The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1)encoded by Epstein-Barr virus(pshLMP1),and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells,we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis,RT-PCR and western blot.pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method.Furthermore,the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors.According to our research,we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells,and also provides a novel application of RNA interference technology against-EBV.     

The comparative and functional study between two construction methods of shRNA expression vector targeted <Emphasis Type="Italic">LMP1</Emphasis> gene encoded by EBV

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.      

Latency type-dependent modulation of Epstein-Barr virus-encoded latent membrane protein 1 expression by type I interferons in B cells

We report that type I interferons (IFNs) upregulate latent membrane protein 1 (LMP-1) expression by direct activation of the ED-L1 promoter in several Epstein-Barr virus (EBV)-carrying Burkitt's lymphoma lines. In EBV-infected primary B cells, IFN-α transiently upregulates LMP-1 mRNA, but not protein levels, followed by downregulation of both, suggesting a novel antiproliferative mechanism of type I IFNs. Furthermore, our results may explain the expression of LMP-1 in memory B cells of systemic lupus erythematosus patients.     

靶向shRNA表达载体构建方式的比较研究

目的:探讨两种构建shRNA表达载体方法的不同,寻找更加稳定方便的靶向shRNA表达载体的构建方式。方法:比较构建shRNA表达载体传统方法双链退火法和新方法双链PCR法的设计原则、构建方法和鉴定结果的不同。结果:两种方法均能得到重组质粒,但是双链PCR法构建效率高且不易引起碱基的缺失和突变。结论:双链PCR法构建shRNA表达载体比传统双链退火法更加稳定,构建成功率较双链退火法高。     

在活体小鼠中筛选lZP3基因RNAi有效靶位点的研究

ZP3作为精子结合的靶对象在卵母细胞受精中起着关键作用,也因此而成为研究哺乳动物受精机理的焦点。本研究参照新疆草原兔尾鼠卵透明带3(zonapellucida3geneofLaguruslagurus,lZP3)的mRNA,选择了针对lZP3mRNA的3个区域合成寡聚核苷酸连接到干扰载体pGenesil-1上,构建了3个针对lZP3mRNA的重组干扰载体,和pCDNA3-lZP3表达载体采用脂质体法共转染Hela细胞以及通过尾静脉大容量快速注射法(Hydrodynamics-basedtransfectionmethod,HD法)共注射小鼠,半定量RT-PCR和real-timePCR检测Hela细胞和小鼠肝脏中lZP3mRNA的表达情况,以筛选干扰lZP3mRNA的有效靶位点。结果表明,通过HD法共注射干扰载体和表达载体后,外源基因mRNA在小鼠肝脏中的表达和共转染Hela细胞后在Hela细胞中的表达情况是一致的,有2个干扰载体可以有效干扰lZP3mRNA的表达。研究还说明,利用HD法以小鼠作为实验材料筛选RNA干扰的有效靶位点是一条切实可行的方法。     

在活体小鼠中筛选IZP3基因RNAi有效靶位点的研究

ZP3作为精子结合的靶对象在卵母细胞受精中起着关键作用,也因此而成为研究哺乳动物受精机理的焦点。本研究参照新疆草原兔尾鼠卵透明带3（zonapellucida3 gene of Lagurus lagurus,IZP3）的mRNA,选择了针对IZP3 mRNA的3个区域合成寡聚核苷酸连接到干扰载体pGenesil-1上,构建了3个针对IZP3 mRNA的重组干扰载体,和pCDNA3-IZP3表达载体采用脂质体法共转染Hela细胞以及通过尾静脉大容量快速注射法（Hydrodynamics—based transfection method,HD法）共注射小鼠,半定量RT—PCR和real—timePCR检测Hela细胞和小鼠肝脏中IZP3mRNA的表达情况,以筛选干扰IZP3mRNA的有效靶位点。结果表明,通过HD法共注射干扰载体和表达载体后,外源基因mRNA在小鼠肝脏中的表达和共转染Hela细胞后在Hel。细胞中的表达情况是一致的,有2个干扰载体可以有效干扰IZP3mRNA的表达。研究还说明,利用HD法以小鼠作为实验材料筛选RNA干扰的有效靶位点是一条切实可行的方法。     

