High-efficiency promoter-dependent transduction by adeno-associated virus type 6 vectors in mouse lung

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues has been shown to depend heavily on the AAV type from which the vector capsid proteins are derived. Among the AAV types studied, AAV6 efficiently transduces cells of the airway epithelium, making it a good candidate for the treatment of lung diseases such as cystic fibrosis. Here we have evaluated the effects of various promoter sequences on transduction rates and gene expression levels in the lung. Of the strong viral promoters examined, the Rous sarcoma virus (RSV) promoter performed significantly better than a human cytomegalovirus (CMV) promoter in the airway epithelium. However, a hybrid promoter consisting of a CMV enhancer, beta-actin promoter and splice donor, and a beta-globin splice acceptor (CAG promoter) exhibited even higher expression than either of the strong viral promoters alone, showing a 38-fold increase in protein expression over the RSV promoter. In addition, we show that vectors containing either the RSV or CAG promoter expressed well in the nasal and tracheal epithelium. Transduction rates in the 90% range were achieved in many airways with the CAG promoter, showing that with the proper AAV capsid proteins and promoter sequences, efficient transduction can be achieved.     

小鼠骨髓源性树突状细胞培养与冻存

背景:树突状细胞因其良好的抗原提呈功能及促T细胞增殖功能,成为肿瘤抗原的良好载体.获得大量的树突状细胞,对于肿瘤疫苗的研制具有重要作用.目的:对小鼠骨髓树突状细胞进行诱导、培养、扩增及冻存,并对比冻存后复苏细胞与未冻存细胞的细胞特性,寻找一种能够大量获得树突状细胞及有效储存的方法.方法:取小鼠骨髓细胞,在含重组小鼠粒-巨噬细胞集落刺激因子(10 μg/L)和重组小鼠白细胞介素4(5 μg/L)的完全培养基中对其进行诱导、培养及扩增.培养第6天,对培养获得的树突状细胞在加有冷冻保护剂DMSO的完全培养液中冻存.复苏后,在培养基中加入脂多糖、对细胞诱导,获得大量的成熟树突状细胞,最终获得的树突状细胞与未进行冻存的细胞在细胞活力、形态学、细胞表型、混合淋巴细胞反应等方面对比.结果与结论:复苏后,(82.2±4.73)%细胞存活,存活的细胞经脂多糖诱导后发育为成熟树突状细胞,在形态学、细胞表型及混合淋巴细胞反应等方面与未经冻存的树突状细胞差异无显著性意义.提示小鼠骨髓源性树突状细胞冻存复苏后,细胞生物学特性与未冻存的细胞无明显差别,用冻存的方法储存树突状细胞,能够避免在不同时期应用细胞时反复进行培养,可以获得大量的同质细胞.     

Genomic stability of self-complementary adeno-associated virus 2 during early stages of transduction in mouse muscle in vivo

Studies have demonstrated that packaging of recombinant adeno-associated virus 2 (rAAV) as self-complementary duplex strand (sc) results in early transgene expression, possibly eliminating rate-limiting second-strand synthesis. In the present study, we evaluated the molecular organization, stability of the sc AAV genome, and transgene expression in the quadriceps muscle of C57BL/6J mice in vivo as compared with single-stranded (ss) AAV. Studies were carried out with rAAV encoding green fluorescent protein (GFP) or human carcinoembryonic antigen (CEA) either as single-stranded or self-complementary duplex strand structures, encapsidated in AAV-2 capsids. Mice were injected with 10(11) particles of the respective viruses and the vector-injected muscles were harvested 1 week, 2 weeks, 3 weeks, or 2 months later. Tissues were processed for total DNA isolation for the analyses of vector genomic configuration and copy number, and for immunostaining of transgene expression. ELISA was done on serum samples to quantitate CEA-specific humoral immune response as a correlate of transgene expression. Results of Southern blot and PCR analyses indicated more disintegration of the monomeric ss AAV DNA in vivo compared with linear sc AAV DNA. The results also indicated efficient conversion of the self-complementary duplex-stranded vector genome to dimer during early time points. As expected, transgene expression was detected at early time points with self-complementary duplex-stranded vector and persisted stably. However, the advantage of higher transgene expression from sc AAV was balanced over time by the single-stranded vector. These data demonstrate that sc AAV provides better stability for transgene structure during the initial stages of transduction and may have better utility in AAV gene therapy in situations, which mandate early transgene expression.     

