短发夹状双链RNA抑制丙型肝炎病毒IRES介导的基因表达

目的研究载体表达的短发夹状双链RNA（shRNA）对丙型肝炎病毒（HCV）IRES介导的基因表达的特异性抑制作用。方法构建HCV IRES调控的绿色荧光蛋白表达载体（pIRES—GFP）和虫荧光索酶表达载体（p5′ UTR—Luc），以及针对HCV IRES的shRNA表达载体（pshRNA-HCV）。共转染HepG2细胞，于转染后24、48、72h观察绿色荧光的强弱，用Western blot检测绿色荧光蛋白的表达，半定量逆转录聚合酶链反应法检测GFP的mRNA水平。双荧光索酶系统检测虫荧光索酶活性。结果pshRNA-HCV作用组绿色荧光强度明显弱于未干扰组，GFP蛋白表达量及虫荧光素酶活性降低60％～70％，半定量逆转录聚合酶链反应显示pshRNA-HCV导致了GFP基因mRNA水平的降低。结论针对HCV IRES的shRNA能够显著和特异地抑制该区域调控的蛋白表达水平及mRNA水平，该研究结果为利用RNA干扰技术治疗HCV感染进行了初步探索。     

shRNA介导mcl-1基因沉默对人肝癌细胞HepG2凋亡的影响

目的观察shRNA介导mcl-1基因沉默对肝癌细胞HepG2的凋亡作用。方法构建靶向mcl-1基因的shRNA表达载体，经脂质体介导将其转入HepG2细胞内，48h后利用RT—PCR和Westernblot检测mcl-1mRNA和蛋白水平，Hoechst染色进行凋亡形态学观察，流式细胞仪检测细胞凋亡率。结果所构建的shRNA表达载体能明显降低HepG2细胞内mcl-1mRNA和蛋白水平。转染shRNA表达载体的细胞经Hoechst染色出现明显凋亡核形态学表现。经流式检测，shRNA组的凋亡率明显高于阴性对照组（7．74％±0．97％vs2．81％±0．46％，P=0．001）。结论shRNA介导mcl-1基因沉默可导致HepG2细胞凋亡，靶向mcl-1基因的RNA干扰可能是肝癌治疗的有效途径之一。     

RNA干扰bcl-2基因的筛选及对肝星状细胞生物活性的影响

目的研究小干扰RNA（shRNA）重组载体介导抑制大鼠肝星状细胞（hepaticstellatecell，HSC）bcl-2基因的表达，初步观察其对HSC生物活性的影响。方法设计有小发夹结构的3条DNA序列构建重组质粒载体pGPU6-GFP，脂质体转染HSC—T6细胞株以荧光定量PCR和Westernblot筛选鉴定，通过CCK-8法及AnnexinV／PI双标记流式细胞术检测、观察其对HSC生长的影响。结果pGPU6-GFP—shRNA1、shRNA2均能抑制bcl-2mRNA和蛋白表达（P〈0．05），pGPU6-GFP—shRNAl转染HSC．T6株72h后对bcl-2基因抑制达80％，且HSC—T6体外生长明碌受到抑制，早期凋亡率为33．34％～44．12％。结论bel-2小发夹RNA重组载体shRNA1能最有效抑制HSC—T6中bcl-2的表达与细胞生长，促进凋亡，为下一步探索肝纤维化基因治疗提供实验依据。     

Survivin特异性shRNA表达载体的构建及评价

目的 构建针对人结肠癌细胞系Lovo的高效率沉默Survivin的shRNA表达载体.方法 合成特异性干扰Survivin的小发卡RNA(shRNA)片段,并与pGenesil2 载体连接,构建Survivin干扰载体(pGenesil2-Survivin shRNA).利用脂质体将其转染Lovo细胞,分为正常细胞对照组和3个shRNA干扰组(pGenesil2-Survivin1、2及3),应用荧光显微镜对转染效果进行评价,应用Western印迹和免疫细胞化学法分析转染后Lovo中Survivin的蛋白表达水平.结果 插入片段测序结果与合成shRNA结果一致;转染效率可达70%左右,转染pGenesil2-Survivin shRNA后,3个干扰组的Lovo细胞中Survivin蛋白表达水平均较正常对照组显著降低(P＜0.01).结论 成功构建了能高效沉默Survivin的RNAi表达载体;且pGenesil2-Survivin高效抑制结肠癌Lovo细胞中Survivin基因表达.     