Stathmin1 RNA干扰载体的构建及功能检测

目的构建用于stathmin1 RNA干扰的重组质粒并验证其干扰效果,初步探讨stathmin1对于黑色素细胞生长的影响。方法两步PCR法构建含有shRNA编码序列的重组质粒,脂质体转染黑色素细胞。RT-PCR、Western印迹等方法检测stathmin1在黑色素细胞中表达水平改变;MTT、流式细胞仪等方法检测黑色素细胞生长状态。结果成功构建3个stathmin1特异性的RNA干扰载体,在黑色素细胞中能有效降低stathmin1在mRNA水平的表达,蛋白质表达水平分别降低为对照组的6％、17％和15％;黑色素细胞的生长由于stathmin1减弱表达而受到明显抑制,凋亡率提高到约60％。结论干扰质粒能有效介导stathmin1的RNA干扰;stathmin1对于黑色素细胞的生长具有重要意义。     

Latent membrane protein 1 of Epstein-Barr virus inhibits as well as stimulates gene expression

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) functionally resembles a constitutively active, CD40-like receptor and contributes to the maintenance of proliferation of EBV-infected primary human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it binds TRAF, TRADD, and JAK molecules to activate NF-kappaB-, AP-1-, and STAT-dependent pathways as does CD40. Yet LMP-1 appears to lack a ligand to regulate its signaling. We have found that LMP-1, when expressed at physiologic levels, inhibits gene expression detectably. Higher levels of LMP-1 expression eventually inhibit both the steady-state level of RNA produced from a BamHI C promoter reporter and general cellular protein synthesis. These findings indicate that LMP-1 can limit its signaling and that this control is manifest at two levels. The domain of LMP-1 that binds TRAF, TRADD, and JAK/STAT molecules is not required for this regulation. A derivative of LMP-1 that contains only its amino-terminal and membrane-spanning domains is sufficient to inhibit reporter activity when the reporter genes are expressed from the BamHI C and LMP-1 promoters. This same derivative of LMP-1 in parallel assays is sufficient to inhibit wild-type LMP-1's stimulation of NF-kappaB-dependent gene expression. We suggest that LMP-1 encodes stimulatory and inhibitory activities; the latter could limit signaling in the apparent absence of ligand-dependent down-regulation.     

RNA干扰敲减FucT VII表达抑制人结肠癌细胞HT-29和HUVECs的粘附能力

 探讨利用RNA干扰（RNAi）技术抑制岩藻糖基转移酶Ⅶ（FucT Ⅶ）表达对人结肠癌细胞HT-29与人脐静脉内皮细胞（HUVEC）粘附能力的影响及其机制.本课题构建3对针对FucT Ⅶ基因的RNAi表达载体,并将其转染入人结肠癌细胞HT-29,Western 印迹检测FucT Ⅶ及其下游产物sLeX蛋白的变化;实时PCR 检测FucT Ⅶ mRNA表达的变化;玫瑰红染色法检测RNAi 对HT-29与HUVECs细胞粘附能力的影响.结果显示,3对FucT Ⅶ siRNA表达载体均可有效抑制HT-29细胞FucT Ⅶ mRNA和蛋白表达,以pSilencer 2.0 FucT Ⅶ 2最为有效;与空白细胞组比较,转染pSilencer 2.0-FucT Ⅶ的HT-29细胞表面sLeX表达水平明显下降,以pSilencer 2.0-FucT Ⅶ 2最为显著;RNA干扰FucT Ⅶ表达后HT 29细胞和HUVEC之间的粘附能力明显受到抑制.研究表明,RNAi靶向沉默HT-29细胞中FucT Ⅶ基因表达可显著降低其下游产物sLeX的合成,进而抑制HT-29细胞与HUVECs的粘附能力.     

