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 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.     

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。     

Effects of HNE-modification induced by Aβ on neprilysin expression and activity in SH-SY5Y cells

The cerebral accumulation of β-amyloid (Aβ) is a consistent feature of and likely contributor to the development of Alzheimer's disease. In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism also plays an important role in Aβ accumulation. We have previously shown that neprilysin (NEP), the major protease which cleaves Aβ in vivo , is modified by 4-hydroxy-nonenal (HNE) adducts in the brain of Alzheimer's disease patients. To determine if these changes affected Aβ, SH-SY5Y cells were treated with HNE or Aβ, and then NEP mRNA, protein levels, HNE adducted NEP, NEP activity and secreted Aβ levels were determined. Intracellular NEP developed HNE adducts after 24 h of HNE treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. HNE-modified NEP showed decreased catalytic activity, which was associated with elevations in Aβ1–40 in SH-SY5Y and H4 APP695wt cells. Incubation of cells with Aβ1–42 also induced HNE adduction of NEP. In an apparent compensatory response, Aβ-treated cells showed increased NEP mRNA and protein expression. Despite elevations in NEP protein, the activity was significantly lower compared with the NEP protein level. This study demonstrates that NEP can be inactivated by HNE-adduction, which is associated with, at least partly, reduced Aβ cleavage and enhanced Aβ accumulation.     

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.     

Repression of anti-proliferative factor <Emphasis Type="Italic">Tob1</Emphasis>in osteoarthritic cartilage

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal (n = 9) and osteoarthritic (n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1β and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage (P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression.