PTTG基因siRNA载体的构建与鉴定

目的构建针对垂体腺瘤细胞系AtT20的高效率沉默PTTG基因的shRNA表达载体。方法合成特异性干扰PTTG基因的小发卡RNA(shRNA)片段,并与pGenesil2载体连接,构建PTTG干扰载体(pGenesil2-PTTG shRNA)。利用脂质体将其转染AtT20细胞,分为正常细胞对照组、阴性对照组和3个shRNA干扰组(pGenesil2-PTTG siRNA1、2及3),应用半定量逆转录聚合酶链式反应(RT-PCR)和Western blot法分析转染后AtT20细胞中PTTG的mRNA和蛋白表达水平。结果酶切证实PTTG-shRNA表达载体构建成功;转染效率可达75%左右。转染pGenesil2-PTTG shRNA后,3个干扰组的AtT20细胞中PTTG的mRNA和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PTTG的RNAi表达载体;且pGenesil2-PTTG siRNA高效地抑制了垂体腺瘤细胞AtT20细胞中PTTG綦因的表达。     

Immunogenic issues concerning recombinant adeno-associated virus vectors for gene therapy

Recombinant adeno-associated virus (rAAV) vectors have emerged as highly promising for use in gene transfer for a variety of reasons, including lack of pathogenicity and wide host range. In addition, all virus-encoded genes have been removed from standard rAAV vectors, resulting in their comparatively low intrinsic immunogenicity. For gene replacement strategies, transgenes encoded by rAAV vectors may induce less robust host immune responses than other vectors in vivo. However, under appropriate conditions, host immune responses can be generated against rAAV encoded transgenes, raising the potential for their use in vaccine development. In this review, we have summarized current understanding of the generation of both undesirable and beneficial host immune responses directed against rAAV and encoded transgenes, and how they might be exploited for optimal use of this promising vector system.     

表达胰高血糖素样肽1重组腺伴随病毒载体的构建

背景：胰高血糖素样肽1(glucagon-like peptide-1, GLP-1)在人体内半衰期过短限制了其应用。 目的：构建可表达GLP-1的重组腺伴随病毒。 方法：将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中,构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒。采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系,用其感染Hela细胞。 结果与结论：重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342 bp的目的片段,说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内。免疫细胞化学结果显示,转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒,阳性率达到70%以上,说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1。     

钙转运蛋白基因siRNA表达载体的构建及鉴定

目的：构建针对钙转运蛋白（CaT1）特异性干扰RNA（siRNA）及其表达载体，并检测其对CaT1基因的干扰作用。方法：设计CaT1靶向的发夹状siRNA，合成两条互补的寡核苷酸链，退火后连接人pSilencer3．1-H1载体，转化扩增后进行序列测定。用脂质体转染法转染人类胃癌细胞BGC，通过RT-PCR检测CaT1基因表达水平的变化。结果：酶切鉴定与DNA测序分析证明CaT1基因的siRNA载体构建正确，RT-PCR结果表明CaT1基因的表达水平明显降低。结论：成功地构建了针对CaT1基因的siRNA载体，转染细胞后可抑制CaT1基因表达。     

Inhibition of NF-kappaB mediated inflammation by siRNA expressed by recombinant adeno-associated virus

NF-kappaB mediated inflammation is a key process to many diseases. RNA interference (RNAi) is the specific suppression of genes by short double-stranded RNA. It was the aim of the present study to modify NF-kappaBdependent inflammation by small interfering RNA (siRNA) expressed by recombinant adeno-associated virus (rAAV). To study the kinetics of rAAV mediated expression of siRNA, the expression of the luciferase gene was targeted and resulted in a significant decrease of luciferase activity as compared to a control vector in the human 293 cell line. The effect was dose dependent and was detectable 24 h after infection. rAAV coding for siRNA against the p65 subunit of NF-kappaBsignificantly reduced the p65 protein. In a cellular model of TNF-alpha induced inflammation, expression of siRNA against p65 significantly suppressed the secretion of IL-8 from BEAS-2B cells. In conclusion, rAAV vectors coding for siRNA are an useful tool for efficient gene silencing in mammalian cells and can be used to modify NF-kappaB mediated inflammation.     