脂联素siRNA对3T3-L1细胞基础葡萄糖转运的影响

目的：构建携带小鼠脂联素（Acrp30）siRNA腺病毒载体,并检测其对小鼠脂肪细胞Acrp30表达以及对3T3-L1脂肪细胞基础葡萄糖转运的影响。方法：设计并化学合成小鼠脂肪细胞Acrp30 siRNA片断,将其亚克隆入AdEaxy XL腺病毒载体系统,在293细胞内包装扩增为重组腺病毒。用此重组腺病毒感染3T3-L1脂肪细胞,用RT-PCR和ELISA检测其Acrp30 mRNA和蛋白表达。采用2Deoxy-[3H]D—glucose掺入法测定脂肪细胞葡萄糖转运。结果：设计并构建了小鼠Acrp30基因特异性siRNA腺病毒载体,该载体感染脂肪细胞后,能显著抑制Acrp30 mRNA和蛋白表达,影响3T3-L1脂肪细胞基础葡萄糖的转运,与对照组相比,差异有显著性意5C（P〈0．05）。结论：构建的Acrp30基因特异性siRNA腺病毒载体能有效的抑制脂联素在3T3-L1脂肪细胞中的表达,从而影响3T3-L1脂肪细胞基础葡萄糖转运。     

HeLa细胞中miRNA-214靶基因Mek3的确定

目的探索miRNA-214在HeLa细胞中的与其靶基因Mek3相互作用。方法通过miRNA靶基因预测网站寻找可能与miRNA-214相互作用的靶基因,合成miRNA-214和对照序列,将miRNA-214、对照序列、Mek3的3’非翻译区（3’UTR）以及突变的Mek3 3’UTR分别克隆到表达载体上,转染HeLa细胞,转染48h后提取蛋白,检测绿色荧光蛋白的表达水平;HeLa细胞转染miRNA-214后,Trizol抽提RNA,通过荧光定量PCR检测Mek3mRNA的表达水平;Western印迹检Mek3的蛋白表达水平。经过以上实验从mRNA和蛋白水平上验证了在HeLa细胞中miRNA-214对靶基因Mek3的作用效应。结果生物信息学方法显示miRNA-214和Mek3存在可能的结合位点。经过实验验证了miRNA-214可以下调Mek3的mRNA和蛋白水平。结论miRNA-214可以负调节靶基因Mek3的表达。     

核受体相关因子1表达下调对多巴胺能细胞酪氨酸羟化酶和神经突起生长的影响

将合成的核受体相关因子1（nuclear receptor-related factor 1,Nurr1）特异性短发夹寡核苷酸（small-hairpin RNA,shRNA）序列插入真核表达载体pSilen Circle（pSC）,构建Nurr1基因特异性shRNA真核表达载体,转染体外培养多巴胺能神经前体细胞系MN9D,分别采用实时荧光定量PCR和Western blot方法检测其对MN9D细胞内源Nurr1的干扰作用及其对酪氨酸羟化酶（tyrosine hydroxylase,TH）表达的影响,并在倒置显微镜下观察MN9D细胞神经突起生长的情况,探讨Nurr1 shRNA表达载体对多巴胺能细胞表型标记物删和以神经突起延长为特征的细胞成熟的影响。结果表明,脂质体组细胞和转染阴性对照质粒的MN9D细胞内Nurr1、TH的表达正常,而转染Nurr1 shRNA真核表达载体（pSC-N1和pSC-N2）的MN9D细胞内Nurr1和TH的mRNA水平明显降低,Nurr1 mRNA的下降率分别为62．3％和45．6％,TH mRNA的下降率分别为76.3％和62．6％。同时Nurr1和TH蛋白的表达亦明显下调,Nurr1蛋白的下降率分别为57．4％和72．0％,TH蛋白的下降率分别为79．1％和70．1％。另外,转染Nurr1 shRNA真核表达质粒的MN9D细胞神经突起延长有所减少,但是与正常细胞无明显差异。结果提示：Nurr1 shRNA真核表达载体能显著下调MN9D细胞内源Nurr1和TH mRNA和蛋白的表达,同时可能对MN9D细胞的神经突起延长有一定的抑制作用。Nurr1 shRNA表达载体的成功构建为多巴胺能神经元发育以及帕金森病相关基因的功能研究奠定了基础。     

shRNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因表达的研究

应用短发夹RNA(Short hairpin RNA,shRNA)表达载体抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达。应用已鉴定的shRNA表达载体pHPV1、pHPV2转染Hela细胞,G418筛选阳性细胞,建立稳定转染细胞株;倒置荧光显微镜检测转染情况;提取细胞内总RNA,RT-PCR方法检测HPV18 E6、E7 mRNA;WesternBlot检测HPV18 E6、E7蛋白表达的变化;采用灰度分析软件对PCR扩增条带与蛋白质条带进行灰度分析。pHPV1实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的31%、38%,E6、E7蛋白分别为阴性对照组的37%、31%;pHPV2实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的54%、77%,E6、E7蛋白分别为阴性对照组的52%、83%。pHPV1、pHPV2表达载体能抑制Hela细胞HPV18 E6、E7的表达,针对外显子区434-452的pHPV1抑制作用更强。     

Truncated human LMP-1 triggers differentiation of C2C12 cells to an osteoblastic phenotype in vitro

LIM mineralization protein-1 （LMP-1） is a novel intracellular osteoinductive protein that has been shown to induce bone formation both in vitro and in viva. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. In this study, we investigated whether a truncated form of human LMP-1 （hLMP-1 [t]）, lacking the three C-terminal LIM domains, triggers the differentiation of pluripotent myoblastic C2C12 cells to the osteoblast lineage. C2C12 cells were transiently transduced with AdS-hLMP-1 （t）-green fluorescent protein or viral vector control. The expression of hLMP-1 （t） RNA and the truncated protein were examined. The results showed that hLMP-1 （t） blocked myotube formation in C2C12 cultures and significantly enhanced the alkaline phosphatase （ALP） activity. In addition, the expressions of ALP, osteocalcin, and bone morphogenetic protein （BMP）-2 and BMP-7 genes were also increased. The induction of these key osteogenic markers suggests that hLMP- 1 （t） can trigger the pluripotent myoblastic C2C12 cells to differentiate into osteoblastic lineage, thus extending our previous observation that LMP-1 and LMP-1 （t） enhances the osteoblastic phenotype in cultures of cells already committed to the osteoblastic lineage. Therefore, C2C12 cells are an appropriate model system for the examination of LMP-1 induction of the osteoblastic phenotype and the study of mechanisms of LMP-1 action.     

人中性粒细胞弹性蛋白酶诱导气道黏液高分 泌细胞模型的建立及其机制的初步研究

目的：以人中性粒细胞弹性蛋白酶(HNE)为诱导因素,研究建立黏蛋白(MUC)5AC和5B高表达的细胞模型,同时对黏蛋白高表达机制进行初步研究。方法：培养人肺腺癌细胞A549,以HNE为刺激因素,EGFR中和抗体、表皮细胞生长因子受体（EGFR）磷酸化阻断剂AG1478为干预因素,分组培养。采用四甲基偶氮唑盐光吸收法（MTT法）检测HNE对细胞活性的影响;逆转录-聚合酶链反应（RT-PCR）检测MUC5AC mRNA、MUC5B mRNA的变化;酶联免疫吸附测定法(ELISA)定量分析MUC5AC和MUC5B蛋白含量的差异;细胞免疫化学以及激光共聚焦技术进一步直观观察MUC5AC、MUC5B、p-EGFR蛋白表达的变化。结果：HNE对A549细胞活力的影响呈剂量依赖性;HNE刺激组的MUC5AC、MUC5B基因转录和蛋白表达水平均明显高于对照组,差异有统计学意义（均P<0.01）;HNE刺激组p-EGFR蛋白表达显著增多,EGFR中和抗体、AG1478能显著降低HNE诱导的MUC5AC高表达,但对MUC5B高表达无干预作用。结论：人肺腺癌细胞A549同时表达MUC5AC和MUC5B,HNE能有效刺激A549细胞高表达MUC5AC和MUC5B,黏蛋白高表达细胞模型的建立为研究气道粘液高分泌疾病提供了实验基础。HNE通过激活EGFR信号转导通路诱导MUC5AC的高表达,但MUC5B高表达机制与之不同,有待进一步研究。     

