RNA干扰GPC3基因表达影响肝癌细胞Huh-7凋亡的机制研究

目的观察RNA干扰GPC3基因表达对人肝癌细胞Huh-7凋亡的影响,并探讨其初步机制。方法设计并合成靶向GPC3的特异性siRNA片段,通过脂质体转染法瞬时转染肝癌细胞Huh-7,48h后验证siRNA的干扰效率;Annexin V/PI染色法观察GPC3基因沉默对细胞凋亡的影响;通过Western blot和定量PCR来检测caspase3的蛋白水平和核酸水平表达。结果设计合成的GPC3siRNA转染后,能够有效抑制Huh-7细胞中GPC3的表达;流式细胞仪检测结果显示,GPC3干扰组细胞在转染后48、72h的凋亡率显著高于空白对照组（P〈0.01）,96h后两组凋亡率差异无统计学意义（P〉0.05）。GPC3干扰组的caspase3在转染后48、72h的表达高于未转染组,96h后两组间caspase3水平差异无统计学意义（P〉0.05）。结论 siRNA静默GPC3基因能促进肝癌细胞凋亡,其机理可能与上调caspase3有关。进而推测,GPC3基因可能成为肝癌靶向治疗的一个新靶点。     

慢病毒介导的GATA6基因沉默对肝癌Huh-7细胞凋亡的影响

目的:研究GATA6基因沉默对肝癌Huh-7细胞凋亡的影响,同时探讨可能的作用机制。方法:构建靶向GATA6基因的慢病毒干扰载体,用流式细胞术确定转染效率,用RT-PCR和Western blotting法检测其对人类肝细胞癌细胞株Huh-7的干扰效果,流式细胞术检测细胞凋亡比例,Western blotting法检测各转染组Huh-7细胞NF-κB及Bcl-2蛋白表达。结果:靶向GATA6基因的慢病毒干扰载体感染肝癌Huh-7细胞后,转染效率为57.4%。GATA6在mRNA和蛋白水平的表达明显下降。GATA6沉默的肝癌Huh-7细胞凋亡比例增加(P0.05),NF-κB和Bcl-2蛋白水平表达降低。结论:利用慢病毒载体干扰技术沉默GATA6基因的表达可以明显促进Huh-7细胞凋亡,其机制可能通过NF-κB信号通路调节凋亡相关蛋白的表达,进而影响细胞的凋亡。靶向GATA6的RNA干扰技术在肝癌的基因治疗中具有一定的研究价值。     

siRNA抑制胃癌细胞BGC-823及裸鼠皮下移植瘤中生存素的表达

目的： 探讨RNA干扰用于胃癌治疗的可行性与特异性。方法: 设计靶向生存素基因的小干扰RNA,并导入胃癌BGC-823细胞株和裸鼠皮下移植瘤,在体内、外诱导RNA干扰,采用MTT、流式细胞检测技术、RT-PCR法、Western bloting、免疫组织化学和TUNEL检测survivin基因表达、细胞周期和细胞凋亡。结果: 重组质粒pTZU6+1-siRNA- survivin导入BGC-823后,survivin mRNA和蛋白表达均明显下调;细胞生长被抑制,细胞周期中S期细胞减少, G1/G0期细胞增加,出现明显细胞凋亡。裸鼠皮下移植瘤体积明显缩小,Survivin表达均下调。体内、外对照组各指标均无明显变化。 结论: RNA干扰明显抑制靶基因survivin在胃癌细胞BGC-823及裸鼠皮下移植瘤中的表达及肿瘤细胞增殖,并诱导细胞凋亡,且特异性好,可能成为肿瘤治疗的新方法。     

应用siRNA抑制K562细胞中c-Myc基因的表达

目的利用小分子干扰RNA(siRNA)技术抑制白血病细胞系K562细胞中c鄄Myc基因的表达,为研究c鄄Myc基因在K562细胞中的作用提供一个新的方法。方法设计c鄄Myc基因特异性小分子干扰RNA,用体外转录方法合成c鄄Myc基因的小分子干扰RNA并转染K562细胞,培养48h后,收集细胞,应用实时荧光定量RT鄄PCR和westernblot方法检测转染细胞中c鄄Myc基因mRNA水平和蛋白表达量的变化,四甲基偶氮唑蓝(MTT)法检测细胞增殖活性。结果转染siRNA后,与对照组相比,实验组c鄄MycmRNA水平和蛋白表达量明显降低,抑制率分别为82.16%和74.0%,MTT法表明实验组的增殖速率明显低于对照组的增殖速率。结论在K562细胞中存在RNA干扰的机制,特异性siRNA能够有效的抑制c鄄Myc基因的表达,为研究c鄄Myc基因在肿瘤细胞中的调节途径提供了一个新的方法。     