应用干扰RNA体外抑制严重急性呼吸综合征冠状病毒N基因的表达

目的构建绿色荧光蛋白（GFP）与严重急性呼吸综合征（SARS）冠状病毒N蛋白的融合表达载体pEGFP-C1-N以及针对SARS冠状病毒N基因的小发卡型shRNA表达载体pshRNAN，观察shRNA对SARS冠状病毒N基因复制和表达的影响。方法将构建成功的pEGFP-C1-N与psh RNA-N共转染293细胞，于转染后24、48、72h观察GFP表达的强弱，采用Western Blot分别检测GFP与N蛋白的表达，逆转录PCR检测N基因的mRNA。结果pshRNA—N作用组绿色荧光强度明显弱于未干扰组，Western Blot和逆转录PCR结果也证实pshRNA—N对N基因和N蛋白的表达有明显抑制作用，而无关序列的shRNA无此作用。结论针对SARS冠状病毒N基因的shRNA具有显著和特异的抑制N蛋白表达的作用。     

慢病毒介导的shRNA干扰人HepG2.2.15细胞Akt2基因表达的研究

目的针对Akt2基因构建shRNA慢病毒干扰载体并评价慢病毒介导的RNA干扰在人HepG2.2.15中的基因沉默效应。方法设计Akt2的RNAi寡聚核苷酸序列,利用慢病毒载体构建Akt2的shRNA载体,转染入大肠埃希菌并观察重组表达状况,利用293T细胞包装得到重组腺病毒,以绿色荧光蛋白(GFP)作为标记,逐孔稀释法确定转染效率及滴度,以实时荧光定量法比较各组对Akt2 mRNA的干扰效果。结果筛选了所构建的3个Akt2靶向序列,包装shRNA慢病毒后转染HepG2.2.15细胞,慢病毒转染后的沉默效率可达85%,比较得出沉默效率最高的靶序列和工作条件。结论本研究成功构建并筛选了针对Akt2的shRNA慢病毒载体,有效抑制HepG2.2.15细胞中Akt2 mRNA的表达。     

慢病毒载体介导绿色荧光蛋白基因转染血管内皮祖细胞的实验研究

目的探讨HIV-1来源的慢病毒载体介导绿色荧光蛋白（GFP）基因转染血管内皮祖细胞（EPCs）的可行性和方法。方法用梯度密度离心法分离人脐带血内皮祖细胞,在EGM-2培养基中培养。用细胞免疫荧光染色和流式细胞仪检测其表达情况。以HIV-1来源的慢病毒为载体、以GFP基因为目的基因转染EPCs,MTT法检测不同病毒滴度（MOI）时细胞增殖情况并观察转染率。结果单个核细胞经EGM-2培养基培养1周后即分化成EPCs。GFP转染后48 h细胞即发出绿色荧光。MOI 1∶10转染组细胞转染率低于MOI 1∶50组（P〈0.05）。MOI 1∶50转染后的细胞与未转染GFP组比较,生长曲线无明显差异（P〉0.05）。MOI 1∶100组转染后细胞的增殖处于停滞状态。结论采用HIV-1来源的慢病毒载体介导GFP基因转染标记EPCs是可行的。以MOI 1∶50进行转染对细胞生长影响小,转染效率高。     

YAP干扰质粒的构建及转染肺腺癌细胞A549的筛选鉴定

目的构建YAP基因小发卡RNA(shRNA),并转染人肺癌A549细胞株,建立稳定低表达YAP基因的肺癌细胞系。方法根据信息检索,分别构建4个shRNA(shRNA1,shRNA2,shRNA3,shRNA4)质粒表达载体和一个阴性对照表达载体(NCRNA),脂质体法转染人肺癌A549细胞株,荧光显微镜下观察绿色荧光蛋白的表达,有限稀释法建立稳定低表达YAP基因的A549细胞系,RT-PCR和Western印迹检测YAP在该细胞中的表达。结果转染的细胞可见绿色荧光蛋白表达,RT-PCR和Western印迹检测到目的基因YAP在转染细胞中为低表达。RT-PCR检测YAP mRNA示shRNA1,shRNA2,shRNA3,shRNA4,NCRNA组和空白对照组的2-ΔΔct值分别为2.775,0.498,0.432,2.834,3.289,3.320。Western印迹检测示shRNA1组,shRNA2组,shRNA3组,shRNA4,NCRNA组,空白对照组的蛋白相对灰度值分别为0.690,0.411,0.354,0.688,0.699,0.712。结论成功构建了靶向YAP基因的shRNA,筛选出了干扰作用最为明显的shRNA,并建立了稳定低表达YAP基因的A549细胞系。     