Optimization of stealth adeno-associated virus vectors by randomization of immunogenic epitopes

Therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity. Molecular engineering was recently used to identify key immunogenic amino acid residues of the viral capsid and generate mutants with decreased antibody recognition. Here we explored the importance of finely tuning amino acid identity at immunogenic sites to optimize vector phenotype. A capsid library was generated by codon randomization at five positions where substitutions were shown to yield antibody evading phenotypes. Screening this library to isolate immune-escaping mutants allowed an exhaustive scan of combinations of the 20 natural amino acids at each position and yielded variants that remained infectious when incubated with serum or IVIG concentrations that completely neutralize AAV-2. Clones obtained replacing different residues at the same positions displayed strikingly different phenotypes, demonstrating that a precise choice of amino acid substitutions is fundamental to optimize immune-escaping, packaging ability, infectivity and tropism.     

Haplotypes of the alpha-1 antitrypsin gene in healthy controls and Z deficiency patients

Alpha-1 antitrypsin (AAT; HUGO symbol, SERPINA1) is one of the major serine protease inhibitors (serpins) in human plasma. Deficiency of AAT is a recognised risk factor for chronic obstructive pulmonary disease (COPD), attributed to uninhibited neutrophil elastase released into the lung tissue during inflammatory states. In this study we used sequencing to screen the exonic regions, 5' and 3' flanking sequence of the AAT gene in order to generate a high density map of single nucleotide polymorphisms (SNPs). 16 SNPs were identified throughout AAT. Haplotypes based on SNPs with a minor allele frequency of > or =5% were estimated using genotypic information from 225 healthy control individuals and 41 AAT deficient Pi-ZZ individuals. AAT shows a large amount of variation in the control population, with 17 haplotypes accounting for 88% of the observed variation. The haplotype distribution of the common deficiency Pi-Z variant of AAT was significantly different when compared to the normal variants. In addition to the haplotype information, we present evidence for a functional effect of a SNP in intron 1.     

Transduction of human adipose-derived mesenchymal stem cells by recombinant adeno-associated virus vectors

Human adipose-derived stem cells (ASCs) are attractive targets for genetic manipulation and cellular therapies. However, current methods of gene transfer are limited by lack of efficiency, toxicity, or safety concerns. Recombinant adeno-associated virus (rAAV) has been extensively assessed as a gene therapy vector and has an excellent safety profile. This study reports the efficient transduction of well-characterized, homogeneous cultures of human ASCs by rAAV serotypes 2, 5, and 6. Transduction with rAAV2 at high multiplicity of infection was associated with reduced cell viability; however, no adverse effect was seen with serotypes 5 and 6. A further increase in transduction efficiency was observed using a rAAV6 Y731F tyrosine capsid mutant. rAAV-transduced ASCs retained their adipogenic potential. Therefore, rAAV serotypes 2, 5, and 6 should be considered the vectors of choice for genetic manipulation of ASCs.     

Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.     

单纯疱疹病毒1型 US12 基因siRNA筛选及其对病毒增殖的影响

目的: 筛选高效沉默单纯疱疹病毒1型(HSV-1) US12 基因的siRNA,研究siRNA沉默 US12 基因后对HSV-1增殖的影响。方法: 构建pEGFP-N1-US12融合表达质粒,设计并化学合成3对靶向 US12 基因的siRNA,与pEGFP-N1-US12融合表达质粒共转染Vero细胞,流式细胞术筛选高效抑制pEGFP-N1-US12融合蛋白的siRNA,实时荧光定量PCR检测siRNA对细胞内US12 mRNA表达水平的抑制效果,空斑减数实验评价siRNA对HSV-1增殖的抑制效果。结果: 共转染实验筛选出高效抑制pEGFP-N1-US12融合蛋白的siRNAS121、siRNAS122及siRNAS123。3对siRNA均能显著降低感染细胞US12 mRNA的表达水平,但空斑减数实验均未发现3对siRNA对HSV-1的增殖有显著抑制效果。结论: 成功构建pEGFP-N1-US12融合表达质粒,筛选到高效抑制 US12 基因表达的3对siRNA,但是对病毒的体外增殖无显著影响,表明 US12 表达的立即早期蛋白ICP47的功能可能与HSV-1体外增殖无直接相关。     

alpha-1 antitrypsin phenotypes by isoelectric focusing in a metropolitan southern Chinese population