Inhibition of hepatitis B virus replication by recombinant small interfering RNAs

RNA interference (RNAi) is a biological phenomenon in which introduction of a small, double-stranded interfering RNAs (siRNAs) into a cell causes a specific degradation of homologous single-stranded RNA. siRNA can be delivered into the cell by different approaches including synthetic RNA, in vitro transcribed RNA and RNA transcribed from polymerase III-based recombinant vectors. As hepatitis B (HB) represents a worldwide health problem, we attempted to develop a fast and easy approach to generation and screening of specific siRNA-targeted HB virus (HBV) genes. Using PCR amplification, specific siRNA expression cassettes (SECs) were developed and used to generate effective siRNAs against HB virus (HBV) replication and gene expression in mammalian cells. After screening, we identified two SECs that expressed siRNAs which efficiently decreased the level of HBV pre-c/c gene expression in transfected Bel-7402 cells by 81.9% and 87.3%, respectively. In addition, the level of HBV DNA was decreased by 83.5% and 85.2% in HepG2 2.2.15 cells, respectively. This study provides (i) a new effective application of RNA interference to study viral gene function and viral replication and (ii) a new tool for the prevention and treatment of human HBV infection.     

Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells

RNA interference (RNAi) using short inhibitory RNAs (siRNAs) has been widely explored for the suppression of cellular mRNA levels to investigate the function of specific genes, including gene function in differentiation and development. The establishment of human embryonic stem cell (hESC) models for differentiation of selected lineages is an area of intense interest and activity. On the basis of our previous work with stable overexpression of enhanced green fluorescent protein (EGFP) in hESC, we used plasmid vector-based siRNA expression to silence EGFP expression in stably-transfected hESC. After hygromycin selection, we derived several cell lines in which EGFP expression was significantly reduced. At the genomic DNA level, there was no difference between the two cell lines and the parental H1EGFP cell line when analyzed with quantitative PCR; however, there were significant differences among the three cell lines at the RNA and protein levels as analyzed with real-time RT-PCR and Western blotting. From these data, we conclude that the decrease in EGFP expression was caused by RNAi, not by genomic DNA loss. Down-regulation of EGFP expression was sustained through multiple passages of both siEGFP cell lines. This simple silencing system will allow novel investigations of target gene function in hESC self-renewal or differentiation, as well as differentiated function in other cell types.     

siRNA对卵巢癌细胞周期及EGFR表达的影响

目的体外构建编码人类表皮生长因子受体（EGFR）的小干扰RNA（siRNA）的质粒表达载体,观察其对卵巢癌Skov-3细胞EGFR的特异性抑制作用及对细胞凋亡和周期的影响。方法构建pSilencer-EGFR siRNA真核基因表达载体,采用脂质体介导的方法将其转染到卵巢癌细胞株Skov-3,实验分为以下3组：空白对照组（未经转染的人卵巢癌Skov一3细胞）、非特异性转染组（转染非特异性质粒载体）及特异性转染组。用RT-PCR的方法检测mRNA水平的变化,用免疫荧光法检测EGFR蛋白水平的变化,流式细胞仪检测其对卵巢癌细胞周期及凋亡的影响。结果成功构建pSilencer—EGFR siRNA真核基因表达载体。psiRNA—EGFR质粒明显下调Skov-3细胞中EGFR的表达,阻断细胞周期在G1期,促进细胞凋亡。结论重组质粒psiRNA—EGFR能有效地抑制Skov-3细胞内EGFR的表达、抑制细胞增殖,其抑制机制可能与引起细胞周期再分布、降低S期细胞比例和促进细胞凋亡有关。     