小鼠骨髓树突状细胞的培养扩增及生物学特性分析

背景:树突状细胞是目前已知功能最强的专职性抗原递呈细胞,但树突状细胞在体内分布广泛且数量较少,获得大量的树突状细胞是研究树突状细胞特性和功能的前提。目的:建立小鼠骨髓源树突状细胞体外培养和扩增方法,观察其形态和相关特征。设计、时间及地点:树突状细胞形态学观察实验,于2006-08/2007-05在中山大学中山眼科中心实验室完成。材料:健康雌性C57BL/6小鼠。方法:在无菌条件下提取C57BL/6鼠骨髓细胞,分离单个核细胞,以小鼠重组粒细胞-巨噬细胞集落刺激因子和白细胞介素4协同诱导下培养,加用磷酸脂多糖刺激,分成磷酸酯多糖-树突状细胞和单纯树突状细胞组,前者在培养加入磷酸酯多糖,后者不加。主要观察指标:应用光镜和电镜下观察树突状细胞的形态,流色细胞仪检测鉴定其生物学特征。结果:体外培养2周后具有典型的树突状细胞形态,光镜下显示细胞表面不规则,呈树突状突起,电镜下可见培养的细胞具有典型树突状细胞的形态特征,流式细胞鉴定为髓系树突状细胞,高表达MHCII类分子及共刺激分子(MHCII,CD80,CD83,CD11c)。单纯树突状细胞组的细胞中度表达MHCⅡ类分子,低水平表达共刺激分子,刺激T细胞增殖的能力较弱;...     

Assessment of ocular transduction using single-stranded and self-complementary recombinant adeno-associated virus serotype 2/8

To date adeno-associated viral (AAV) vectors are the only gene therapy vectors that have been shown to efficiently transduce photoreceptor cells and have thus become the most commonly used vector for ocular transduction. Various AAV serotypes have been evaluated in the eye, the first of which was AAV2, which is able to transduce photoreceptors, retinal pigment epithelium (RPE) and retinal ganglion cells. AAV serotypes 1 and 4, as well as AAV2 pseudotyped with these capsids, only transduce the RPE. AAV serotype 5 and AAV2/5 transduce the photoreceptors as well as RPE, but not retinal ganglion cells. Here, we assessed the capacity of the novel serotype AAV2/8 to transduce various ocular tissues of the adult murine retina by administering AAV2/8 green fluorescent protein intravitreally, subretinally and intracamerally. We also determined the kinetics and efficiency of self-complementary AAV (scAAV) vectors of serotypes 2/2, 2/5 and 2/8 and compared them with single-stranded AAV (ssAAV). We found that ssAAV2/8 transduces photoreceptors and RPE more efficiently than ssAAV2/2 and ssAAV2/5, and that scAAV2/8 had faster onset and higher transgene expression than ssAAV2/8. This improved transduction efficiency might facilitate the development of improved gene therapy protocols for inherited retinal degenerations, particularly those caused by defects in photoreceptor-specific genes.     