腺病毒介导的shRNA下调SHP2表达对人肝星状细胞LX-2凋亡的影响

目的探讨腺病毒介导的短发夹RNA (shRNA)下调含SH2结构域的蛋白酪氨酸磷酸酶2(SHP2)对体外培养的人肝星状细胞LX-2凋亡的影响。方法将携带靶向SHP2的shRNA并表达绿色荧光蛋白(GFP)的重组腺病毒Ad-shRNA/SHP2及仅表达GFP的对照空病毒Ad-GFP分别转染体外培养的LX-2细胞;实时荧光定量PCR检测LX-2细胞的SHP2 mRNA表达;用Western blot检测LX-2细胞的SHP2、Bax及Bcl-2蛋白表达;TUNEL及膜联蛋白-V/碘化丙啶双标记流式细胞术检测LX-2细胞凋亡。实验分组:(1)对照组:以DMEM代替腺病毒转染LX-2细胞;(2)Ad-GFP组:转染空病毒Ad-GFP;(3)Ad-shRNA/SHP2组:转染重组腺病毒Ad-shRNA/SHP2。多组间均数比较用单因素方差分析, 组间比较采用LSD检验。结果靶向SHP2的shRNA显著下调LX-2细胞的SHP2蛋白及mRNA表达(P < 0.05);TUNEL及膜联蛋白-V/碘化丙啶双标记流式细胞术检测结果显示, 与对照组LX-2细胞凋亡率(3.077%±0.731%, 9...     

携带Pyk2基因的shRNA质粒构建及其在Lovo结肠癌细胞系中的表达

目的:构建重组质粒PGCsi-Pyk2 shRNA,并检测所引起的Pyk2 mRNA和蛋白在Lovo结肠癌细胞系中的表达.方法:设计合成3对pkv2基因shRNA序列,形成双链后将其依次连入带有U6启动子并含有潮霉素B的pGcsi空载体,构建成能产生Pyk2短发卡RNA的质粒:采用双酶切和测序分析鉴定插入基因的序列:脂质体介导重组质粒pGCsi-Pyk2 shRNA稳定转染Lovo细胞系并通过潮霉素B筛选,获得比较单一的转染细胞;分别采用RT-PCR、Western blot等检测转染前后Pyk2的水平表达.结果:酶切鉴定和测序分析表明重组表达质pGCsi-Pyk2 shRNA构建无误:绿色荧光照相及PCR表明质粒转染成功:RT-PCR和Western blot均表明,与转染空质粒PGCsi组和转染仅含免疫荧光基因组细胞相比,转染重组表达质pGCsi-Pyk2 shRNA细胞t'Pyk2mRNA及蛋白表达水平均明显降低.结论:成功构建重组质pGCsi-Pyk2 shRNA.并证明他能降4Pyk2在Lovo胞株系中的表达,为进一步研究Pyk2何调控Hic-5/ARA55,Paxillion等下游靶基因的表达和参与结肠癌表观遗传学发生机制奠定了基础.     

琼枝麒麟菜多糖对白血病细胞株HL-60凋亡的影响

目的观察琼枝麒麟菜多糖(EGP)对白血病细胞株HL-60凋亡的影响。方法分别采用100、200、400 mg/L的EGP处理HL-60细胞。采用荧光显微镜观察HL-60细胞形态、MTT法测定细胞株的增殖抑制情况,流式细胞仪测定细胞凋亡率,逆转录—聚合酶链反应(RT-PCR)检测凋亡相关基因Bcl-2、Fas表达情况。结果200、400 mg/kg的EGP作用后HL-60细胞出现典型的凋亡形态学变化,HL-60细胞抑制率增加;凋亡抑制基因Bcl-2表达下调,促凋亡基因Fas表达上调。结论琼枝麒麟菜多糖能诱导HL-60细胞发生典型凋亡。     

