短发卡RNA

摘要： 目的 探讨短发卡RNA(shRNA)对细胞内H1N1及H7N9甲型流感病毒(IVA)的抑制作用.方法 合成12种针对H1N1亚型流感病毒保守区域的短发卡RNA(shRNA),观察了这些shRNA对不同亚型流感病毒在细胞中复制的影响.结果 转染了质粒pLKD-M-121、pLKD-M-935、pLKD-NP-391、pLKD-NP-1291、pLKD-PA-1365和pLKD-PA-1645的MDCK细胞的H1N1病毒载量显著低于H1N1阳性对照组和空质粒组(P＜0.05).转染了质粒pLKD-M-935、pLKD-NP-391、pLKD-PA-1365、pLKD-PA-1645、pLKD-M-121和pLKD-NP-1291的MDCK细胞的H7N9病毒载量显著低于对照组和空质粒组(P＜0.05).结论 针对H1N1亚型的流感病毒基质蛋白(M2),核壳体蛋白(NP)和聚合酶A(PA)片段的shRNAs可以抑制H1N1和H7N9亚型流感病毒的复制.

摘要： 目的探讨短发卡RNA(shRNA)对细胞及小鼠体内甲型流感病毒(IVA)抑制作用。方法合成12种针对IVA保守区域的shRNA,观察这些shRNA对IVA在细胞中复制的影响。将转染了shRNA的质粒以气溶胶形式雾化吸入小鼠,然后以流感病毒感染小鼠并检测小鼠肺组织流感病毒载量。结果转染了质粒PLKD-M-121、PLKD-M-935、PLKD-NP-391、PLKDNP-1291、PLKD-PA-1365、PLKD-PA-1645的犬肾细胞中IVA载量均显著低于对照组及空质粒组(P <0. 05)。染PLKD-NP-391小鼠肺部IAV载量明显低于对照组和空质粒组(P <0. 05)。结论针对流感病毒基质蛋白(M2)、核壳体蛋白(NP)和聚合酶A(PA)片段的shRNA可以抑制流感病毒的产生,以气溶胶形式吸入聚乙烯亚胺/PLKD-NP-391复合物可以抑制小鼠肺部流感病毒的产生。

摘要： 本研究旨在筛选抑制B组柯萨奇病毒(coxsackievirus B,CVB)3D聚合酶表达的短发卡RNA(short hairpin RNA,shRNA)并构建其慢病毒表达载体,以抑制CVB的3D聚合酶表达.设计并合成3对针对3D基因的RNA干扰序列及相应的对照组序列,通过定量反转录-聚合酶链反应(quantitative reverse transcription-polymerase chain reaction,RT-qPCR)和蛋白免疫印迹法筛选出干扰效率最高的一对,合成shRNA序列.退火后,应用基因重组技术构建pLVTHM-3DshRNA重组质粒,经酶切鉴定及DNA测序后,将重组正确的pLVTHM-3DshRNA与psPAX2、pMD2.G共转染293T细胞,包装慢病毒并鉴定其对CVB33D基因表达的干扰效果.测序结果显示,成功构建了pLVTHM-3DshRNA重组慢病毒载体,并收获了包装后的慢病毒,病毒滴度为5×107 TU.感染HeLa细胞和BALB/c小鼠后,3D聚合酶表达量下降.本研究成功构建了慢病毒Lenti-sh3D,其能有效下调CVB 3D聚合酶的表达,为进一步抗CVB致病机制的研究奠定了基础.

