共查询到20条相似文献，搜索用时 31 毫秒
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Quesada-Pascual F Jimenez-Flores R Flores-Langarica A Silva-Sanchez A Calderon-Amador J Mendez-Cruz R Limon-Flores AY Estrada-Parra S Santos-Argumedo L Estrada-Garcia I Flores-Romo L 《Veterinary immunology and immunopathology》2008,124(3-4):220-229
Armadillos are apparently important reservoirs of Mycobacterium leprae and an animal model for human leprosy, whose immune system has been poorly studied. We aimed at characterizing the armadillo's langerhans cells (LC) using epidermal sheets instead of tissue sections, since the latter restrict analysis only to cut-traversed cells. Epidermal sheets by providing an en face view, are particularly convenient to evaluate dendritic morphology (cells are complete), spatial distribution (regular vs. clustered), and frequency (cell number/tissue area). Lack of anti-armadillo antibodies was overcome using LC-restricted ATPase staining, allowing assessment of cell frequency, cell size, and dendrites extension. Average LC frequency in four animals was 528 LC/mm(2), showing a rather uniform non-clustered distribution, which increased towards the animal's head, while cell size increased towards the tail; without overt differences between sexes. The screening of antibodies to human DC (MHC-II, CD 1a, langerin, CD86) in armadillo epidermal sheets, revealed positive cells with prominent dendritic morphology only with MHC-II and CD86. This allowed us to test DC mobilization from epidermis into dermis under topical oxazolone stimulation, a finding that was corroborated using whole skin conventional sections. We hope that the characterization of armadillo's LC will incite studies of leprosy and immunity in this animal model. 相似文献
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Gene silencing with short interfering RNA (siRNA) expression vectors is a powerful method for the analysis of gene functions. For the expression of siRNA in mammalian cells, mammalian U6 small nuclear RNA (snRNA) promoters are widely used. However, the mammalian U6 promoter might not function well in other species. In this study, we cloned four putative chicken U6 promoters by PCR and analyzed their functions. First, we screened the chicken genomic database using the human U6 snRNA gene and identified four candidate sequences. The sequences contained some control elements in their promoter regions, but as we could not rule out that they were pseudogenes, we amplified these sequences and used them as promoters for short hairpin RNA (shRNA) expression. Using the firefly luciferase (Luc) gene as a target, transient expression assays were performed with chicken ovary-derived cells. All four putative chicken U6 promoters exhibited suppressive activity toward Luc, and so could act as a promoter for expression of the snRNA gene in the chicken genome. The promoter activity was not as strong as that of a commercially available siRNA expression vector. This probably reflects artificial sequences between the promoters and synthetic DNA encoding shRNA. 相似文献
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Yoon S Yoo HJ Shim NR Baek SY Kim BS Kim JB Jun EJ Son YK Lee SY Yoo YH 《Anatomia, histologia, embryologia》2003,32(2):80-88