Differential internalization and nuclear uncoating of self-complementary adeno-associated virus pseudotype vectors as determinants of cardiac cell transduction

Recently it was shown that several new pseudotyped adeno-associated virus (AAV) vectors support cardioselective expression of transgenes. The molecular mechanisms underlying this propensity for cardiac cell transduction are not well understood. We comparatively analyzed AAV vector attachment, internalization, intracellular trafficking, and nuclear uncoating of recombinant self-complementary (sc) AAV2.2 versus pseudotyped scAAV2.6 vectors expressing green fluorescence protein (GFP) in cells of cardiac origin. In cardiac-derived HL-1 cells and primary neonatal rat cardiomyocytes (PNCMs), expression of GFP increased rapidly after incubation with scAAV2.6-GFP, but remained low after scAAV2.2-GFP. Internalization of scAAV2.6-GFP was more efficient than that of scAAV2.2-GFP. Nuclear translocation was similarly efficient for both, but differential nuclear uncoating rates emerged as a key additional determinant of transduction: 30% of all scAAV2.6-GFP genomes translocated to the nucleus became uncoated within 48 h, but only 16% of scAAV2.2-GFP genomes. In contrast to this situation in cells of cardiac origin, scAAV2.2-GFP displayed more efficient internalization and similar (tumor cell line HeLa) or higher (human microvascular endothelial cell (HMEC)) uncoating rates than scAAV.2.6-GFP in non-cardiac cell types. In summary, both internalization and nuclear uncoating are key determinants of cardiac transduction by scAAV2.6 vectors. Any in vitro screening for the AAV pseudotype most suitable for cardiac gene therapy - which is desirable since it may allow significant reductions in vector load in upcoming clinical trials--needs to quantitate both key steps in transduction.     

重组9型腺相关病毒转染小鼠骨髓间充质干细胞

背景：重组9型腺相关病毒对心肌细胞具有良好的亲和力,是目前研究基因治疗心肌梗死的理想载体。目的：观察携带增强型绿色荧光蛋白基因的重组9型腺相关病毒（r AAV9-e GFP）对小鼠骨髓间充质干细胞的转染效率。方法：将携带增强型绿色荧光蛋白基因的重组9型腺相关病毒以不同感染复数（1×105,1×106,1×107）转染体外培养的第4代小鼠骨髓间充质干细胞,并在转染后1-7 d连续用荧光倒置显微镜观察骨髓间充质干细胞中增强型绿色荧光蛋白阳性表达情况,寻找最佳感染条件;采用流式细胞仪检测最佳感染复数下携带增强型绿色荧光蛋白基因的重组9型腺相关病毒对小鼠骨髓间充质干细胞的转染效率。结果与结论：转染后第1天,感染复数为1×107组可见增强型绿色荧光蛋白开始表达,转染后第2天,感染复数为1×105、1×106组开始表达;各组增强型绿色荧光蛋白表达强度随感染复数值的增高而增强,同时增强型绿色荧光蛋白的表达强度随着时间延长而逐渐增强;转染后第5天达到高峰,此时感染复数为1×107组转染效率约8%,结果表明携带增强型绿色荧光蛋白基因的重组9型腺相关病毒对小鼠骨髓间充质干细胞转染效率较低。     