摘要： Objective To evaluate the influence on the invasion ability of T24 bladder cancer cell lines after silencing of NANOG gene.Methods Human urinary bladder cancer cells T24 were cultured and transfected with short hairpin RNA (shRNA) to silence NANOG gene.Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and enzyme linked immunosorbent assay (ELISA) were performed to detect the expression level of matrix metalloproteinase (MMP)-2,MMP-9 and inhibitor of matrix metalloproteinases-1 (TIMP-1).Wound-healing assay and invasion test were performed to determine the invasion capability of T24 cell lines.Results When NANOG gene was silenced,the expression of MMP-2 and MMP-9 was down-regulated (for MMP-2 by 63.21％,P ＜ 0.05;for MMP-9 by 73.55％,P ＜ 0.05).ELISA showed that the expression of MMP-9 in supernatant of T24 cells was down-regulated by 21.07％ (P ＜0.05).Scratch test and invasive test showed that the invasive ability of cells decreased after NANOG gene was silenced (all P ＜ 0.05).Conclusion NANOG may modulate the invasion of bladder cancer cells through regulating the expression of MMP-2 and MMP-9.%目的 检测T24膀胱癌细胞的NANOG基因被沉默后影响T24细胞的侵袭能力.方法 以人膀胱癌细胞株T24细胞为靶细胞,转染短发卡RNA(shRNA)沉默T24细胞的NANOG基因.通过实时定量反转录聚合酶链反应(RT-qPCR)技术和酶联免疫吸附试验(ELISA)检测基质金属蛋白酶(MMP)-2、MMP-9和基质金属蛋白酶抑制因子(TIMP)-1表达的变化,通过划痕实验和侵袭实验检测T24细胞侵袭力的变化.结果 当NANOG基因被沉默后,RT-qPCR检测T24细胞中MMP-2、MMP-9的mRNA表达下调(MMP-2下调63.21％,P＜ 0.05;MMP-9下调73.55％,P＜0.05),ELISA结果显示T24细胞上清中MMP-9的蛋白水平表达下调(21.07％,P＜O.05).划痕实验和侵袭实验表明在NANOG基因被沉默后,细胞的侵袭能力下降(均P＜0.05).结论 NANOG基因可能通过调节膀胱癌细胞MMP4及MMP-9的表达来介导膀胱癌细胞向周围组织的侵袭.

摘要： 目的 构建敲减小鼠海马神经元兴奋性氨基酸转运体3(excitatory amino acid transporter 3,EAAT3)重组腺相关病毒(recombinant adeno-associated virus,rAAV)载体,建立一种小鼠海马EAAT3敲减动物模型.方法 基于SLC 1A1 (solute carrier family 1 member 1)基因mRNA序列设计4条shRNA(short hairpin RNA),将其模板DNA与酶切后的AAV-GP-1(pAAV-U6-EGFP)载体质粒连接、转化并测序鉴定得到重组质粒,将重组质粒、pHelper包装质粒和pAAV-DJ辅助质粒共同转染至AAV-293细胞包装得到rAAV;用所得rAAV侵染HT-22细胞,72 h后RT-qPCR检测SLC1A1 mRNA表达水平;36只成年C57小鼠随机分为6组,每组6只,予海马注射rAAV-shRNA-SLC1A1-2-EGFP病毒液1μL/侧,分别于注射后即刻、24h、7d、14d、21d、28 d取双侧海马组织,Western blot检测EAAT3表达水平.结果 经测序重组质粒核苷酸序列正确,包装所得病毒浓缩滴度为1×1013 TU/ml;rAAV感染HT-22细胞72 h后SLC1A1表达量明显降低且显著低于阴性对照(P＜0.01,n=3);小鼠海马注射rAAV-shRNA-SLC 1A 1-2-EGFP 7 d后EAAT3相对表达量下降(P＜0.01),注射后21 d、28 d表达进一步降低(P＜0.01).结论 成功构建介导shRNA表达的重组腺相关病毒系统,建立了一种有效的小鼠海马EAAT3敲减动物模型.