质粒介导的RNAi抑制端粒酶活性

为了探讨靶向人端粒酶反转录酶（hTERT）基因的小干扰RNA（siRNA）表达载体是否具有抑制HeLa细胞端粒酶活性的能力，人工合成2条64个核苷酸（nt）的片段，其中19nt与hTERT基因1789-1807位碱基同源，将其退火、连接到质粒psUPER中，构建psUP—hTE。在psUP—hTE基础上，把该质粒的启动子和64nt插入片段酶切、克隆到质粒pEGFP—C1中生成pEGFP—hTE。从而获得新霉素抗性筛选质粒。将pEGFP—hTE用脂质体转染HeLa细胞，G418筛选后获得抗性克隆并将其收获、传代，用不同方法检测hTERT的mRNA和蛋白表达水平、HeLa细胞的端粒酶活性以及细胞的增殖能力。结果显示，pEGFP—hTE转染的HeLa细胞与对照组比较，hTERT的mRNA水平下降及蛋白表达减少、细胞端粒酶活性降低38％，但细胞增殖能力没有明显改变。以上结果表明，pEGFP—hTE能通过RNA干扰（RNAi）途径特异性抑制HeLa细胞hTERT基因的表达，从而有效抑制细胞端粒酶话性，这可能将为肿瘤生物治疗提供一条新的途径。     

RNA干扰抑制小鼠OX40基因表达的实验研究

目的研究靶向小鼠OX40基因的二聚体小干扰RNA(siRNA-OX40)对靶基因表达的沉默作用。方法建立并筛选稳定表达OX40基因的293T转染细胞株,用脂质体转染法将体外合成的siRNA-OX40转染上述细胞株,采用实时荧光定量PCR检测靶基因OX40 mRNA表达情况。结果用不同浓度的siRNA-OX40转染细胞48h,10nmol/L浓度对293T细胞靶基因表达的抑制作用最强(抑制率76.4%),1nmol/L浓度仍有一定的抑制作用(39.4%);25、10nmol/L siRNA转染293T细胞后,均在24h内起效(24.9%,20.5%),抑制作用至少能维持72h(74.4%,38.8%)。结论siRNA-OX40对293T细胞外源性靶基因表达有较强的特异性沉默作用,siRNA在24h内起效,48～72h达高峰,抑制作用至少能维持72h。本研究结果为下一步在动物或临床进行siRNA干扰试验提供了实验依据。     

shRNA表达质粒抑制耐阿霉素的人乳腺癌细胞株(MCF-7/AdrR)绿色荧光蛋白表达的研究

目的：探讨靶向增强型绿色荧光蛋白（EGFP）小发夹结构RNA（Short hairpin RNA，shRNA）对耐阿霉素的人乳腺癌细胞（MCF-7／AdrR）中EGFP基因表达的抑制作用。方法：构建针对EGFP基因的shRNA表达载体，与pcDNA3．0 EGFP质粒共转染MCF-7／AdrR细胞，分别用激光共聚焦扫描显微镜（Laser confocal scanning microscope，LCSM）和流式细胞术检测干扰效果。结果：瞬时共转染pSilencer^TM3．1-H1 neo EGFP shBNA质粒和pcDNA3．0 EGFP质粒后，荧光显微镜观察结果显示MCF-7／AdrR细胞中发出荧光的细胞数量减少，荧光强度明显降低。流式细胞术检测阳性细胞百分率降低至35．6％，差异有显著性。结论：靶向EGFP的shRNA表达质粒能有效而特异抑制EGFP在MCF-7／AdrR中的表达，MCF-7／AdrR细胞中存在RNA干扰现象，为进一步利用RNA干扰技术逆转乳腺癌多药耐药性的研究奠定基础。     

VEGF基因特异性siRNA转染后对人乳腺癌细胞增殖和凋亡的影响

目的：探讨利用干扰RNA（RNAi）抑制血管内皮生长因子（VEGF）基因表达对乳腺癌细胞MCF-7增殖和凋亡的影响。方法：设计针对VEGF基因的小干扰RNA（siRNA）,合成DNA模板,体外转录siRNA。以脂质体转染法将双链siRNA导入MCF-7细胞后,用MTT比色法检测siRNA对MCF-7细胞增殖的影响。通过Hoechst33258染色观察MCF-7细胞的凋亡。用流式细胞术检测细胞周期的改变,RT-PCR检测VEGF mRNA表达的变化,免疫细胞化学法检测VEGF蛋白的表达。结果：所设计的两个靶位点siRNA,均能有效地抑制MCF-7细胞的增殖,诱导细胞凋亡,使细胞周期阻滞于G0/G1期,VEGFmRNA及其蛋白的表达明显减少;而作为阴性对照的错义序列组siRNASCR则没有上述效应。结论：体外转录合成的siRNA可抑制MCF-7细胞中VEGF基因的表达,抑制细胞增殖,促进细胞凋亡。     