小鼠骨髓来源树突状细胞吲哚胺2,3-过氧化酶表达对T细胞增殖的影响

目的:诱导树突状细胞产生吲哚胺2,3-过氧化酶,从而调节T细胞反应,可能是诱导器官移植免疫耐受的理想途径.观察小鼠骨髓来源的树突状细胞本身及其在多种因素刺激活化状态下吲哚胺2,3-过氧化酶的表达变化,分析其对T细胞增殖的影响.方法:实验于2005-01/08在解放军第二军医大学免疫研究所完成,动物实验方法符合动物伦理学要求.①培养C57BL/6小鼠骨髓来源的树突状细胞,并分别利用脂多糖80 μg/L、CD40L500 μg/L、γ-干扰素50 μg/L及三者联合作用于培养7 d的上述细胞,利用反转录聚合酶链反应检测各组树突状细胞中吲哚胺2,3-过氧化酶mRNA的表达.②培养BALB/C小鼠脾脏来源的T淋巴细胞做为静息T淋巴细胞,并采用抗CD3单抗及抗CD28单抗各1 mg/L刺激T淋巴细胞作为活化T淋巴细胞.将树突状细胞及γ–干扰素作用下的树突状细胞与静息T淋巴细胞及活化T淋巴细胞共同培养,利用混合淋巴细胞反应法测定其刺激T细胞增殖的能力.结果:①培养7 d的树突状细胞及在脂多糖、CD40L刺激下的树突状细胞均未见吲哚胺2,3-过氧化酶mRNA的表达,而γ–干扰素及γ–干扰素 脂多糖 CD40L三者联合刺激下的树突状细胞可检测到吲哚胺2,3-过氧化酶 mRNA的表达.②与树突状细胞相比,γ–干扰素活化的树突状细胞可明显抑制T细胞增殖(P < 0.01),加入色氨酸可使对T细胞增殖的抑制作用有所恢复,同树突状细胞比较差异亦具有显著性意义(P < 0.01).结论:活化的树突状细胞可以产生吲哚胺2,3-过氧化酶,通过降解色氨酸发挥抑制T细胞增殖的作用,可能在移植排斥反应中发挥效应.     

慢病毒介导可溶性肿瘤坏死因子受体1在小鼠骨髓未成熟树突状细胞中的表达(英文)

背景:肿瘤坏死因子α是介导树突状细胞成熟的重要细胞因子之一,可溶性肿瘤坏死因子受体1与其结合可阻断肿瘤坏死因子α的作用,维持树突状细胞于不成熟状态,诱导免疫耐受.目的:构建含有人sTNFR1的慢病毒表达载体,观察其在未成熟树突状细胞中的表达.方法:以人外周血单个核细胞总RNA为模板,RT-PCR扩增出sTNFR1基因片段,亚克隆至慢病毒转移质粒pXZ208,通过IRES连接eGFP报告基因,建立双顺反子慢病毒转移质粒,命名为pXZ9-sTNFR1,DNA测序鉴定.采用脂质体转染293 FT细胞,根据报告基因eGFP测定病毒滴度.采用小剂量粒-巨噬细胞集落刺激因子+白细胞介素4体外培养扩增C57BL/6小鼠骨髓来源树突状细胞.培养第5天,以pXZ9-sTNFRl重组慢病毒上清感染未成熟树突状细胞,RT-PCR检测感染后sTNFRl转录,Westernblot法检测sTNFR1蛋白表达,观察sTNFR1基因修饰及脂多糖刺激后树突状细胞的表型特征.结果与结论:成功构建重组质粒pXZ9-sTNFR1,转染293 FT细胞24 h后观察到eGFP表达,病毒滴度在10~6U/L以上.RT-PCR显示pXZ9-sTNFR1感染的未成熟树突状细胞sTNFR1呈阳性表达,Western blot检测到sTNFR1蛋白存在于感染后未成熟树突状细胞和培养上清中.培养第5天的树突状细胞低表达CD40、CD86、CD80和MHCⅡ类分子,脂多糖刺激后,高表达MHCⅡ类分子和CD40、CD80、CD86分子,显示出成熟型树突状细胞表型特征,sTNFR修饰的树突状细胞MHC Ⅱ类分子和CD40、CD80、CD86分子表达水平无变化.提示:①成功构建了负载sTNFR1基因片段及含eGFP报告基因的慢病毒载体,获得了高滴度的重组慢病毒颗粒.②经慢病毒高效转导的未成熟树突状细胞sTNFR1 mRNA及蛋白稳定地表达,可以保护未成熟树突状细胞不被外源性脂多糖刺激活化,维持树突状细胞于非成熟状态.     

