超声靶向微泡破裂法介导RNA干扰沉默survivin基因对宫颈癌HeLa细胞凋亡的影响  

Effects of silencing survivin gene by RNA interference on cell apoptosis using ultrasound targeted microbubble destruction techniques

作　　者：陈智毅[1] 梁琨[2] 谢明星[3] 张静[3] 

机构地区：[1]广州医学院第三附属医院医学超声科,广东广州510150 [2]广州医学院第三附属医院妇产科,广东广州510150 [3]华中科技大学同济医学院附属协和医院超声影像科湖北省分子影像重点实验室,湖北武汉430022

出　　处：《肿瘤》2009年第7期626-630,共5页Tumor

摘　　要：目的:通过超声靶向微泡破裂(ultrasound targeted microbubble destruction,UTMD)法介导基因转染,观察RNA干扰对宫颈癌HeLa细胞survivin基因的沉默效应和诱导细胞凋亡的作用。方法:构建靶向survivin基因的短发夹状RNA(short hairpin RNA,shRNA)表达质粒,将其和微泡共同处理后加入HeLa细胞并予以超声辐照,以空白细胞、单纯质粒、单纯超声辐照、质粒+超声辐照和质粒+Lipofectamine转染作为对照;采用荧光显微镜和FCM法评估基因转染效率,同时对超声辐照参数进行优化;应用FCM法和Hoechst染色及DNA ladder分析法检测细胞凋亡,应用RT-PCR检测survivin mRNA水平的变化。结果:经酶切鉴定及测序分析证实重组质粒构建成功。HeLa细胞采用UTMD处理后,基因转染率显著增加(P<0.01),并在超声强度为1.0W/cm2、辐照3min的条件下最为显著(P<0.01)。PT-PCR检测结果显示,质粒与微泡共同作用加超声辐照组的survivin mRNA抑制率为(83.33±2.73)%,均显著高于各对照组(P<0.01);FCM法检测结果显示,该组细胞的凋亡率同样显著高于各对照组(P<0.01);Hoechst染色及DNA ladder检测结果显示,只有经脂质体或UTMD转染处理后的细胞中可检测到明显的细胞凋亡形态及凋亡梯带,其余各组均未检测到明显凋亡。结论:UTMD可有效增强表达质粒的转染效率,是一种新的、有应用前景的非病毒基因转移体系。UTMD介导RNA干扰沉默survivin基因可诱导明显的细胞凋亡,为肿瘤基因治疗及研究提供了新的方法。Objective：To transfect genes using ultrasound targeted microbubble destruction （UTMD） techniques and observe the effects of RNA interference on cervical cancer （HeLa） cell line in silencing survivin gene and inducing apoptosis. Methods： Recombinant expression plasmid of short hairpin RNA（shRNA） targeting survivin gene was constructed. It was co-treated with microbubbles and transfected to cultured HeLa cells followed hy exposure to ultrasound （P＋UTMD group）. Moreover, blank control group （C）, plasmid group （P） , ultrasound exposure group （US） , plasmid and uhrasound exposure group （P ＋ US） , plasmid ＋ Lipofectamine group （ P ＋ L） were used as controls, respectively. Transfection efficacy was evaluated by observing the red fluorescence in the cells by fluorescent microscopy and flow cytometry（FCM）. Ultrasound intensity and exposure time were optimized. Cell apoptosis was investigated using flow cytometry analysis, Hoechst staining, and DNA ladder method. Expression of survivin mRNA was assessed by RT-PCR. Results： Restrictive enzyme digestion and sequencing analysis verified that the recombinant plasmid was successfully constructed. UTMD signifi-cantly increased gene transfection efficacy in cultured HeLa cells （P 〈0.01 ）. Gene transfer was the most prominent at ultrasound intensity of 1.0 W/cm^2 and exposure time of 3 rain （ P〈0.01）. RT-PCR showed that the expression of survivin mRNA in P ＋ UTMD group was inhibited by （83.33±2.73）%. The differences were significant compared with any other groups （ P 〈 0.01 ）. FCM analysis showed that the apoptosis ratio in P ＋ UTMD group was significantly increased as compared with other groups （ P 〈 0.01 ）. Hoechst staining and DNA ladder showed that apparent apoptosis and DNA ladder were detected only in P＋UTMD and P ＋ L groups. Conclusions： UTMD effectively enhances the transfeetion efficacy of expression plasmid, it is a novel and effective non-viral gene transfer system and has promi

关 键 词：宫颈肿瘤 RNA 小分子干扰 实验室技术和方法 基因 suvvivin 

分 类 号：R737-33[医药卫生—肿瘤]