放射线诱导的HtrA2基因表达对人葡萄膜黑色素OCM-1细胞的影响  

The effect of up-regulation of HtrA2 gene expression via radiation in human uveal melanoma cells

作　　者：雷荣[1] 李娟[2] 余天[1] 张帆[3] 

机构地区：[1]武汉爱尔眼科医院泪道科,湖北武汉430063 [2]华中科技大学同济医学院附属协和医院干细胞中心,湖北武汉430022 [3]华中科技大学同济医学院附属同济医院骨科,湖北武汉430030

出　　处：《中国癌症杂志》2014年第2期112-118,共7页China Oncology

基　　金：国家自然基金资助项目(No:81202093)

摘　　要：背景与目的:葡萄膜黑色素瘤(uveal melanoma,UM)是成人最常见的眼内原发性恶性肿瘤,其恶性程度高,转移早。放射治疗是多年来葡萄膜黑色素瘤最常用的方法之一,但辐射抗性和受照部位正常组织的损伤是长期困扰UM放疗的问题。本实验运用放射线可调控的HtrA2基因联合疗法,探讨其对UM的抑制作用。方法:含Egr1启动子的pIRES-Egr1-Omi/HtrA2(pEgr1-HtrA2)质粒体外转染入OCM-1细胞,转染后的细胞暴露于不同剂量的放射线中。将移植瘤模型小鼠分为对照组、放射组、基因组和基因放射组,每组10只。应用实时定量PCR(qRT-PCR)和蛋白质印迹法(Western blot)检测HtrA2基因的mRNA表达和蛋白表达。流式细胞仪检测联合处理后的OCM-1细胞凋亡情况。通过克隆形成实验检测HtrA2基因对放射敏感性的影响。在动物实验中测量联合治疗后肿瘤体积。结果:转染pEgr1-HtrA2质粒后,放射组HtrA2 mRNA表达在8 Gy达到高峰,且HtrA2蛋白表达较放射前明显增加(P<0.05),OCM-1细胞凋亡显著增加。克隆形成实验显示,HtrA2基因可增强OCM-1细胞放射敏感性。与对照组相比,基因放射组肿瘤生长被显著抑制(P<0.05)。结论:转染pEgr1-HtrA2质粒后可在放射线调控下增加HtrA2基因表达,并能增强OCM-1细胞对放射的敏感性,抑制肿瘤细胞生长。Background and purpose： Uveal melanoma （UM） is the most common primary intraocular malignancy in adult. Due to a high tendency for early metastasis the treatment of UM is very difficult. This study aimed to explore an effective approach for the treatment of patients with UM, we designed a strategy that combined HtrA2 gene therapy and radiation therapy. Methods： plRES-Egrl-Omi/HtrA2 （pEgrl-HtrA2） recombinant plasmids were constructed and transfected into human UM cells （OCM-1） in vitro. The transfected ceils were exposed to irradiation. HtrA2 mRNA and protein levels were detected by qRT-PCR and Western blot respectively. Assays that evaluated the apoptosis inducibility caused by Htr.42 gene therapy combined with radiation was performed by flow cytometry. Followingly, the effects of HtrA2 overexpression on the in vitro radiosensitivity of uveal melanoma cells were investigated by clonogenic formation assay. The in vivo effects of HtrA2 gene therapy combined with radiation therapy were evaluated in different groups. Results： The recombinant plasmids could be successfully transferred into OCM-1 cells and transfection of pEgrl-HtrA2 plasmids combined with radiotherapy caused dramatically elevation of HtrA2 compared with non-irradiation cells in mRNA and protein levels, which was associated with increased apoptosis.Furthermore, we observed that the transfection of pEgrl-HtrA2 could significantly enhance radiosensitivity of OCM-1 cell in vitro. In mice bearing xenograft tumors, pEgrl-HtrA2 combined with radiation therapy significantly inhibited tumor growth compared with the other treatment groups （P〈0.05）. Conclusion： Our findings indicate that radiation-inducible gene therapy may have potential to be a more effective and specific therapy for uveal melanoma because the therapeutic gene can be spatially or temporally controlled by exogenous radiation.

关 键 词：葡萄膜黑色素瘤 细胞凋亡 HtrA2基因 基因放射治疗 

分 类 号：R730.55[医药卫生—肿瘤]