摘要： 目的 构建人泛素连接酶Cbl-b基因短发卡RNA(shRNA)重组慢病毒载体,探讨其对人黑素瘤A375细胞生物学行为的影响.方法 设计并合成3条沉默Cbl-b基因的特异性shRNA及1条阴性对照shRNA,构建慢病毒载体.将A375细胞分成5组,即分别用3条特异性shRNA转染的CBLB-shRNA-1组、CBLB-shRNA-2组、CBLB-shRNA-3组,阴性对照组(阴性对照shRNA转染),空白对照组(转染空载体).应用实时荧光定量PCR和Western印迹检测转染后72 h各组A375细胞沉默效率;CCK8法检测转染后24、48、72及96 h细胞增殖能力;流式细胞仪检测转染后72 h细胞凋亡情况、细胞周期,Transwell法检测转染后72 h细胞侵袭能力.结果 成功构建3条Cbl-b shRNA慢病毒载体,Western印迹示CBLB-shRNA-3蛋白沉默效率最高.CCK8检测表明,CBLB-shRNA-3组A375细胞增殖能力在转染后72 h和96 h较阴性对照组、空白对照组明显降低(均P＜0.01).流式细胞仪检测示,CBLB-shRNA-3组凋亡率[(22.73±6.58)％]明显高于阴性对照组[(6.08±1.35)％]和空白对照组[(6.34±1.07)％,均P＜0.01].CBLB-shRNA-3组G1期细胞比例明显高于阴性对照组和空白对照组(P＜ 0.01),而S期细胞比例明显低于阴性对照组和空白对照组(P＜0.01).Transwell检测示,阴性对照组、空白对照组和CBLB-shRNA-3组转染后72 h时穿膜细胞数分别为76.60±1.82、73.20±3.83、19.60±1.14,差异有统计学意义(F=794.50,P＜0.01).结论 成功构建能高效沉默Cbl-b蛋白的CBLB-shRNA-3重组shRNA慢病毒载体,其可促进A375细胞凋亡,抑制A375细胞增殖、细胞周期进程和侵袭能力.%Objective To construct a recombinant lentiviral vector carrying the casitas B-lineage lymphoma (Cbl)-b short hairpin RNA (shRNA),and to evaluate its effect on the biological behavior of A375 melanoma cells in vitro.Methods Three specific shRNAs targeting Cbl-b gene and a negative control shRNA were designed and synthesized,and recombinant lentiviral vectors were constructed.A375 cells were divided into 5 groups to be transfected with 3 kinds of lentiviral vector expressing Cbl-b genespecific shRNAs (CBLB-shRNA-1 group,CBLB-shRNA-2 group and CBLB-shRNA-3 group),a lentiviral vector containing negative control shRNA (negative control group),and an empty vector (blank control group).Real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine the silencing efficiency at 72 hours after transfection.Cell counting kit-8 (CCK-8)assay was conducted to evaluate cellular proliferative activity at 24,48,72 and 96 hours after transfection,flow cytometry to detect cell apoptosis and cell cycle at 72 hours after transfection,and Transwell invasion assay to assess cellular invasive activity at 72 hours after transfection.Results Three recombinant lentiviral vectors containing Cbl-b shRNA were constructed successfully.As Western blot analysis revealed,the CBLB-shRNA-3 showed the highest silencing efficiency.CCK-8 assay indicated that the proliferative activity of A375 cells was significantly lower in the CBLB-shRNA-3 group than in the negative control group and blank control group at 72 and 96 hours after transfection(all P ＜ 0.01).Flow cytometry showed that the apoptosis rate of A375 cells was significantly higher in the CBLB-shRNA-3 group (22.73％ ± 6.58％) than in the negative control group (6.08％ ± 1.35％,P ＜ 0.01) and blank control group (6.34％ ± 1.07％,P ＜ 0.01).The CBLB-shRNA-3 group showed a significantly higher proportion of A375 cells at G1 phase,but a significantly lower proportion of A375 cells at S phase compared with the negative control group and blank control group(all P ＜ 0.01).Transwell assay showed that there were significant differences in the number of A375 cells crossing the artificial basement membrane (matrigel) at 72 hours after transfection among the negative control group,blank control group and CBLB-shRNA-3 group (76.60 ± 1.82,73.20 ± 3.83,19.60 ± 1.14,respectively;F =794.50,P ＜ 0.01).Conclusions A recombinant CBLB-shRNA-3-expressing lentiviral vector which can efficiently silence Cbl-b gene has been successfully constructed.It can inhibit the proliferation,cell cycle progression and invasive activity of A375 cells,but promote the apoptosis of A375 cells.

