extracted by RNAiso reagent for RT-PCR detecting the effect of RNAi on genetic level. Soluble subcellular protein components were extracted fractionally by ProteoExtract Subcellular Proteome Extraction Kit after PBS washing, centrifugation and spallation, membrane protein component was analysed by Western Blot detecting the effect of RNAi on protein level.3. 24 hours after transfection, morphology investigation with inverted phase contrast microscope was adopted.4. EC109 cell line grew in 96-well plates, after optimization of cell transfection conditions, proliferation changes by methylthiazolyl tetrazolium (MTT) was adopted after transfection.5. Collecting cells in six pore plate, Flow cytometry was adopted to detect cell apoptosis changes after transfection.Results:1. It is confirmed that TNFR1 has a high expression in EC109 cell line by RT-PCR and Western Blot.2. We silenced the expression of TNFR1 successfully by transferring TNFR1-siRNA to EC109 cell line.3. After RNAi, three groups showed no diffenrences in morphology; The MTT results were similar between blank control group and negative control group within 32 hours after RNAi (P>0.05); there had a increasing proliferation in experimental group (P<0.05); The flow cytometry results were similar between blank control group and negative control group after RNAi (P>0.05); there had a decreased apoptosis in experimental group (P<0.05).Conclusions:1. TNFR1 expressed highly in EC109 cell line.2. TNFR1 gene silencing effect esophageal carcinoma cell on proliferation and apoptosis; it suggests TNFR1 plays an important role in cancer genesis and development.3. TNFR1 is inclined to be a role mediating apoptosis in esophageal carcinoma EC109 cell line.更多还原