靶向VEGF165 RNA干涉对HeLa细胞凋亡的影响

RNA干涉(RNA interference,RNAi)是指序列特异性的转录后基因沉默,它已经发展成为高效、特异阻断目的基因表达的有效工具[1].据此,我们针对人VEGF165基因mRNA序列,设计了2个干涉靶位点,体外转录合成siRNA,通过脂质体转染入HeLa细胞,分别体内、体外观察对HeLa细胞凋亡的影响.     

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。     

短双链RNA对柯萨奇B组3型病毒体外感染的抑制作用研究

目的利用RNA干扰技术，在细胞、蛋白和基因水平上观察短双链RNA对柯萨奇病毒体外感染Hela细胞的增殖抑制作用、对病毒RNA复制和蛋白合成的影响。方法应用病毒致细胞病变作用保护实验、空斑形成减少实验、Western Blot、RT-PCR等方法，在体外检测其抗CVB3病毒作用。结果设计、合成的8条SiRNA中，针对基因组2B、VP4、2A、3C区的SiRNA-2、SiRNA-3、SiRNA-6、SiRNA-7具有不同程度的抑制病毒作用。其中，病毒基因组2B的SiRN．2作用效果最好，对Hela细胞CVB3感染72h后致细胞病变和空斑形成的抑制率为95％，抑制病毒蛋白的合成，病毒基因的复制水平也显著降低，在病毒0．1、0．01、0．001、0．0001 MOI感染水平上对CVB3致细胞病变作用的抑制率分别为30％、70％、88％和99％（48h）。结论针对CVB3基因组2B的SiRNA-2具有较强的抑制柯萨奇B3型病毒感染的作用。     

siRNA对胃癌细胞系BGC-823生长抑素基因表达的抑制效应

目的：研究siRNA对胃癌细胞BGC-823生长抑素(SOM)分泌的抑制效应。方法：设计4条针对SOM基因不同位点的寡核苷酸序列,应用RiboMAXT7体外转录合成siRNA并转染胃癌细胞系BGC-823,经RT- PCR、免疫细胞化学法检测SOM mRNA和蛋白的表达水平,筛选抑制效果最佳的序列,MTT法检测BGC-823细胞的增殖变化。结果：转染后24、48及72h,SOM基因的表达被抑制,抑制效率有差异,并呈浓度及时间依赖性。结论：体外合成siRNA抑制胃癌细胞系SOM基因的表达,增强了细胞的增殖能力。     

应用RNA干扰技术抑制人胃癌细胞环氧合酶-2基因的表达研究

目的 研究应用RNA干扰技术进行抑制胃癌SGC-7901细胞COX-2的表达研究。方法 依据siRNA设计原则,针对人COX-2的mRNA序列,设计并合成3条21bp的双链RNA,瞬时转染人胃癌细胞SGC-7901,Western blot验证基因沉默效率以筛选合适的siRNA序列;根据筛选出的有效序列,合成编码短发卡RNA的双链寡核苷酸,定向克隆到含有U6 RNA聚合酶Ⅲ启动子的pSilencer? 2.1-U6 neo 真核表达载体,稳定转染胃癌细胞SGC-7901中,经G418筛选,获得克隆细胞。 结果 筛选出一条有效的干扰序列,位于291-311bp,成功构建表达载体,COX-2在蛋白水平的表达明显被抑制。结论：利用化学合成siRNA进行初筛,再构建shRNA表达载体,可快速实现目的基因的表达抑制。     

双向启动子小干扰RNA载体干扰效率的验证

RNA干扰技术是目前抑制基因功能较为有效的热点技术。通常用于表达小干扰RNA(siRNA)的载体多利用单个RNA聚合酶Ⅲ启动子转录连接在一起的2条互补回文序列,形成袢状结构的siRNA分子。这种方法需要较长的引物而造价较高,且容易错配,难于合成和测序;此外,袢的长度及序列的差异可以影响siRNA抑制基因表达的效率[1]。一种新型的siRNA表达载体逐渐获得人们的重视。这种载体利用2个RNA聚合酶Ⅲ,从目的序列的两端同时进行互为反向的转录,形成有功能的siRNA[2]。为了正确评价这种载体所表达的siRNA抑制基因功能的效率,我们构建了表达针对…     