This study was undertaken to investigate the immunohistochemical characterization of different subpopulations of macrophages and dendritic cells (DCs) of the spleen, thymus, tongue and heart in cyclophosphamide (CY)-induced immunosuppressed rat. After CY treatment, remarkably, ED1+, ED2+ and ED3+ macrophage subpopulations, in general exhibited signs of cellular activation such as an increase in number and size of cell, and an upregulation of the ED1, ED2 and ED3 reactive surface molecule expression in all the organs studied, except for some macrophage subpopulations including ED1+ macrophages in the non-lymphoid tissues. Subpopulations of DCs showed a differential sensitivity to CY. Lymphoid DCs were more sensitive to CY than non-lymphoid interstitial DCs. CY induced a conspicuous upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in the vascular endothelial cells, splenic marginal zone and thymic cortex. In this study, we demonstrated the in vivo effects of CY treatment on subpopulations of macrophages and DCs as well as on ICAM-1 expression in the rat spleen, thymus, tongue and heart. Moreover, our results shed more light on the activation effects of CY on certain subpopulations of macrophages, on the differential sensitivity of DCs to CY between the immature and mature ones, on the functional role of different subpopulations of macrophages, and on the significance of upregulated ICAM-1 expression in the splenic marginal zone and thymic cortex after CY treatment. 相似文献
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甘露聚糖结合凝集素C(mannose binding lectin C,MBL-C)是C型(Ca2+依赖型)凝集素超家族的成员,其作为一种急性期蛋白,具有抗细菌感染的功能,参与机体的天然免疫反应。为鉴定出结合在MBL2基因启动子区(1 009 bp)的重要转录因子,探寻该基因的转录调控机制,本研究选取海南黑山羊MBL2基因的启动子序列1 009 bp,采用DNA重组技术克隆6个转录起始位点上游1 009 bp的启动子5'端侧翼缺失序列,克隆片段经双酶切后连接至pGL3-Basic载体。重组质粒转染至293T细胞中,结合双荧光素酶活性检测系统筛选MBL2基因的核心启动子区域。通过在线生物信息学软件预测山羊MBL2基因的核心启动子区域的转录因子结合位点,利用点突变技术构建转录因子结合位点缺失的载体,转染293T细胞后结合双荧光素酶活性检测系统分析其转录活性。结果表明,海南黑山羊MBL2基因的核心启动子区域位于转录起始位点上游-304~-45 bp范围内,在线软件分析该区域存在RELA、NF-κB2、MZF1等3种转录因子结合位点。双荧光素酶报告分析结果表明,RELA和NF-κB2的结合位点缺失后均使山羊MBL2基因的转录活性极显著下降(P < 0.01)。结果提示,RELA和NF-κB2对山羊MBL2基因的转录活性可能具有重要的正调控作用。该研究为进一步探寻海南黑山羊MBL2基因的功能提供理论依据。 相似文献
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K Tanabe W Hiraoka M Kuwabara F Sato T Narita M Niikura 《The Japanese journal of veterinary research》1989,37(3-4):181-185
The cytotoxic effects of acyclovir, which is a purine nucleoside analogue and is known as an antibiotic substance, were examined on three lines of rat skin fibroblast FR cells; normal FR cells, FRtk- cells which are deficient in the activity of thymidine kinase (tk) and FRtk-HSVtk+ cells which were prepared by introducing herpes simplex virus' tk gene to FRtk- cells. When FRtk-HSVtk+ cells growing exponentially were incubated in the presence of acyclovir for 4 h, the surviving fractions of the cells decreased in a concentration-dependent manner. Whereas, decrease of the surviving fractions was almost indiscernible in both FR cells and FRtk- cells at the whole ranges of drug-concentrations tested. These results indicate that acyclovir is phosphorylated by the herpes simplex virus' tk and becomes toxic to FRtk-HSVtk+ cells. This also means that FRtk-HSVtk+ cells are useful for the investigation of the biological activity of nucleoside analogues. 相似文献
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【目的】 鉴定绵羊趋化因子C-C基序配体19(C-C motif chemokine ligand 19,CCL19)基因启动子的核心启动子区域和关键转录因子,探究该基因在转录调控方面的作用机制。【方法】 选取绵羊CCL19基因5'-侧翼序列1 000 bp,PCR扩增启动子的7个不同长度的截短片段,并连接至pGL3-Basic质粒;将重组质粒与pRL-TK质粒共转染到293T细胞中,结合双荧光素酶报告基因检测系统分析不同截短片段的相对荧光活性。利用在线预测软件分析和筛选CCL19基因核心启动子区域内的转录因子结合位点。采用定点突变技术构建转录因子结合位点缺失的荧光素酶报告载体,与pRL-TK质粒共转染到293T细胞,分析转录因子结合位点缺失质粒的相对荧光活性。【结果】 成功构建了7个不同长度(pGL3-P、pGL3-P1、pGL3-P2、pGL3-P3、pGL3-P4、pGL3-P5及pGL3-P6)的CCL19基因启动子片段的荧光素酶报告载体;采用双荧光素酶报告基因检测系统鉴定出转录起始位点上游－256/－186 bp为CCL19基因启动子核心启动子区域,表明该区域对CCL19基因转录调控有重要作用。生物信息学分析预测到该区域存在POU5F1(-201/-189 bp)、ZBTB26(-228/-217 bp)、FOXI1(-239/-228 bp)、GLI2(-255/-243 bp)和SP2(-219/-211 bp) 5个转录因子的结合位点,并成功构建了转录因子结合位点缺失的荧光素酶报告载体。双荧光素酶报告基因检测系统分析显示,POU5F1转录因子的结合位点缺失后绵羊CCL19基因转录活性极显著降低(P<0.01),FOXI1、ZBTB26、SP2转录因子结合位点缺失后绵羊CCL19基因转录活性均极显著升高(P<0.01)。【结论】 试验成功构建CCL19基因启动子荧光素酶报告载体,确定CCL19基因启动子的核心启动子区域为转录起始位点上游－256/－186 bp,并鉴定出转录因子POU5F1结合位点可能是CCL19基因转录的重要调控位点,为下一步研究绵羊CCL19基因在先天性免疫、适应性免疫和淋巴细胞迁移等方面的功能提供理论基础。 相似文献
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Winnicka A Klucinski W Kawiak J Hoser G Sikora J 《Journal of veterinary medicine. A, Physiology, pathology, clinical medicine》2000,47(7):385-394
The aim of this study was to determine the effect of Baypamun on selected lymphocyte subpopulations and granulocyte phagocytic activity mediated by lectin-like receptors in goats of normal immune status and in goats experimentally immunosuppressed with dexamethasone. Eighteen goats in total were used. Blood samples were collected 24 h before immunomodulation and 5 and 10 days after immunomodulation. Animals in group I were immunostimulated with Baypamun for 2 days and immunosuppressed with Dexafort for the next 2 days. Animals in group II received Dexafort first and then Baypamun. The number of leucocytes in total and in subpopulations was determined by flow cytometry. Application of Baypamun before (group I) or after (group II) immunosuppression caused significant (P < 0.001) and lasting changes in the percentage of CD2+, CD4+ and CD8+ T cells. Significant but transient changes were observed in CD19+ (B) and WC1-N2+ (null) cells. Results show that application of Baypamun to modulate non-specific defence is advisable following immunosuppression (group I). When Baypamun was applied after immunosuppression (group II), although there was no reaction during the initial phase, an increase in the activity of cells responsible for non-specific immunity was noticeable after 10 days. 相似文献
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为探讨催乳激素对体外培养的山羊乳腺上皮细胞脂肪酸合酶（fatty acid synthase，FASN）基因转录活性的调控作用，试验分别用不同浓度的胰岛素（insulin，INS）、雌激素（estradiol，E2）、催乳素（prolactin，PRL）及不同激素组合（INS+PRL、E2+INS+PRL）处理山羊乳腺上皮细胞24 h，提取细胞总RNA，采用实时荧光定量PCR检测催乳激素对FASN基因mRNA表达水平的影响。细胞转染山羊FASN基因启动子报告基因载体，同样用不同浓度的胰岛素、雌激素、催乳素及激素组合处理24 h，利用双荧光素酶报告基因系统检测催乳激素对FASN基因启动子活性的影响。结果发现，用雌激素、催乳素处理山羊乳腺上皮细胞后，FASN基因启动子活性及mRNA水平极显著或显著上调（P<0.01；P<0.05），雌激素浓度为10、100 μmol/L和催乳素浓度为0.1和1 μg/mL时效果最为明显，而胰岛素对FASN基因的启动子活性及mRNA水平没有显著影响（P>0.05）。用不同激素组合（INS+PRL、E2+INS+PRL）处理细胞均能显著上调FASN基因启动子活性及mRNA表达水平（P<0.05）。结果表明，雌激素和催乳素能够调控FASN基因的转录活性，为进一步研究泌乳过程中FASN基因的分子调控机制提供理论依据。 相似文献
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Robbin MG Wagner B Noronha LE Antczak DF de Mestre AM 《Veterinary immunology and immunopathology》2011,140(1-2):90-101