AIMS/BACKGROUND: alpha-1 antitrypsin (alpha1AT) is an abundant protease inhibitor in human plasma. Its phenotypic variability has been reported to be associated with pulmonary emphysema and chronic liver diseases. However, alpha1AT deficiency is an uncommon condition in the Chinese population. The aim of this study was to describe the phenotypic distribution of alpha1AT in a southern Chinese population. METHODS: A total of 1085 healthy blood donors underwent alpha1AT phenotyping by isoelectric focusing. RESULTS: Two thirds (66.1%) were homozygous for either M1 or M2, whereas 32.6% were heterozygous for two different M phenotypes. The frequency of allelic variants was only 0.007, and deficiency variants were absent. Compared with earlier studies on southern Chinese populations, this study found a lower frequency of M2, and a higher number of allelic variants, including E, L, N, P, and S. This phenomenon can be attributed to population migration and mixing. CONCLUSIONS: An understanding of the alpha1AT pattern is important for evaluating the predisposition of the population to selected clinical diseases.     

靶向S1P2基因的siRNA有效片段慢病毒载体的筛选

目的:筛选能显著抑制原代培养的自发性高血压大鼠(spontaneously hypertensive rats,SHR)阴茎海绵体平滑肌细胞内1-磷酸鞘氨醇受体2(sphingosine-1-phosphate receptor 2,S1P2)基因表达的siRNA慢病毒载体。方法:SHR及SD大鼠各5只用于原代培养阴茎海绵体平滑肌细胞并分成6组,每组5个样本,每个样本约1.0×105个细胞,分别为携带靶向S1P2基因的siRNA-1~3号靶点的SHR慢病毒转染组(SHR siRNA-1、SHR siRNA-2和SHR siRNA-3组)、携带慢病毒空载体的SHR转染组(SHR GFP组)、SHR未转染对照组(SHR control组)和SD大鼠对照组(SD control组)。将靶向S1P2的siRNA片段的慢病毒载体,以感染复数(MOI)=60转染各组SHR阴茎海绵体平滑肌细胞,于转染72 h后荧光显微镜下观察细胞荧光表达情况,用Western blot分析各组S1P2、ROCK1、ROCK2和eNOS在阴茎海绵体平滑肌细胞内的表达变化,RT-PCR检测各组阴茎海绵体平滑肌细胞S1P2、ROCK1和ROCK2的mRNA的表达。结果:荧光显微镜下观察SHR siRNA-1、SHR siRNA-2、SHR siRNA-3和SHR GFP组细胞转染效率均80%;SHR GFP组与SHR control组相比,S1P2、ROCK1和ROCK2的mRNA和蛋白表达无明显差异,SHR siRNA-1、SHR siRNA-2、SHR siRNA-3和SD control组均显著低于SHR control组(P0.05)。而SHR siRNA-1、SHR siRNA-2、SHR siRNA-3和SHR GFP组eNOS蛋白表达与SHR control组相比均无显著差别,但SD control组显著高于SHR control组(P0.05)。结论:3组针对S1P2的siRNA慢病毒载体均能显著抑制SHR阴茎海绵体平滑肌细胞内S1P2的表达,下调ROCK1和ROCK2表达,其中靶向S1P2的siRNA-1抑制效率最高。     