下调VEGF基因表达对乳腺癌细胞MCF-7的增殖抑制作用

目的利用RNA干扰技术特异性抑制乳腺癌细胞MCF-7的VEGF基因表达,研究乳腺癌的治疗新方法.方法体外合成带有T7启动子的VEGF基因DNA片段,转录针对VEGF mRNA的siRNA,使用脂质体转染的方法导入细胞,观察转染后乳腺癌细胞MCF-7的增殖变化,MTT法检测细胞存活率,RT-PCR检测转染后VEGF mRNA表达水平的变化,ELISA检测蛋白表达的下降效果.结果所设计的两个靶位点siRNA能有效抑制乳腺癌细胞生长;VEGF mRNA的表达也受到有效抑制,明显减少;同时,对应的VEGF蛋白水平也显著降低.阴性对照的错义序列组siRNA则没有这种效果.结论应用RNA干扰技术可以有效抑制肿瘤细胞的增殖.     

siRNA和shRNA在syt基因沉默中的一致性

目的通过小干扰RNA(small interference RNA,siRNA)和短发夹RNA(short hairpin RNA,shRNA)对PC12细胞中Synaptotagmin(syt)基因表达沉默效果的比较,确认siRNA在基因沉默中与shRNA的等效性.方法用T7 RNA聚合酶体外合成的针对syt Ⅰ与Ⅸ的siRNA和在H1.1载体上构建的shRNA表达质粒转染PC12细胞,用标记荧光蛋白和免疫荧光检测shRNA和siRNA的转染率,用Western印迹方法检测沉默效果.结果siRNA和shRNA在PC12细胞中沉默syt Ⅰ和Ⅸ的表达时,有一致的沉默效率.结论siRNA可作为基因沉默中快速筛选的工具,可在RNA干扰的应用中与shRNA 配合使用.     

用小双链RNA抑制转化入HUVEC中绿色荧光蛋白基因的表达

目的：建立稳定表达绿色荧光蛋白(GFP)的人脐静脉血管内皮细胞（HUVECs），研究小干扰RNA(siRNA)对HUVECs中GFP表达的抑制作用。 方法： 用lipofectamine2000将编码GFP的质粒pN3-EGFP转入HUVECs中。用G418筛选并维持已转化pN3-EGFP的HUVEC(HUVEC-GFP)。利用T7 RNA转录试剂盒，制备可抑制GFP基因表达的siRNA（GFPsiRNA）和无关对照的RNA（control siRNA）。用oligofectamine将siRNA转入HUVEC-GFP中。继续培养48 h后，检测HUVEC-GFP中GFP蛋白和mRNA表达水平。 结果： 用G418筛选获得了HUVEC-GFP细胞株，可以观察到GFP的稳定表达。HUVEC-GFP转化siRNA后48 h，GFP的荧光强度明显下降，而对照组的荧光强度无明显下降。半定量RT-PCR检测显示，GFPsiRNA对GFP mRNA表达有较强的抑制作用，抑制率达40%，而control siRNA对GFP mRNA表达水平无明显的抑制作用。 结论： 利用体外转录合成的siRNA能有效地抑制HUVECs中GFP的表达。     

RNAi对B系淋巴瘤Namalwa细胞中VEGF表达和增殖的影响

目的 应用RNA干扰 (RNA interference,RNAi) 技术抑制VEGF的表达,观察其对B系淋巴瘤细胞株Namalwa生物学行为的影响.方法 设计3条针对人VEGF mRNA的siRNA序列,经脂质体转染至Namalwa细胞.采用RT-PCR、Western blot法检测转染后Namalwa细胞VEGF mRNA和蛋白的表达.结果 siRNA-2组VEGF mRNA及其蛋白的表达明显减少(P<0.01),siRNA-2组细胞体外增殖能力减弱(P<0.05).结论 RNAi技术可明显抑制Namalwa细胞VEGF的表达及细胞的体外增殖.     

RNAs specifically affect gene expression in a length,position and sequence dependent manner

We aim to explore if RNA regulating gene expression is affected by length, sequence and position of RNA. HeLa cells were co-transfected with modulator plasmids (derived from pcDNA3.1 vector containing different length regulating sequences that produce RNAs) and reporter plasmids (derived from pEGFP-C1 vector); In addition, HeLa cells were transfected with plasmids that possess different sequences of downstream or adjacent genes of GFP reporter gene. We found that long inserting sequences of modulator plasmids induced stronger GFP gene activation than short inserting sequences. Changing of downstream sequences of GFP gene induced significant effects on GFP gene expression. Short sequences of adjacent genes of GFP activated GFP gene. Bioinformatics analysis of genes which is highly expressed in differentiating cells (thymocyte cells, germinal center B-cells) and quiescent cells (T cells, B cells) shows that differentiating cells produce longer RNA than quiescent cells. These findings demonstrate that the length, sequence and producing position of RNAs are important factors for RNA regulating gene expression.     

Mosquito mitochondrial transfer RNAs for valine,glycine and glutamate: RNA and gene sequences and vicinal genome organization 19851230

Summary We report the sequences of 3 transfer RNAs from mosquito (Aedes albopictus) mitochondria, those for valine (anticodon UAC), glutamic acid (anticodon UUC) and glycine (anticodon UCC), as well as sequences for the corresponding genes and for some neighboring mitochondrial genes. TRNA^va1 is notable for its high level of , tRNA^glu for its low level of G and C, and tRNA^gly is notable in that it appears as two species widely separated in gel electrophoresis, differing only in modification status. TRNA^glu is the first sequenced insect mitochondrial tRNA that would be expected to engage in U^* · R wobble (where U^* is a modified U in the first position of the anticodon, and R is G or A in the third position of codons), if the insect system followed the modified wobble rules proposed for mammalian and fungal mitochondria; and the sequence determined does fit the proposal. The gene for tRNA^va1 follows immediately that for 12S ribosomal RNA. The gene for tRNA^glu occurs in a cluster of 6 tRNA genes that is separated from the gene for tRNA^gly by a short reading frame. Features of the DNA sequences are discussed with reference to Drosophila, and mammalian, mitochondrial genome organization.     

子宫内膜癌microRNA表达谱的初步研究

目的 筛选子宫内膜癌与正常子宫内膜组织中的差异mieroRNA.方法 收集3例患者的新鲜子宫内膜癌组织及正常子宫内膜组织,利用miRNA芯片对其进行实验分析,并采用茎环逆转录荧光定量PCR方法验证芯片结果的准确性.结果 相对正常子宫内膜组织,子宫内膜癌组织中表达上调的miRNA基因68个,下调的miRNA基因81个.采用茎环逆转录荧光定量PCR对显著上调的hsa-miR-205,hsa-miR-200b和hsa-miR200c以及显著下调的hsa-miR-495,hsa-miR-216b的验证结果与芯片检测结果一致.结论 筛选获得了子宫内膜癌的miRNA的差异表达谱,可能在子宫内膜癌的发病机制中发挥潜在作用。     

核糖核酸干扰

The phenomenon of RNA interference (RNAi) is highly conserved mechanism in the organism evolution. As a immune system , RNAi is a ubiquitous mechanism against invading microorganism in plant and animal cells. Recently, it has been found that RNAi is the process by which double-strand RNA (dsRNA) directs sequence - specific degradation of messenger RNA and the mediations of sequence specific messenger RNA degradation are 21 - and 23 - nucleotide small interfering RNAs that generate by ribonuclease from endogenous longer dsRNA or by transfectious technics from heterologous dsRNA. Over the past few years,the way in which cells respond to dsRNA by silencing homologous genes has revealed a new regulating paradigm in biology.     

The pattern of synthesis and distribution of membrane bound RNA in brain synaptosomes

Summary The pattern of RNA synthesis and its distribution in subcellular fractions of goldfish brain was studied using uridine-5H³ as the precursor. About 14% of the total RNA synthesized was found to be associated with the synaptosomal fraction after a 3 hr labelling time. The sedimentation characteristics of this RNA was compared with that of the nuclear and cytoplasmic components of brain. The results suggest that the synaptosomal RNA is a membrane-bound fraction with distinctive properties.This research was supported by grants from NIH (NS09407) and the Grant Foundation.     

Dicer酶及其在RNA干扰中的作用

RNA干扰(RNAInterference,RNAi)是双链RNA引起的同源mRNA特异性降解的现象。Dicer酶是核糖核酸酶Ⅲ(RibonucleaseⅢ,RNaseⅢ)家族的成员,在RNA干扰的起始阶段起着重要作用。Dicer酶最早在果蝇中发现,包括Dicer-1和Dicer-2,分别以Dicer-1/R3D1-L、Dicer-2/R2D2复合物的形式作用于微小RNA(microRNA,miRNA)和小干扰RNA(smallinterferingRNA,siRNA)的产生。Dicer酶除了在RNAi中发挥重要作用外,还在调节动物生殖发育中起作用。     

Purification of retrovirus genomic RNA suitable for chemical radioiodination

An efficient method for the purification of genomic RNA from the retrovirus, caprine arthritis-encephalitis virus, is described. The method utilizes proteinase K, extraction with sodium perchlorate and chromatography on oligo(dT)-cellulose and results in highly purified RNA capable of being chemically iodinated with Na125 I to high specific radioactivity. The iodinated RNA exhibits 80-90% precipitability in 5% trichloroacetic acid and is greater than or equal to 99% sensitive to hydrolysis by ribonuclease. Several alternative methods which are effective for the preparation of eukaryotic ribosomal RNA are unreliable for purification of retrovirus RNA suitable for radioiodination.     

Evolution of virus-derived sequences for high-level replication of a subviral RNA

Turnip crinkle virus (TCV) and its 356-nt satellite RNA satC share 151 nt of 3'-terminal sequence, which contain 8 positional differences and are predicted to fold into virtually identical structures, including a series of four phylogenetically inferred hairpins. SatC and TCV containing reciprocal exchanges of this region accumulate to only 15% or 1% of wild-type levels, respectively. Step-wise conversion of satC and TCV 3'-terminal sequences into the counterpart's sequence revealed the importance of having the cognate core promoter (Pr), which is composed of a single hairpin that differs in both sequence and stability, and an adjacent short 3'-terminal segment. The negative impact of the more stable TCV Pr on satC could not be attributed to lack of formation of a known tertiary interaction involving the 3'-terminal bases, nor an effect of coat protein, which binds specifically to TCV-like Pr and not the satC Pr. The satC Pr was a substantially better promoter than the TCV Pr when assayed in vitro using purified recombinant TCV RdRp, either in the context of satC or when assayed downstream of non-TCV-related sequence. Poor activity of the TCV Pr in vitro occurred despite solution structure probing indicating that its conformation in the context of satC is similar to the active form of the satC Pr, which is thought to form following a required conformational switch. These results suggest that evolution of satC following its initial formation generated a Pr that can function more efficiently in the absence of additional TCV sequence that may be required for full functionality of the TCV Pr.     

RNA干扰分子机制研究进展

RNA干扰 (RNA interference,RNAi)即双链 RNA(double- stranded RNA,ds RNA)介导的同源m RNA特异性降解过程。作为一种简单有效的影响基因表达和一定程度上替代基因敲除的遗传学工具 ,RNAi已在秀丽新小杆线虫、黑腹果蝇、拟南芥、红色面包霉菌等多种模式生物体中得到广泛证实。同时 ,RNAi的分子机制的研究也不断取得进展 ,包括对基因转录后水平、翻译水平、基因组甲基化及沉默信号的传递等层次的研究。清晰地阐明 RNAi作用机理 ,将为大规模的基因系统筛查、新基因的发现、人类肿瘤及难治性疾病的基因治疗提供重要的理论依据和有力的工具。     

抑制Itk表达对Jurkat细胞分泌细胞因子的影响

目的 研究在Jurkat细胞上敲减Itk蛋白的表达对细胞增殖及炎症相关的细胞因子产生的影响,为Itk作为小分子药物靶标提供可行性实验依据.方法 针对Itk基因设计合成三个shRNAs,通过与pEGFP-C1-hItk质粒共转染,观察其抑制Itk-GFP融合蛋白的表达情况,筛选有效序列包装成慢病毒颗粒.将慢病毒颗粒感染Jurkat细胞,观测Itk蛋白的表达水平、细胞增殖情况及细胞因子的变化.结果 Itk-shRNA1感染Jurkat细胞后Itk基因的mRNA水平与细胞对照组及感染shRNAnon的对照组相比,敲减率约55%,差异有统计学意义P＜0.05.Itk蛋白的敲减导致Jurkat细胞在受刺激时增殖降低,以未感染病毒的Jurkat细胞受刺激后酶标仪检测的A值为1,Itk-shRNA1感染组平均比值为0.54,shRNAnon对照组平均比值为0.83,两组差异有统计学意义,P＜0.05.Itk-shRNA1感染组中IL-2、IL-5、IL-10及IFN-γ等细胞因子水平均较shRNAnon对照组低,差异有统计学意义,提示Itk蛋白的表达减少导致Jurkat细胞产生细胞因子减少.结论 抑制Itk表达能有效降低淋巴细胞的增殖,同时减少与炎症相关细胞因子分泌.