慢病毒介导的RNA干扰沉默Slug基因对结肠癌HCT116细胞增殖和凋亡的影响

目的:探讨利用RNA干扰(RNA interference,RNAi)技术沉默Slug基因,观察对结肠癌HCT116细胞增殖和周期的影响。方法:构建Slug基因特异性siRNA慢病毒载体,感染结肠癌HCT116细胞,设立空白对照组、阴性对照组及SlugsiRNA三组,应用Real-time PCR和Western blot方法分别从基因和蛋白质水平检测各组干扰质粒对Slug基因的干扰效果,MTT法检测Slug基因在siRNA作用下的细胞增殖率,流式细胞仪检测细胞凋亡周期变化情况。结果:转染Slug siRNA后,结肠癌HCT116细胞中Slug基因mRNA和蛋白表达明显受到抑制(P<0.05);MTT检测,干扰组细胞增殖水平明显低于阴性对照组;流式细胞仪检测细胞G1期细胞百分比(52.3±0.6)高于阴性对照组(45.1±0.3,P<0.05)。结论:Slug siRNA能明显下调靶基因Slug的表达,在体外可抑制结肠癌HCT116细胞的生长并促进其凋亡。     

Poly(beta-amino ester) as a carrier for si/shRNA delivery in lung cancer cells

Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNA interference (RNAi) studies. In the present study, we evaluated biodegradable poly(beta-amino ester) (PAE) carrier composed of low molecular weight polyethylenimine and poly(ethylene glycol) for si/shRNA delivery in lung cancer cells. PAE carrier successfully delivered EGFP (enhanced green fluorescence protein) siRNA (siGFP) and silenced EGFP expression. The silencing achieved with PAE carrier was found to be nearly 1.5 times superior and safer than standard PEI25K. Also, our PAE carrier exhibited superior Akt1 shRNA delivery (shAkt) and thereby silenced oncoprotein Akt1 efficiently. PAE-shAkt mediated Akt1 knock-down hindered cancer cell growth in Akt1 specific manner. Superior shAkt delivery and low cytotoxicity of PAE carrier promoted Akt1 knock-down specific apoptosis, while low delivery efficiency and high cytotoxicity of PEI25K carrier mainly exhibited undesirable necrosis. Moreover, basic cancer properties like cell proliferation, malignancy and metastasis were reduced more efficiently using PAE-shAkt system. These findings demonstrated the potential of PAE as an alternative to PEI25K in si/shRNA-based RNAi studies.     

RNA干扰沉默IGF—IR基因治疗卵巢癌的实验研究

目的研究RNA干扰技术沉默IGF—IR在卵巢癌靶向治疗中的意义。方法以IGF—IR为目的基因，设计合成siRNA重组质粒；利用Real—timePCR和Westernblot方法，分别从mRNA水平及蛋白水平检测IGF—IR的表达；通过MTF实验检测细胞增殖情况。结果本实验成功构建了三个表达载体pSihIGF—IR-1、pSihIGF—IR-2和pSihIGF—IR-3，转染卵巢癌OVCAR3细胞，48h后，IGF—IR的mRNA及蛋白水平均明显降低，并且显著抑制了OVCAR3细胞的体外增殖。结论应用RNA干扰技术，能够高效、特异的沉默IGF—IR基因的表达，抑制肿瘤生长。针对卵巢癌IGF—IR的RNAi技术，可能为卵巢癌的临床治疗开拓-条新的途径。     