摘要： 目的 利用带有多个启动子的质粒pGenesil10-3p,构建针对HSV-2靶基因UL27和UL54的双短发卡RNA(Duo-shRNA)表达载体,探讨双shRNA表达载体对双靶基因的干扰作用以及对HSV-2病毒增殖的影响.方法 Duo-shRNA通过脂质体转染HEK293细胞后接种HSV-2,采用四唑盐(MTT)比色法检测细胞存活率,终点滴定法检测细胞上清液中病毒滴度,QPCR(Quantitative Real-time PCR,QPCR)检测对靶基因mRNA的抑制率,蛋白质印迹(Western blot,WB)检测靶蛋白的表达量.结果 结果表明Duo-shRNA对于细胞存活率、抑制HSV-2复制以及干扰靶基因表达方面都优于单-shRNA,可以达到多个单基因-shRNA联合作用的干扰效果.结论 利用双启动子的方法构建了针对HSV-2的UL27和UL54的shRNA质粒表达载体,更好的抑制HSV-2病毒靶基因mRNA表达水平以及病毒在HEK293细胞中增殖,实现了多个单基因的shRNA干扰效应的相互作用.

摘要： Objective To block the expression of Yes-associated protein 1 (YAP1) gene in human bladder cancer cell line T24 using RNA interference,and to observe the changes of cell proliferation,migration and invasion.Methods The YAP1 expression was detected in T24 cells.The recombinant plasmid targeting YAP1 gene with eukaryotic transcription plasmid pGreenPuro.YAPlshort hairpin RNA (shRNA) plasmids of 4 different sequences and YAP1 shRNA normal control (NC) were transfected into T24 cells respectively.After 48 h,the expression level of YAP1 was detected by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR),and the shRNA plasmid with the best silencing effect of YAP1 gene was screened for following research.The methyl thiazol tetrazolium (MTT) assay was used to detect the effect of shRNA plasmid on the proliferation of T24 cells,the effect of shRNA plasmid on cell migration ability was detected by scratch test,and the invasion ability of T24 cells was analyzed by Transwell assay.Results RT-qPCR detection showed that T24 cells expressed YAP1 gene.YAP1 shRNA plasmid was successfully transfected into T24 cells by Lipofectamine 2000.RT-qPCR detection showed that YAP1 shRNA3 plasmid had the most significant effect on silencing YAP1 of T24 cells (the ratio of silencing was 75.5％),and then T24 cells were interfered with YAP1 shRNA3 plasmid.MTT assay showed that the proliferation ability of T24 cells transfected with YAP1 shRNA3 plasmid was less than control group (the proliferation ratio of interference group was 85.9％ of the control group,P =0.000).The migration ability of T24 cells transfected with YAP1 shRNA3 was weaker than that in control group [24-h scratch width was (218.50 ± 3.50) μm and (86.5 ± 4.50) μm respectively,P =0.003],and the invasion ability was also weaker than that in control group (the mean number of cells penetrating Transwell was 62 ∶ 345,P =0.000).Conclusion RNA interference can significantly reduce the expression of YAP1 gene in human bladder cancer cell line T24,and inhibit the proliferation,migration and invasion of tumor cells.%目的 用RNA干扰技术阻断人膀胱癌T24细胞中Yes相关蛋白1(YAP1)基因的表达,观察细胞增殖、迁移和侵袭能力的变化.方法 检测人膀胱癌T24细胞中YAP1表达水平;采用真核转录质粒pGreenPuro构建针对YAP1基因的重组转染质粒.将4种不同序列的YAP1短发卡RNA(shRNA)质粒以及含与YAP1无关序列的对照质粒(YAP1 shRNA NC)分别转染T24细胞,48 h后行实时定量反转录聚合酶链反应(RT-qPCR)检测其YAP1表达水平,筛选YAP1基因沉默效果最好的shRNA质粒干扰T24细胞;使用噻唑蓝(MTT)检测shRNA质粒对T24细胞增殖能力的影响、划痕实验检测其对迁移能力的影响、Transwell小室体外侵袭实验检测侵袭能力的影响.结果 RT-qPCR检测T24细胞阳性表达YAP1基因,用Lipofectamine 2000成功将不同YAP1 shRNA质粒转染入T24细胞;RT-qPCR检测发现YAP1 shRNA3质粒沉默T24细胞YAP1表达效果最显著(抑制率为75.5％),随后用YAP1 shRNA3质粒干扰T24细胞,MTT检测结果显示转染YAP1 shRNA3质粒组T24细胞增殖能力小于空白对照组(干扰组增殖率为空白对照组的85.9％,P=0.000);转染YAP1 shRNA3的T24细胞迁移能力较空白对照组低[24h平均划痕宽度分别为(218.50±3.50) μm和(86.5±4.50) μm,P=0.003];转染YAP1 shRNA3的T24细胞侵袭能力较空白对照组弱(穿透Transwell小室的平均细胞数62∶ 345,P=0.000).结论 用RNA靶向干扰能明显降低人膀胱癌T24细胞内的YAP1基因表达,并抑制肿瘤细胞的增殖、迁移和侵袭能力.