Recombinant self-complementary adeno-associated virus serotype vector-mediated hematopoietic stem cell transduction and lineage-restricted, long-term transgene expression in a murine serial bone marrow transplantation model

Although conventional recombinant single-stranded adeno-associated virus serotype 2 (ssAAV2) vectors have been shown to efficiently transduce numerous cells and tissues such as brain and muscle, their ability to transduce primary hematopoietic stem cells (HSCs) has been reported to be controversial. We have previously documented that among the ssAAV serotype 1 through 5 vectors, ssAAV1 vectors are more efficient in transducing primary murine HSCs, but that viral second-strand DNA synthesis continues to be a rate-limiting step. In the present studies, we evaluated the transduction efficiency of several novel serotype vectors (AAV1, AAV7, AAV8, and AAV10) and documented efficient transduction of HSCs in a murine serial bone marrow transplantation model. Self-complementary AAV (scAAV) vectors were found to be more efficient than ssAAV vectors, and the use of hematopoietic cell-specific enhancers/promoters, such as the human beta-globin gene DNase I-hypersensitive site 2 enhancer and promoter (HS2-betap) from the beta-globin locus control region (LCR), and the human parvovirus B19 promoter at map unit 6 (B19p6), allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. The proviral AAV genomes were stably integrated into progenitor cell chromosomal DNA, and did not lead to any overt hematological abnormalities in mice. These studies demonstrate the feasibility of the use of novel scAAV vectors for achieving high-efficiency transduction of HSCs as well as erythroid lineage-restricted expression of a therapeutic gene for the potential gene therapy of beta-thalassemia and sickle cell disease.     

Delivery of MDR1 small interfering RNA by self-complementary recombinant adeno-associated virus vector.

Small interfering RNAs (siRNAs) are potentially powerful tools for therapeutic gene regulation. DNA cassettes encoding RNA polymerase III promoter-driven hairpin siRNAs allow long-term expression of siRNA in targeted cells. A variety of viral vectors have been used to deliver such cassettes to cells. Here we report on the development and use of a self-complementary recombinant adeno-associated virus (scAAV) vector for siRNA delivery into mammalian cells. We demonstrate that this modified vector efficiently delivers siRNA into multidrug-resistant human breast and oral cancer cells and suppresses MDR1 gene expression. This results in rapid, profound, and durable reduction in the expression of the P-glycoprotein multidrug transporter and a substantial reversion of the drug-resistant phenotype. This research suggests that scAAV-based vectors can be very effective agents for efficient delivery of therapeutic siRNA.     

冻融肺癌细胞对骨髓树突状细胞的免疫调节作用

背景:冷冻免疫是近年来倍受关注的课题,但冷冻对细胞免疫功能的影响一直缺乏深入的研究,特别是在细胞凋亡和树突状细胞方面.目的:观察体外培养的肺癌NCI-H446细胞经氩氦刀冻融处理后细胞形态和细胞表面免疫表型的变化,及其能否有效激发骨髓树突状细胞产生特异性抗瘤效应.设计、时间及地点:细胞免疫水平的对照实验,于2002-08/2003 04在解放军海军总医院血液实验室及解放军军事医学科学院细胞与基因治疗中心完成.材料:小细胞肺癌细胞系NCI-H446细胞购自上海科学院细胞库.方法:取体外培养的肺痛NCI-H446细胞经氩氦刀处理后,在体外树突状细胞培养过程中,加入冻融的肺癌细胞,分别观察肺癌细胞的形态结构、混合淋巴细胞反应及细胞毒T淋巴细胞杀癌细胞效应.主要观察指标:培养树突状细胞的细胞形态和细胞表面免疫表型变化,培养细胞的混合淋巴细胞反应结果,培养细胞的凋亡情况.结果:骨髓树突状细胞体外培养后,符合树突状细胞特征.在培养过程中加入冻融的肺癌细胞,不影响树突状细胞细胞表面抗原的表达,混合淋巴细胞反应增强,细胞毒T淋巴细胞可引起肺癌细胞凋亡.结论:经氩氦刀处理的肺痛细胞溶解,胞膜不完整,体外能有效激发骨髓树突状细胞产生特异性抗瘤效应.     