DC-SIGN荧光融合蛋白的构建、表达和生物学功能初探

目的 构建绿色荧光蛋白（EGFP）标记的DC-SIGN分子（DC-SIGN-EGFP融合蛋白）,并在哺乳动物细胞中获得功能性表达。方法 PCR方法获得DC-SIGN分子和EGFP分子的cDNA,分别克隆入真核表达载体pEGFP-N1和真核表达载体pCI,使EGFP荧光蛋白分别位于DC-SIGN蛋白的C末端和N末端。在转染COS-7细胞后,在激光共聚焦显微镜和流式细胞仪检测重组分子的表达及融合蛋白在细胞定位情况。激光共聚焦显微镜检测融合蛋白的功能情况。结果 获得了预期的重组表达质粒,经DNA测序证实开放阅读框正确,转染COS-7细胞后绿色荧光蛋白位于细胞膜,仅EGFP位于DC-SIGNN-末端的融合蛋白可被抗DC-SIGN抗体结合。EGFP位于DC-SIGN N-末端的融合蛋白可有效摄取DC-SIGN受体的高亲和力配体le（x）寡糖。结论 所构建的DC-SIGN-EGFP融合蛋白在细胞内表达后具有细胞表面受体分子的特征性分布,可被抗DC-SIGN抗体检测及摄取特异性配体,为进一步深入研究DC-SIGN分子功能提供了良好的模型。     

增强型绿色荧光蛋白基因转染对原代培养的人软骨细胞细胞周期的影响

目的:观察转染增强型绿色荧光蛋白 (EGFP) 基因后原代培养的人鼻中隔软骨细胞的细胞周期的变化,建立原代培养的人鼻中隔软骨细胞的示踪方法。方法:在大肠杆菌中扩增pEGFP-N1 质粒,通过Amaxa细胞核转染仪将pEGFP-N1质粒转入原代培养的人鼻中隔软骨细胞,应用激光共聚焦显微镜观察其转染过程及瞬时表达情况,流式细胞仪检测其转染效率和细胞周期的变化。结果:增强型绿色荧光蛋白基因在转染24 h后得到了明显表达,48 h后流式细胞仪检测其表达率为35.37％,细胞周期没有明显的改变,且未影响软骨细胞的贴壁过程。结论:经pEGFP-N1质粒转染的原代培养的人鼻中隔软骨细胞仍能在体外存活,对软骨细胞的生长没有明显的影响,pEGFP-N1是转染原代培养的人鼻中隔软骨细胞较为理想的瞬时表达载体,也是组织工程化软骨形成过程的良好示踪剂。     

Inhibition of NF-kappaB mediated inflammation by siRNA expressed by recombinant adeno-associated virus

NF-kappaB mediated inflammation is a key process to many diseases. RNA interference (RNAi) is the specific suppression of genes by short double-stranded RNA. It was the aim of the present study to modify NF-kappaBdependent inflammation by small interfering RNA (siRNA) expressed by recombinant adeno-associated virus (rAAV). To study the kinetics of rAAV mediated expression of siRNA, the expression of the luciferase gene was targeted and resulted in a significant decrease of luciferase activity as compared to a control vector in the human 293 cell line. The effect was dose dependent and was detectable 24 h after infection. rAAV coding for siRNA against the p65 subunit of NF-kappaBsignificantly reduced the p65 protein. In a cellular model of TNF-alpha induced inflammation, expression of siRNA against p65 significantly suppressed the secretion of IL-8 from BEAS-2B cells. In conclusion, rAAV vectors coding for siRNA are an useful tool for efficient gene silencing in mammalian cells and can be used to modify NF-kappaB mediated inflammation.     

Identification of active siRNAs against IGF-IR of porcine coronary smooth muscle cells in a heterologous cell line

Testing of short interfering RNAs (siRNAs) to knock-down gene expression is complicated in primary differentiated cells due to their limited availability and short in vitro life span. The objective of this study was to bypass these limitations by testing selected siRNAs in a heterologous cell line. A plasmid containing a fragment of porcine IGF-I receptor (pIGF-IR) gene cloned downstream of EGFP sequence was constructed and cotransfected with pIGF-IR/siRNAs in HEK 293T human cell line. The abatement of EGFP fluorescence, measured at mRNA and protein level, was compared with that induced by a EGFP/siRNA. Among the three pIGF-IR/siRNAs tested, one was active against the hybrid reporter gene as EGFP/siRNA, while the other two were inactive. When transfected in primary porcine coronary smooth muscle cells (PC-SMCs) and tested by real-time PCR, immunocytofluorimetry and cell motility assay, pIGF-IR/siRNAs confirmed the results obtained in HEK 293T cells, thus establishing that the search of active siRNAs addressed to a gene of a cell and/or a species of interest can be done in a heterologous cell line.