shRNA干扰Rab9 GTPase表达对麻疹病毒野生株体外增殖的抑制作用

目的构建Rab9 GTPase短发夹RNA（shRNA）表达载体,观察其对Rab9 GTPase基因表达和麻疹病毒野生株体外增殖的抑制作用。方法参照GenBank中Rab9 GTPase基因序列设计合成2对Rab9 GTPase基因特异性shRNA,定向克隆人表达载体,构建重组表达载体,酶切鉴定和序列分析证实后脂质体法转染U937细胞,然后感染麻疹病毒野生株,逆转录聚合酶链反应（RT-PCR）和免疫印迹技术（Western blot）检测转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平;标准蚀斑试验测定病毒滴度;流式细胞仪检测细胞凋亡率的变化;RT-PCR检测转染细胞内双链RNA依赖蛋白激酶（PKR）和2′-5′寡腺甙酸合成酶（OAS-1）的mRNA水平。结果酶切和序列分析证实,成功构建了靶向Rab9 GTPase基因的shRNA表达载体。2个shRNAs均可特异性抑制U937细胞内Rab9 GTPase mRNA和蛋白质的表达,最高抑制率分别为（90.5±0.2）％和（92.1±0.3）％;蚀斑试验结果表明,shRNAs可以有效抑制麻疹病毒野生株体外增殖,其抑制率可达到90％以上;流式细胞仪检测转染后细胞的凋亡率无明显变化;RT-PCR检测PKR和OAs-1的mRNA水平转染前后无明显变化。结论成功构建Rab9 GTPase特异性shRNA表达载体。shRNAs通过特异性抑制Rab9 GTPase基因表达抑制麻疹病毒野生株体外增殖。     

ShRNA-mediated gene silencing of β-catenin inhibits growt of human colon cancer cells

AIM: To observe the gene silencing mediated by the specific shRNA targeted against β-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205. METHODS: Two shRNA plasmid vectors against β-catenin were constructed and transfected into Colo205 cells with LipofectamineTM2000. The down-regulations of β-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two β-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry. RESULTS: These two shRNA vectors targeted against β-catenin efficiently suppressed the expression of β-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level. The shRNA-mediated gene silencing of β-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing. CONCLUSION: These specific shRNAs targeted against β-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against β-catenin is of potential value in gene therapy of colon cancer.     

Effect of vector-expressed shRNAs on hTERT expression

AIM: To study the effect of short hairpin RNAs (shRNAs) expressed from DNA vector on hTERT expression. METHODS: Oligonucleotides coding for four shRNAs against hTERT were cloned into a mammalian shRNA expression vector pUC18U6 to form pUC18U6ht1-4, which were then introduced into HepG2 cells by using liposome-mediated transfection. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir, which expressed shRNA against green fluorescent protein (GFP), were used as controls. hTERT mRNA in the transfected cells were quantified by using real-time fluorescent RT-PCR. RESULTS: Among the four shRNAs against hTERT, two decreased the hTERT mRNA level. Compared with the controls, pUC18U6ht which expressed the two shRNAs reduced hTERT mRNA by 39% and 49% (P<0.05). CONCLUSION: hTERT expression is inhibited by the shRNAs expressed from the DNA vector.     

沉默SIRT1基因表达对胰腺癌细胞PANC1增殖和凋亡的影响

目的 应用RNA干扰技术沉默胰腺癌PANC1细胞的SIRT1基因表达,观察其对细胞增殖和凋亡的影响.方法 构建靶向SIRT1基因表达的短发夹RNA(shRNA)真核表达质粒pGC-shRNA,转染胰腺癌细胞PANC1.设对照shRNA(shRNA-C)转染组和未转染对照组.实时定量PCR和免疫细胞化学法检测转染后细胞SIRT1 mRNA及蛋白的表达;MTT法检测细胞的增殖率;ELASA法检测细胞caspase-3和caspase-9活性;Western bloting检测细胞Bax、Bcl-2蛋白表达.结果 与未转染组相比,shRNA组转染后48 h,PANC1细胞SIRT1 mRNA及蛋白表达的抑制率分别为(76.2%±10.4)%和(80.1±11.6)%;细胞增殖抑制率为(45.1±6.5)%;caspase-3和caspase-9酶活性显著增高;Bax蛋白表达上调,Bcl-2蛋白表达下调.结论 应用RNA干扰技术能有效沉默PANC1细胞SIRT1基因的表达,其机制可能与caspasa酶活性升高及Bax表达上调、Bcl-2表达下调有关.     