Light-activated gene transduction of recombinant adeno-associated virus in human mesenchymal stem cells

Deficiencies in skeletal tissue repair and regeneration lead to conditions like osteoarthritis, osteoporosis and degenerative disc disease. While no cure for these conditions is available, the use of human bone marrow derived-mesenchymal stem cells (HuMSCs) has been shown to have potential for cell-based therapy. Furthermore, recombinant adeno-associated viruses (rAAV) could be used together with HuMSCs for in vivo or ex vivo gene therapy. Unfortunately, the poor transduction efficiency of these cells remains a significant obstacle. Here, we describe the properties of ultraviolet (UV) light-activated gene transduction (LAGT) with rAAV in HuMSCs, an advance toward overcoming this limitation. Using direct fluorescent image analysis and real-time quantitative PCR to evaluate enhanced green fluorescent protein (eGFP) gene expression, we found that the optimal effects of LAGT with limited cytotoxicity occurred at a UV dose of 200 J/m(2). Furthermore, this UV irradiation had no effect on either the chondrogenic or osteogenic potential of HuMSCs. Significant effects of LAGT in HuMSCs could be detected as early as 12 h after exposure and persisted over 21 days, in a time and energy-dependent manner. This LAGT effect was maintained for more than 8 h after irradiation and required only a 10-min exposure to rAAV after UV irradiation. Finally, we show that the production of secreted TGFbeta1 protein from rAAV-TGFbeta1-IRES-eGFP infected to HuMSCs is highly inducible by UV irradiation. These results demonstrate that LAGT combined with rAAV is a promising procedure to facilitate gene induction in HuMSCs for human gene therapy.     

Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells

Synthetic oligodeoxynucleotides (ODNs) had been employed in gene modification and represent an alternative approach to 'cure' genetic disorders caused by mutations. To test the ability of ODN-mediated gene repair in bone marrow-derived mesenchymal stem cells (MSCs), we established MSCs cell lines with stably integrated mutant neomycin resistance and enhanced green fluorescent protein reporter genes. The established cultures showed morphologically homogenous population with phenotypic and functional features of mesenchymal progenitors. Transfection with gene-specific ODNs successfully repaired targeted cells resulting in the expression of functional proteins at relatively high frequency approaching 0.2%. Direct DNA sequencing confirmed that phenotype change resulted from the designated nucleotide correction at the target site. The position of the mismatch-forming nucleotide was shown to be important structural feature for ODN repair activity. The genetically corrected MSCs were healthy and maintained an undifferentiated state. Furthermore, the genetically modified MSCs were able to engraft into many tissues of unconditioned transgenic mice making them an attractive therapeutic tool in a wide range of clinical applications.     

Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human alpha1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding beta-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.     

Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6.

Vectors based on recombinant adeno-associated viruses (rAAV) have emerged as tools of choice for gene transfer to skeletal muscle. rAAV vectors demonstrate efficient, safe, and stable transduction. Multiple serotypes of AAV exist, but vectors based on serotype 2 (rAAV2) are the most thoroughly characterized and frequently employed. Here, we characterize transduction of the skeletal musculature using rAAV vectors pseudotyped with serotype 6 capsid proteins (rAAV6). We demonstrate that rAAV6 vectors can efficiently transduce the skeletal musculature of mice at levels >500-fold higher than is achievable with rAAV2 vectors and can readily saturate individual muscles following direct injection. Further, rAAV6 vectors are capable of transducing the diaphragm and intercostal muscles of mice after a simple injection into the intrathoracic cavity and are capable of widespread transduction throughout the musculature of mice injected in the intraperitoneal space as newborn pups. These results demonstrate that rAAV6 vectors hold great potential for use in gene delivery protocols targeting the skeletal musculature.     