摘要： 本发明公开了针对哺乳动物R‑Spondin3基因靶点的小干扰RNA、短发卡RNA及载体和应用。所述小干扰RNA包含正义链和反义链。基于该小干扰RNA可通过化学合成法合成短发卡RNA。短发卡RNA可进一步与病毒表达载体或非病毒表达载体相连，形成一种针对哺乳动物R‑Spondin3基因靶点的载体。其中病毒表达载体为慢病毒载体或腺病毒载体，非病毒表达载体为质粒载体。上述的短发卡RNA的载体可用于制备肝纤维化基因治疗药物。本发明设计的针对R‑Spondin3基因靶点的RNA干扰片段能促使激活的肝星状细胞回退到静止状态或凋亡，从而有效促进肝纤维化的恢复过程。

摘要： 本发明公开了针对哺乳动物R‑Spondin2基因靶点的小干扰RNA、短发卡RNA及载体和应用。所述小干扰RNA包含正义链和反义链。基于该小干扰RNA可通过化学合成法合成短发卡RNA。短发卡RNA可进一步与病毒表达载体或非病毒表达载体相连，形成一种针对哺乳动物R‑Spondin2基因靶点的载体。其中病毒表达载体为慢病毒载体或腺病毒载体，非病毒表达载体为质粒载体。上述的短发卡RNA的载体可用于制备肝纤维化基因治疗药物。本发明设计的针对R‑Spondin2基因靶点的RNA干扰片段能促使激活的肝星状细胞回退到静止状态或凋亡，从而有效促进肝纤维化的恢复过程。

摘要： 本发明公开了针对哺乳动物R‑Spondin1基因靶点的小干扰RNA、短发卡RNA及载体和应用。所述小干扰RNA包含正义链和反义链。基于该小干扰RNA可通过化学合成法合成短发卡RNA。短发卡RNA可进一步与病毒表达载体或非病毒表达载体相连，形成一种针对哺乳动物R‑Spondin1基因靶点的载体。其中病毒表达载体为慢病毒载体或腺病毒载体，非病毒表达载体为质粒载体。上述的短发卡RNA的载体可用于制备肝纤维化基因治疗药物。本发明设计的针对R‑Spondin1基因靶点的RNA干扰片段能促使激活的肝星状细胞回退到静止状态或凋亡，从而有效促进肝纤维化的恢复过程。

摘要： 本发明公开了一种针对NFAT3基因靶点的短发卡RNA、重组载体及应用，所述基因靶点的序列如SEQ ID NO：3所示，所述RNA的正义链序列如SEQ ID NO：1所示，所述RNA的反义链序列如SEQ ID NO：2所示。针对NFAT3基因靶点设计了短发卡RNA序列，并构建了相应的重组载体，该重组载体能在宿主细胞内转录得到NFAT3‑shRNA，经宿主细胞加工后可生成小分子干扰RNA，并靶向NFAT3的信使RNA，降低NFAT3的表达，导致基因沉默效应，有效抑制黑色素瘤细胞的增殖，具有抗癌应用价值，为进一步研究NFAT3在皮肤癌细胞中的功能及靶向NFAT3的药物研发提供了基础。

