【作者】 翁德胜；

【导师】 丁彦青；

【作者基本信息】 第一军医大学 ， 病理学， 2004， 硕士

【摘要】 鼻咽癌(nasopharyngeal carcinoma, NPC)是中国南方数省高发的鼻咽部恶性上皮源性肿瘤。目前的研究表明，NPC发病是一个多因素、多阶段的过程。表皮生长因子受体(epidermal growth factor receptor, EGFR)在肿瘤的发生发展的各个阶段均扮演着重要的角色，人体多种肿瘤中EGFR均处于高表达状态。在NPC患者中EGFR表达阳性率明显高于对照组，并且肿瘤分化越差，EGFR表达阳性率越高；NPC有颈淋巴结转移者EGFR表达明显高于无颈淋巴结转移者。说明EGFR与NPC的发生、癌细胞分化和肿瘤进展有一定关系。 RNA干扰(RNA interference, RNAi)是指内源性或外源性双链RNA(double stranded RNA, dsRNA)介导的细胞内mRNA发生特异性降解，从而导致靶基因的表达沉默，产生相应的功能表型的缺失现象。这一现象属于转录后基因调控机制。目前RNAi已应用于基因敲除、基因表达调控、信号转导和基因治疗等方面的研究，与以往基因沉默的研究方法相比，RNAi能够更加高效特异地进行特定基因的抑制。利用这一崭新的技术使鼻咽癌细胞EGFR表达沉默，观察细胞水平变化，能够探讨EGFR与NPC之间的相关性，且具有高效、方便的优点。 本课题采用体外转录的方法合成EGFR特异性siRNA，将其瞬时转染人鼻咽癌细胞株CNE1和5—8F，检测转染后EGFR表达水平的变化，筛选有效干扰序列，并通过观察EGFR表达沉默后鼻咽癌细胞的生长情况，初步探索EGFR与鼻咽癌的相关性。 课题进行了如下工作： 1．在www.oligoengine.com上进行设计，得到三条siRNA序列。利用T7启动子体外转录合成单链RNA，退火后得到dsRNA，对dsRNA进行消化并纯化，获得高纯度的EGFR 5 iRNA。 2.分别以10nM、20nM、40nM和60nM的三对SIRNA用脂质体瞬时转染CNEI、5一SF，提取转染后24h、48h、96h和144h细胞总RNA和蛋白质，用半定量RT一PCR和Western Blot测定EGFR表达水平变化。RT一PCR结果显示，51 RNAI能够有效地沉默两种细胞的EGFR表达，抑制效率在24h时可达71.7%和67.5%，siRNAZ和SIRNA3则无法有效干扰EGFR表达。RT一PCR和Western Blot显示，在一定范围内随SIRNA浓度增加，对EGFR抑制效应增强，40nM时抑制效率最高。实验结果还表明，体外转录合成的siRNA作用时间有限，超过48h后效应减弱。 3.筛选出有效的SIRNA序列和最佳作用浓度后，将40nM SIRNAI转染两种鼻咽癌细胞，用台盼蓝染色计数法绘制细胞生长曲线，用流式细胞仪检测细胞生长周期变化。结果显示，EGFR表达沉默后，CNEI及5一8F生长速度明显下降。流式细胞仪检测发现，RNAi抑制EGFR表达能够诱导鼻咽癌细胞停滞在G1期。 本研究创新之处在于: 1.运用体外转录法合成SIRNA，成功建立了RNAi抑制鼻咽癌EGFR表达的新技术，为快速沉默特定基因表达提供了新方法。 2.首次在细胞水平证实，RNAi沉默EGFR表达可抑制鼻咽癌细胞生长，并诱导癌细胞停止在G1期。 3.对EGFR 5 iRNA在鼻咽癌细胞株CNEI和5一8F中的作用条件进行了优化，证实其最佳作用浓度为40nM，干扰效应在48h内最强 我们的工作为探索鼻咽癌发生发展机制和基因治疗提供了新思路。更多还原

【Abstract】 Nasopharyngeai carcinoma (NPC) is a human malignant tumor derived from nasopharyngeal epithelial cells with high incidence in several provinces of Southern China. The development of NPC is a multifactor and multistage process. Epidermal growth factor receptor (EGFR) plays important roles in occurrence and development of human tumors. Some studies revealed that EGFR over expressed in many malignant tumors. EGFR expression is detected more frequently in NPC patients than in normal people, and the poorly-differentiated NPC cases are more likely to be EGFR positive. The NPC cases with neck lymph node metastasis show obviously higher EGFR positive rate than those without metastasis. This indicates that EGFR maybe closely related to NPC occurrence, differentiation and evolvement.RNA interference (RNAi) refers to phenomenon that endogenous or exogenous double-stranded RNA (dsRNA) mediate degradation of specific mRNA, which results in silence of target genes and brings about the absence of specific functional phenotype. RNAi regulates gene expression after transcription. This new technology is now used to research gene knock-out, signal pathway, gene regulation and gene therapy. Compared to those methods inducing gene silence, RNAi is more specific and more effective. RNAi has many advantages in study of triggering EGFR silence of NPC cells. It can help us to observe the change of the cells and explore the possible relation between EGFR and NPC more conveniently.Using in vitro transcribed synthesizing method, we constructed threeEGFR specific siRNAs. To screen effective siRNA, we transiently transferred these siRNAs into NPC cells CNE1 and 5-8F, then detected the EGFR expression level by RT-PCR and Western Blot. After the EGFR expression of the cells was silenced, we detected the growth and cell cycle changes.We did the following work in this paper:Firstly, we designed 3 siRNAs sequences according to the principles on the web site www.oligoengine.com. After synthesis of DNA templates, single stranded RNAs were transcribed in vitro from the templates using T7 promoter. High quality siRNAs were obtained after annealing, digestion and purification.Secondly, we transiently transferred 10M, 20nM, 40nM and 60nM siRNAs into CNE1 and 5-8F respectively by lipofectamine. Total RNA and protein were extracted after 24h, 48h, 96h and 144h. EGFR expression levels were detected by semi-quantitative RT-PCR and Western Blot. The results of RT-PCR indicated that only No.1 siRNA can suppress EGFR effectively. 24h after siRNA transfection, CNE1 and 5-8F EGFR mRNA content was decreased by approximately 71.7% and 67.5% respectively. The results of RT-PCR and Western Blot showed that siRNA silencing effect ascended with the increasing of tranfection concentration to some extent. The effect got to top when siRNA transfection concentration was 40nM. We also found that the silencing effect can only last for less than 96h.Finally, we transiently transferred 40nM No. 1 siRNA into the NPC cells. The cells were harvested and counted by trypan blue exclusion every day after transfection. 24h and 48h after transfection, the cells were harvested and cell cycle distribution were analyzed using FACS. Transfection with siRNA targeted EGFR resulted in reduction of CNE1 and 5-8F cells proliferation. FACS results indicated cell cycle arrest at Gl phase but not apoptosis in association with EGFR silencing. The innovation of our research is:First, we established a new method to inhibit the expression of EGFR inNPC cells using siRNA synthesized by in vitro transcribing, which provided a new way for specific gene silencing research.Second, for the first time, we demonstrated at cell level that EGFR silencing can reduce NPC cell proliferation and arrest cell cycle at Gl phase.Finally, through optimizing experiments we found that the best working siRNA transfection concentration of EGFR in NPC cells is 40nM, and the good silencing effect can only last for about 48h.The finding of this study sheds light on the possible use of RNA interferen更多还原