含癌胚抗原片段的重组腺相关病毒转染树突状细胞疫苗的体外杀伤作用

目的：观察研究人外周血单核细胞来源的树突状细胞（DC）转染含癌胚抗原（CEA）片段的重组腺相关病毒后所诱导的特异性T细胞对直肠癌细胞株LOVO和SW480的体外杀伤作用。方法：抽取HLA表型为A11的健康志愿者外周血，分离单核细胞，体外培养，使用含CEA片断的重组腺相关病毒转染未成熟DC，诱导特异性T细胞。检测体外培养的DC和CTL活性，并使用MTT法检测细胞毒性T细胞（CTL）对LOVO细胞的杀伤作用。结果：转染或未转染的体外培养的成熟DC高表达CD40、CD86、IL-12，诱导的细胞毒性T细胞高表达IFN-γ；转染后DC诱导特异性细胞毒性T细胞可有效识别并杀伤HLA-A11阳性的LOVO细胞。结论：重组腺相关病毒转染DC，不明显改变DC表型和刺激淋巴细胞增殖、分化功能，可诱导自体细胞毒性T细胞增殖，含CEA片断的腺相关病毒转染DC诱导自体细胞毒性T细胞对LOVO细胞有明显杀伤作用，DC疫苗可以作为直肠癌患者免疫治疗的有效补充。     

Antigen-independent induction of histamine synthesis by immunoglobulin E in mouse bone marrow-derived mast cells

Immunoglobulin (Ig)E-mediated activation of mast cells has long been thought to occur only when Fc(epsilon)RI receptor-bound IgE is cross-linked via multivalent antigens. However, recent studies have raised the possibility that mast cells may be activated by the binding of IgE to the Fc(epsilon)RI receptor in the absence of antigen. Here we demonstrate that IgE binding without antigen induces the expression of histidine decarboxylase (HDC) in mouse interleukin (IL)-3-dependent bone marrow-derived mast cells (BMMCs). The induction of HDC by the binding of IgE was found to require an influx of extracellular calcium ions, which was attenuated by pretreatment with U73122, a phospholipase C inhibitor. Furthermore, the increase in HDC activity upon sensitization with IgE was completely suppressed by pretreatment of BMMCs with protein kinase C inhibitors, such as H7, staurosporine, and G?6976. In addition, immediate activation of the tyrosine kinase Lyn was not detectable upon treatment with IgE. These results suggest that the binding of IgE to its receptor in the absence of antigen results in de novo synthesis of HDC in BMMCs through a signaling pathway distinct to that operating during antigen-stimulated Fc(epsilon)RI activation.     

RelB shRNA慢病毒感染小鼠骨髓树突状细胞的免疫学效应

背景:沉默树突状细胞发育成熟的关键基因核因子кB/RelB可构建新型致耐受树突状细胞? 目的:探讨RelB shRNA转染小鼠骨髓树突细胞的生物免疫学功能的影响.方法:利用重组粒细胞-巨噬细胞集落刺激因子和重组白细胞介素4联合诱导小鼠骨髓树突状细胞;慢病毒载体将RelB shRNA转染致小鼠骨髓树突细胞后,分为未成熟树突状细胞、脂多糖刺激成熟、RelB基因沉默及脂多糖刺激RelB沉默的4组树突状细胞进行观察.结果:体外培养第 6 天脂多糖刺激组细胞表面可见大量细长的类似树枝的突起,其他3组细胞形态特征相似,呈圆形、皱缩状态,这3组细胞表面MHC-II类分子、CD86和CD40分子表达水平相当,但低于脂多糖刺激组;3组混合淋巴细胞反应中刺激T细胞增殖能力差异无显著性意义(P > 0.05),但均较脂多糖刺激组显著降低(P < 0.01);RelB基因沉默的树突状细胞分泌Th1细胞因子γ-干扰素和白细胞介素2的能力较低,分泌Th2白细胞介素10和白细胞介素4的能力较高(P < 0.01),Th1/Th2细胞因子的比例与未成熟树突状细胞类似.说明RelB shRNA经慢病毒转染骨髓源性树突状细胞后,在细胞形态、表面分子表达、免疫学功能等方面均具有与未成熟树突状细胞相似的特点,且不能被脂多糖刺激成熟.