【作者】 唐薇薇；

【导师】 郑洪；

【作者基本信息】 遵义医学院 ， 病理学与病理生理学， 2009， 硕士

【摘要】 背景与目的:近年来,人们对肿瘤的研究逐步向分子领域延伸,以期通过基因治疗达到治愈肿瘤的目的。细胞凋亡调控的紊乱是肿瘤发生、发展的一个重要环节,其中凋亡抑制蛋白家族（Inhibitor of apoptosisproteins,IAPs）是一个研究热点。Survivin基因是IAPs中的一个新成员,具有抑制细胞凋亡和促进细胞增殖的活性,通过直接或间接抑制caspase3和caspase7活性等途径阻断各种刺激诱导的细胞凋亡。RNA干扰（RNAi）是当前应用在逆向遗传学研究中高效的基因沉默技术,能高效特异地阻断基因的表达。目前,有关siRNA沉默survivin基因对卵巢癌H08910PM细胞凋亡作用的研究还鲜有报道。本实验意在研究Survivin-shRNA对survivin的沉默效应在卵巢癌细胞H08910PM中的作用,为卵巢癌基因治疗奠定一定的基础。方法:研究对象为人卵巢癌H08910PM细胞株。（1）设计并构建针对survivin的siRNA真核表达质粒载体;（2）培养H08910PM细胞,以脂质体法转染细胞,设计空白对照组（Blank组）、阴性对照组（NC组,pGPU6-GFP-NC质粒转染细胞）、转染试剂对照组（MOCK组）、sur-shRNA组（pGPU6-GFP-survivin-shRNA质粒转染细胞）;（3）实时荧光定量PCR检测survivin mgAN表达水平;（4）流式细胞术检测细胞凋亡和细胞周期情况;（5）Western blot检测survivin蛋白的表达量。结果:（1）重组质粒表达载体pGPU6-GFP-survivin-shRNA经酶切、测序鉴定,证实插入载体目的基因片段大小与插入方向正确;（2）通过脂质体介导转染人卵巢癌H08910PM细胞,在荧光显微镜下观察到绿色荧光。（3）实时荧光定量PCR结果证实干扰组的HO-8910PM细胞在48h和72h survivin mRNA表达量下降了38%和57%,与blank组相比有统计学意义（P＜0.05）,48h到72h干扰片段的沉默效应呈增强趋势;（4）流式细胞术检测,细胞的转染率为52.26%,在48h和72h sur-shRNA组细胞凋亡率（30.78±0.68和38.47±0.84）高于Blank组（2.15±0.78和2.74±0.55）（P＜0.05）;NC组（2.87±0.81和3.67±0.29）,MOCK组（3.38±0.56和3.88±0.25）和Blank组比无差异（P＜0.05）:（5）PI单染流式细胞仪检测细胞周期结果证实sur-shRNA组细胞周期发生改变,转染后G₀/G₁期细胞数显著增加,与Blank组相比出现G₀/G₁期阻滞现象,G₀/G₁期升高而G₂/M期下降（P＜0.05）。（6）Western blot结果证实了转染pGPU6-GFP-survivin-shRNA的H08910PM细胞SUrvivin蛋白的量明显下调。结论:（1）针对survivin基因的pGPU6-GFP-survivin-shRNA表达载体构建成功。（2）针对survivin基因的shRNA可明显降低卵巢癌H08910PM细胞survivin基因mRNA及蛋白的表达。（3）针对survivin基因的shRNA可促进H08910PM细胞的凋亡,将细胞阻滞于G₀/G₁期。本实验构建的重组质粒能有效沉默survivn基因的表达,并能促进H08910PM细胞的凋亡和抑制细胞增殖,为卵巢癌基因治疗奠定了一定的基础。更多还原

【Abstract】 Background and objective:In recent years,people research on cancer gradually extended to the field of molecular,with a view to cure through gene therapy to achieve the purpose of tumor.Disordered regulation of apoptosis are the tumor occurrence and development of an important aspect.Inhibitor of apoptosis protein family,which is a research hotspot.Survivin gene is a new member of the inhibitor of apoptosis protein family,which can inhibit apoptosis and promote cell proliferation activity,directly or indirectly through inhibition caspase3 and caspase7 activity to block apoptosis of a variety of ways induced.RNA interference is gene silencing technology which used in the reverse genetics studies,can be effective in blocking specific gene expression.At present,the siRNA silencing survivin gene on ovarian cancer cell apoptosis HO8910PM study also has been reported rarely.The experiment intended to study the survivin-siRNA silencing effect on human ovarian cancer HO8910PM cells,and lay the foundation for the ovarian carcer gene treatment.Methods:The study used the human ovarian cancer cell HO8910PM;（1） A eukaryotic expression vector targeting on survivin siRNA was designed and constructed;（2） Cultured HO8910PM cells,then transfected into cells by Lipofectamine TM²⁰⁰⁰,and designed blank control group（black）,negative control group（NC,pGPU6-GFP- NC transfected into cells）,Lipofectamine TM²⁰⁰⁰ transfection group（mock）,interference group（pGPU6-GFP-survivin-shRNA transfected into cells）;（3） Expression level of survivin mRNA was assayed by real-time fluorescence quantitative PCR technology;（4）The apoptosis and cycle of cells were assessed by flow cytometry;（5） Expression level of survivin protein was assayed by Western bloting.Results:（1） After the enzyme cutting and sequence identification,it was verified that the size and direction of objective gene segment inserting vector.（2） Liposome-mediated transfection of human ovarian cancer HO8910PM cell,green fluorescence was observed under in fluorescence microscopy.（3） The result of real time fluorescence quantitative RT-PCR indicated that expression of survivin mRNA of HO-8910PM cells transfects with the pGPU6-GFP-survivin-shRNA after 48 hours and 72 hours dropped by 38%and 57%,compared with the blank control had difference（P＜0.05）,and the effect of silencing gradually enhances at 48 hours to 72 hours;（4）The FCM results showed that,the transfection rate was56.26%,at 48 hours to 72 hours the apoptotic rate in sur-shRNA group（30.78±0.68 and 38.47±0.84） higher than those in blank control group（2.15±0.78 and 2.74±0.55）（P＜0.05）,NC group（2.87±0.81 and 3.67±0.29 ） and mock group（3.38±0.56 and 3.88±0.25） compara with blank control group had no difference（P＜0.05）;（5） The FCM of PI simple staining indicated that sur-shRNA group could cause the number of cells to raise at the time of G₀/G₁ and decrease at the time of G₂/M（P＜0.05）;（6） Western bloting results showed that expression of survivin proteinum of HO-8910PM cells transfects with the PGPU6/GFP/Neo/survivin-siRNA decreased obviously.Conclusion:（1） Construct the shRNA expression vector of pGPU6-GFP-survivin -shRNA successfully;（2） Survivin shRNA targeting to survivin can effectively inhibit the expression of survivin mRNA and protein.（3） Survivin shRNA targeting to survivin can promote apoptosis of HO8910PM cells and enable the cell cycle of HO8910PM cells to appear G₀/G₁ phase arrest.The recombinant plasmid of this experiment can make survivn gene expression silence effectively,and promote the apoptosis of HO8910PM cells,inhibit cell proliferation.So it can selltle foundation for the ovarian carcer gene treatment.更多还原