RNAi沉默EGFR基因对卵巢癌放疗敏感性影响.docVIP

RNAi沉默EGFR基因对卵巢癌放疗敏感性影响 　 作者：林远洪 吴永忠 李少林 郭启帅 罗茜 【摘要】 目的 探讨表皮生长因子受体(EGFR)基因对卵巢癌放疗敏感性的增强作用。方法 构建针对EGFR基因序列特异性短发夹状RNA(shRNA)表达载体，应用卵巢癌SKOV3细胞建立裸鼠移植瘤模型，随机分为对照组、单纯放疗组、基因放疗组，采用6 MV X线照射裸鼠瘤组织，监测瘤体积，测量瘤质量，取瘤组织行RTPCR、Western印迹、免疫组化、电镜检测。结果 治疗后三组瘤体积差异均显著(均Plt;0.01)，单纯放疗组、基因放疗组的抑瘤率分别为28.1%、46.1%，基因放疗组EGFR基因的表达明显下调，透射电镜下瘤组织凋亡、坏死最明显。结论 动物实验证实RNAi沉默EGFR基因明显提高卵巢癌的放疗敏感性。 【关键词】 EGFR基因;RNA干扰;放疗;卵巢癌 　　【Abstract】 Objective To explore the effects of epidermal growth factor receptor (EGFR) gene on the radiosensitivity of ovarian cancer by RNAi technology. Methods EGFR gene sequencespecific shRNA expression vector were constructed. Tumoranimal model with ovarian cancer SKOV3 cells were established and the nude mice were randomly divided into control, radiotherapy and generadiotherapy groups. The ovarian cancer tissue was irradiated by 6 MV Xray. Tumor volume and tumor weight were measured. RTPCR, Western blot, immunohistochemistry and electron microscopy were used to detected cancer tissue. Results There were obvious difference among three groups in tumor volume (Plt;0.01). The inhibitingtumor rate of the radiotherapy and the generadiotherapy groups were 28.1% and 46.1% respectively. The expression of EGFR gene in generadiotherapy group was significantly decreased and the tumor cell apoptosis, necrosis were all obvoius.Conclusions Silenced EGFR gene by RNAi can significantly improve the radiosensitivity of ovarian cancer. 　　【Key words】 EGFR gene;RNA interference;Radiotherapy;Ovarian cancer 　 表皮生长因子受体(EGFR)的高表达与肿瘤恶性程度、转移、放化疗敏感度下降密切相关，可作为诊断及判断预后的指标，许多阻滞和下调EGFR的方法被用来抑制肿瘤增殖以改善预后，并已显示出初步疗效及良好的安全性，因此EGFR成为目前公认的肿瘤基因治疗的重要靶位〔1〕。本实验选择放疗相对不敏感的卵巢癌SKOV3细胞为研究对象，以目前分子靶向治疗中的EGFR基因为靶点，采用RNA干扰(RNAi)技术，探讨EGFR基因与卵巢癌放疗敏感性的关系，实验初步显示了RNAi技术在卵巢癌基因放疗中的潜在作用，为肿瘤治疗提供新策略。 　　1 材料与方法 　　1.1 主要材料 卵巢癌SKOV3细胞株购自中科院上海细胞生物研究所;BALB/C雌性裸鼠购自北京维通利华公司。脂质体lipofectamine2000购自Invitrogene公司;去内毒素质粒提取试剂购自Omega公司;鼠抗EGFR单克隆抗体购自Santa Cruz公司;蛋白提取试剂盒购自赛百盛公司;免疫组化试剂盒及SDSPAGE凝胶购自博士德公司;总RNA提取试剂购自华舜公司;RTPCR试剂盒购自宝生公司。 　　1.2 方法 　　1.2.