摘要： 本发明涉及分子生物学、基因工程技术以及生物医药领域，具体涉及一种针对SOX4基因靶点的小干扰RNA、短发卡RNA及重组载体和应用。小干扰RNA包括SEQ ID NO.1所示的正义链和SEQ ID NO.2所示的反义链。短发卡RNA包括SEQ ID NO.3所示的正义链和SEQ ID NO.4所示的反义链。包含短发卡RNA的重组载体能在宿主细胞内转录得到SOX4‑shRNA，经宿主细胞加工后可生成小分子干扰RNA，并靶向SOX4的信使RNA，降低SOX4的表达，导致基因沉默效应，有效抑制食管鳞癌细胞的增殖并促进化疗药物阿霉素诱导的食管鳞癌细胞的衰老，具有抗肿瘤应用价值，为进一步研究SOX4在肿瘤细胞衰老逃逸中的功能及靶向SOX4的药物研发提供了基础。

摘要： 本发明公开了针对哺乳动物R‑Spondin3基因靶点的小干扰RNA、短发卡RNA及载体和应用。所述小干扰RNA包含正义链和反义链。基于该小干扰RNA可通过化学合成法合成短发卡RNA。短发卡RNA可进一步与病毒表达载体或非病毒表达载体相连，形成一种针对哺乳动物R‑Spondin3基因靶点的载体。其中病毒表达载体为慢病毒载体或腺病毒载体，非病毒表达载体为质粒载体。上述的短发卡RNA的载体可用于制备肝纤维化基因治疗药物。本发明设计的针对R‑Spondin3基因靶点的RNA干扰片段能促使激活的肝星状细胞回退到静止状态或凋亡，从而有效促进肝纤维化的恢复过程。

摘要： 本发明公开了针对哺乳动物R-Spondin1基因靶点的小干扰RNA、短发卡RNA及载体和应用。所述小干扰RNA包含正义链和反义链。基于该小干扰RNA可通过化学合成法合成短发卡RNA。短发卡RNA可进一步与病毒表达载体或非病毒表达载体相连，形成一种针对哺乳动物R-Spondin1基因靶点的载体。其中病毒表达载体为慢病毒载体或腺病毒载体，非病毒表达载体为质粒载体。上述的短发卡RNA的载体可用于制备肝纤维化基因治疗药物。本发明设计的针对R-Spondin1基因靶点的RNA干扰片段能促使激活的肝星状细胞回退到静止状态或凋亡，从而有效促进肝纤维化的恢复过程。

摘要： 本发明公开了针对哺乳动物R-Spondin2基因靶点的小干扰RNA、短发卡RNA及载体和应用。所述小干扰RNA包含正义链和反义链。基于该小干扰RNA可通过化学合成法合成短发卡RNA。短发卡RNA可进一步与病毒表达载体或非病毒表达载体相连，形成一种针对哺乳动物R-Spondin2基因靶点的载体。其中病毒表达载体为慢病毒载体或腺病毒载体，非病毒表达载体为质粒载体。上述的短发卡RNA的载体可用于制备肝纤维化基因治疗药物。本发明设计的针对R-Spondin2基因靶点的RNA干扰片段能促使激活的肝星状细胞回退到静止状态或凋亡，从而有效促进肝纤维化的恢复过程。

摘要： 本发明公开了一种针对NFAT3基因靶点的短发卡RNA、重组载体及应用，所述基因靶点的序列如SEQ ID NO：3所示，所述RNA的正义链序列如SEQ ID NO：1所示，所述RNA的反义链序列如SEQ ID NO：2所示。针对NFAT3基因靶点设计了短发卡RNA序列，并构建了相应的重组载体，该重组载体能在宿主细胞内转录得到NFAT3‑shRNA，经宿主细胞加工后可生成小分子干扰RNA，并靶向NFAT3的信使RNA，降低NFAT3的表达，导致基因沉默效应，有效抑制黑色素瘤细胞的增殖，具有抗癌应用价值，为进一步研究NFAT3在皮肤癌细胞中的功能及靶向NFAT3的药物研发提供了基础。

摘要： 描述了包含用于抑制基因表达(例如病毒介导的基因表达)的短发卡RNA(shRNA)的方法、组合物和试剂盒。