肺耐药蛋白在急性白血病中的表达及意义

目的 探讨急性白血病（AL）患者骨髓单个核细胞肺耐药蛋白（LRP）的表达及意义。方法 采用LRP单克隆抗体、流式细胞技术分别测定15例单纯缺铁性贫血患者（对照组）和65例AL患者（AL组）LRP的表达率。结果 LRP的表达率在初治和复发／难治急性淋巴细胞白血病（ALL）患者分别高于初治和复发／难治急性髓细胞白血病（AML）患者（P均〈0．05）;AML中M5亚型表达率高于M3亚型（P〈0．05）。初治及复发／难治LRP（＋）者缓解率明显低于LRP（-）者（P〈0．05）。结论 LRP的表达与AL患者临床预后密切相关,化疗前检测LRP表达率有助于个体化治疗和预测疗效。     

ROS在齐墩果酸诱导HL-60细胞凋亡中的作用

目的 观察ROS在齐墩果酸(OA)诱导HL-60细胞凋亡中的作用.方法 分瓶培养细胞,实验设空白对照组和12、24、48 h的OA用药组.收集细胞,提取细胞总蛋白,制备去线粒体细胞浆蛋白,Western印迹法检测caspase-9表达.流式细胞术检测ROS水平.结果 OA处理HL-60细胞后,ROS含量增加,并引起caspase-9的激活.结论 OA使ROS量增加,通过线粒体凋亡信号通路诱导HL-60细胞凋亡.     

Regulation of 17-AAG-induced apoptosis: role of Bcl-2, Bcl-XL,and Bax downstream of 17-AAG-mediated down-regulation of Akt,Raf-1, and Src kinases

17-allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone function of heat shock protein-90 (Hsp-90) and promotes the proteasomal degradation of its misfolded client proteins. Here, we demonstrate that treatment of the human acute myeloid leukemia HL-60 cells with 17-AAG attenuates the intracellular levels of a number of Hsp-90 client proteins, including Akt, c-Raf-1, and c-Src. Also, 17-AAG induced the mitochondrial release and cytosolic accumulation of cytochrome c (cyt c) and second mitochondria-derived activator of caspases (Smac)/DIABLO, resulting in the activation of caspase-9 and caspase-3 and apoptosis. Treatment with 17-AAG triggered the B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax) conformational change associated with apoptosis, while Bax-deficient cells were resistant to 17-AAG-induced apoptosis. In addition, in HL-60/Bcl-2 and HL-60/Bcl-xL cells, which ectopically express Bcl-2 and Bcl-xL respectively, 17-AAG-induced Bax conformational change, cytosolic accumulation of cyt c and Smac/DIABLO, and apoptosis were markedly inhibited. Although the rate of 17-AAG-mediated decline in Akt, c-Raf-1, and c-Src levels was blunted, the total decline was not compromised in HL-60/Bcl-2 and HL-60/Bcl-xL cells. Cotreatment with HA14-1, a nonpeptidic ligand that can bind and inhibit the antiapoptotic activity of Bcl-2, significantly overcame the resistance to 17-AAG-induced apoptosis in HL-60/Bcl-2 cells. Together, these findings indicate that although 17-AAG treatment causes the levels of a number of survival-signaling protein kinases to decline, the downstream engagement of the mitochondrial pathway of apoptosis is regulated by the activity of the Bcl-2 family of proteins. Also, neutralizing the antiapoptotic effect of Bcl-2 would further enhance the antileukemia activity of 17-AAG.     

大蒜素对卵巢癌耐顺铂细胞株SKOV-3/DDP增殖的影响及机制

目的探讨大蒜素对卵巢癌耐顺铂细胞株SKOV-3/DDP凋亡的影响及机制。方法将SKOV-3/DDP细胞随机分为四组,大蒜素组加入40μg/mL的大蒜素,顺铂组加入40μg/mL的顺铂,联合组加入大蒜素和顺铂各40μg/mL,对照组不干预。各组分别培养24、48 h。采用MTT法检测各组细胞增殖抑制率;RT-PCR法测定Bax、Bcl-2mRNA表达;Western blot法测定Bax、Bcl-2蛋白表达。结果顺铂组、大蒜素组、联合组各时点(24 h、48 h)增殖抑制率明显高于对照组,联合组明显高于大蒜素组、顺铂组,P均<0.05。顺铂组、大蒜素组、联合组各时点Bax mR-NA和蛋白水平明显高于对照组,大蒜素组、联合组Bcl-2 mRNA和蛋白水平明显低于对照组,P均<0.05;顺铂组48 h Bcl-2蛋白明显高于对照组,P<0.05;联合组各时点Bax mRNA和蛋白水平明显高于、Bcl-2 mRNA和蛋白水平明显低于对照组,P均<0.05。结论大蒜素诱导SKOV-3/DDP凋亡作用优于顺铂,两者联合具有协同作用,其机制可能为上调Bax表达和下调Bcl-2表达有关。     

Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi

Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.