CDK1靶向shRNA质粒载体的构建及鉴定

目的:设计并构建CDKI -shRNA质粒表达载体,转染肝癌HepG2细胞,验证shRNA对肝癌HepG2细胞CDK1基因的沉默效应,便于进一步研究CDK1的功能.方法:设计针对CDK1 mRNA靶序列的小发夹状RNA,化学合成含茎环结构的正义链与反义链,退火形成带内切酶粘/平末端的双链后,与酶切后的pSIREN-RetroQ-ZsGreen载体片段进行连接、转化.经测序等方法鉴定重组克隆.将重组载体质粒经脂质体包裹转染肝癌HePG2细胞,realtime PCR及Western blot检测RNA干扰效果.结果:重组克隆经测序证实插入的序列正确,realtime PCR及Western blot检测证实设计的3条RNA干扰序列有效沉默了肝癌HepG2细胞中的内源性CDKI.结论:所制备的CDK1-shRNA在肝癌HepG2细胞中可获得高效转染,并能产生特异性的基因沉默效应,以CDK1为靶向的shRNA能够有效下调CDK1基因的表达,对周期依赖激酶CDK1功能的研究奠定基础.     

Mutation detection in the alpha-1 antitrypsin gene (PI) using denaturing gradient gel electrophoresis

A method for mutation detection in the alpha-1 antitrypsin gene (protease inhibitor 1; PI) has been developed using denaturing gradient gel electrophoresis of PCR amplified gene fragments. Using this experimental approach, all common phenotypes and mutations could be detected. Denaturing gradient gel electrophoresis (DGGE) was compared with standard isoelectric focusing (IEF) in 20 potential alpha1-antitrypsin deficient patients and their relatives. The genotype determined by DGGE was found to be more reliable in some cases than IEF, which is essential for a proper diagnosis of alpha-1 antitrypsin malfunctioning.     

沉默c-myc基因对Jiyoye细胞增殖与凋亡的影响

目的 运用RNA干扰（RNAi）技术,探讨经慢病毒载体介导的小干扰RNA（siRNA）转染Jiyoye细胞对肿瘤细胞增殖及凋亡的影响,以期为靶向c-myc基因的白血病和恶性淋巴瘤基因治疗提供体外实验依据。方法 设计、合成一对针对c-myc基因的c-myc-3寡聚核苷酸链和一对阴性对照c-myc- neg寡聚核苷酸链。退火,然后连接到pLVX干扰载体上,包装,慢病毒介导转染人Jiyoye细胞株。转染后培养72 h,流式细胞术检测各组细胞转染率,pLVX-c-myc-3转染Jiyoye细胞,MTT法检测细胞增殖;Annexin V-异硫氰酸荧光素/碘化吡啶（FITC/PI）双染流式细胞术检测细胞凋亡。结果 ①转染72 h后,流式细胞术鉴定阳性细胞比例,c-myc-neg组与c-myc-3组比较,差异无统计学意义（P 〉 0.05）[（54.3 ± 4.2） % vs（52.8 ± 2.9） %];两组细胞平均荧光强度差异、细胞转染率差异无统计学意义。②转染168 h生长曲线显示,转染pLVX-c-myc实验组细胞生长缓慢,与阴性对照组和空白对照组比较,差异有统计学意义（P 〈 0.05）。转染pLVX-c-myc 72 h后,实验组细胞抑制率为（46.40 ± 1.41） %,阴性对照组细胞抑制率为（17.03 ± 1.32） %,差异均有统计学意义（P 〈 0.05）。③转染pLVX-c-myc 72 h后,实验组早期凋亡细胞比例（25.6 ± 4.2） %,阴性对照组早期凋亡细胞（15.1 ± 4.2） %,空白对照组早期凋亡细胞为（12.7 ± 1.8） %,差异有统计学意义（P 〈 0.05）。实验组晚期凋亡细胞比例（11.5 ± 4.7） %,阴性对照组为（9.3 ± 4.7） %,空白对照组为（8.14 ± 2.8） %,差异有统计学意义（P 〈 0.05）。结论 设计合成构建的干扰序列pLVX-c-myc-3,可沉默c-myc基因,抑制Jiyoye细胞增殖,诱导细胞凋亡,为进一步探讨沉默c-myc基因在白血病和淋巴瘤靶向治疗中的作用提供了实验依据。