CD21-Dependent Infection of an Epithelial Cell Line, 293, by Epstein-Barr Virus

Epstein-Barr virus (EBV) is invariably present in undifferentiated nasopharyngeal carcinomas, is found sporadically in other carcinomas, and replicates in the differentiated layer of the tongue epithelium in lesions of oral hairy leukoplakia. However, it is not clear how frequently or by what mechanism EBV infects epithelial cells normally. Here, we report that a human epithelial cell line, 293, can be stably infected by EBV that has been genetically marked with a selectable gene. We show that 293 cells express a relatively low level of CD21, that binding of fluorescein-labeled EBV to 293 cells can be detected, and that both the binding of virus to cells and infection can be blocked with antibodies specific for CD21. Two proteins known to form complexes with CD21 on the surface of lymphoid cells, CD35 and CD19, could not be detected at the surface of 293 cells. All infected clones of 293 cells exhibited tight latency with a pattern of gene expression similar to that of type II latency, but productive EBV replication and release of infectious virus could be induced inefficiently by forced expression of the lytic transactivators, R and Z. Low levels of mRNA specific for the transforming membrane protein of EBV, LMP-1, as well as for LMP-2, were detected; however, LMP-1 protein was either undetectable or near the limit of detection at less than 5% of the level typical of EBV-transformed B cells. A slight increase in expression of the receptor for epidermal growth factor, which can be induced in epithelial cells by LMP-1, was detected at the cell surface with two EBV-infected 293 cell clones. These results show that low levels of surface CD21 can support infection of an epithelial cell line by EBV. The results also raise the possibility that in a normal infection of epithelial cells by EBV, the LMP-1 protein is not expressed at levels that are high enough to be oncogenic and that there might be differences in the cells of EBV-associated epithelial cancers that have arisen to allow for elevated expression of LMP-1.     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.     

脂联素siRNA对3T3-L1细胞基础葡萄糖转运的影响(英文)

目的:构建携带小鼠脂联素(Acrp30)siRNA腺病毒载体,并检测其对小鼠脂肪细胞Acrp30表达以及对3T3-L1脂肪细胞基础葡萄糖转运的影响。方法:设计并化学合成小鼠脂肪细胞Acrp30 siRNA片断,将其亚克隆入AdEaxy XL腺病毒载体系统,在293细胞内包装扩增为重组腺病毒。用此重组腺病毒感染3T3-L1脂肪细胞,用RT-PCR和ELISA检测其Acrp30 mRNA和蛋白表达。采用2 Deoxy-[3H]D-glucose掺入法测定脂肪细胞葡萄糖转运。结果:设计并构建了小鼠Acrp30基因特异性siRNA腺病毒载体,该载体感染脂肪细胞后,能显著抑制Acrp30 mRNA和蛋白表达,影响3T3-L1脂肪细胞基础葡萄糖的转运,与对照组相比,差异有显著性意义(P<0.05)。结论:构建的Acrp30基因特异性siRNA腺病毒载体能有效的抑制脂联素在3T3-L1脂肪细胞中的表达,从而影响3T3-L1脂肪细胞基础葡萄糖转运。