Several distinct T lymphocyte subpopulations with immunoregulatory activity have been described in a number of mammalian species. This study performed a phenotypic analysis of cells expressing regulatory T cell (Treg) markers in the peripheral blood of a cohort of 18 horses aged 6 months to 23 years, using antibodies to both intracellular and cell surface markers, including Forkhead box P3 (FOXP3), CD4, CD8, CD25, interferon gamma (IFNγ) and interleukin 10 (IL-10). In peripheral blood, a mean of 2.2 ± 0.2% CD4+ and 0.5 ± 0.1% CD8+ lymphocytes expressed FOXP3. The mean percentage of CD4+FOXP3+ cells was found to be significantly decreased in horses 15 years and older (1.5%) as compared to horses 6 years and younger (2.7%), but did not differ between females and males and ponies and horses. Activation of peripheral blood mononuclear cells by pokeweed mitogen resulted in induction of CD25 and FOXP3 expression by CD4+ cells, with peak expression noted after 48 and 72 h in culture respectively. Activated CD4+FOXP3+ cells expressed IFNγ (35% of FOXP3+ cells) or IL-10 (9% FOXP3+ cells). Cell sorting was performed to determine FOXP3 expression by CD4(+)CD25(-), CD4(+)CD25(dim) and CD4(+)CD25(high) subpopulations. Immediately following sorting, the percentage of CD4+FOXP3+ cells was higher within the CD4(+)CD25(high) population (22.7-26.3%) compared with the CD4(+)CD25(dim) (17% cells) but was similar within the CD4(+)CD25(dim) and CD4(+)CD25(high) cells after resting in IL-2 (9-14%). Fewer than 2% of cells in the CD4(+)CD25(-) population expressed FOXP3. These results demonstrate heterogeneity in equine lymphocyte subsets that express molecules associated with regulatory T cells. CD4+FOXP3+ cells are likely to represent natural Tregs, with CD4+FOXP3+IL-10+ cells representing either activated natural Tregs or inducible Tregs, and CD4+FOXP3+IFNγ+ cells likely to represent activated Th1 cells. 相似文献
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中国流行株HIV—1B亚型Gag重组鸡痘病毒构建及其免疫原性 总被引：1，自引：0，他引：1
在以鸡痘病毒(FPV)282E4株为基础构建的重组表达质粒pUTAL的ATI-P7.5复合启动子下游,插入编码中国流行株HIV-1 B亚型核心蛋白gag基因,构建了重组表达质粒pUTALG.重组质粒与FPV共转染CEF细胞,进行了同源重组.通过BUdR加压筛选,X-gal染色,获得重组鸡痘病毒vUTALG.Westernblot检测结果表明,重组病毒表达了Gag蛋白,裂解后其相对分子质量分别为55×103、48×103、24×l03和l 7×103.以重组病毒vUTALG免疫小鼠,与FPV对照比较,小鼠脾T淋巴细胞CD4+T细胞数和CD4+/CD8+值均有上升趋势,ConA、LPS诱导的脾细胞增殖能力增强,并能诱导CTL和HIV-1特异的血清抗体反应,说明该重组鸡痘病毒能提高小鼠细胞免疫和体液免疫水平. 相似文献
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猪瘟病毒感染对猪外周血T淋巴细胞亚群及TNF-α和IFN-γ的影响 总被引：2，自引：0，他引：2
为探讨猪瘟病毒(CSFV)弱毒株T株和野毒株G株感染对猪外周血T淋巴细胞亚群、TNF-α和IFN-γ的影响,本研究应用流式细胞术和ELISA等方法检测CSFV感染猪与未感染猪的白细胞凋亡、CD4+与CD8+T淋巴细胞亚群数量的动态变化以及TNF-α和IFN-γ的动态变化。结果表明,猪感染CSFV T株和G株第4 d和第7 d后CD4+T淋巴细胞比例分别为28.6%、26%和26%、20%,未感染前分别为33.4%和36.8%。猪感染CSFV T株和G株第4 d、第7 d后CD8+T淋巴细胞比例分别为41%、32%和38%、25%,感染前分别为43.8%和48.8%。外周血白细胞凋亡的检测结果显示,猪感染CSFV T株和G株第7 d后,白细胞凋亡比例分别为8.35%和9.89%,未感染的猪为1.63%。ELISA检测结果表明,猪感染CSFV T株和G株第7 d后,TNF-α的产生量分别为553.4 pg/mL和594.2 pg/mL;IFN-γ的产生量分别为8.2 pg/mL和9.8 pg/mL,未感染猪分别为498 pg/mL、12.5 pg/mL。以上结果提示,CSFV感染会引起机体免疫相关细胞及免疫分子发... 相似文献
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ZBED6是锌指蛋白家族的一员,在胎盘哺乳动物中极其保守,可通过对IGF2的调控参与骨骼肌生长。为进一步探究ZBED6基因自身的表达调控机制,本研究以民猪基因组DNA为模板,通过常规PCR扩增ZBED6基因启动子区系列截短片段,构建克隆质粒,通过双酶切和连接反应定向连入pGL3-basic载体,利用PK15细胞和双荧光素酶检测系统测定重组质粒的相对荧光素酶活性;利用在线软件预测启动子区的转录因子结合位点,使用重叠PCR定点缺失转录因子结合位点,构建突变载体并在PK15细胞中检测突变载体的相对荧光素酶活性。结果表明:ZBED6基因启动子区-2053^-1777 bp存在多个转录因子结合位点,尤其是-1808^-1777 bp,该片段缺失造成启动子活性下降(P<0.01);利用在线软件在该区间预测到3个转录因子HINFP、Adf-1和CREB3,经实验验证后发现这3个转录因子均可调控ZBED6基因的转录,其中Adf-1效果最为明显。据此推测,民猪ZBED6基因的转录调控机制较为复杂,其启动子区存在HINFP、Adf-1和CREB3等多个调控元件的结合位点。 相似文献
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【目的】 分析鹅p21基因的结构和启动子活性，探讨p21基因的转录调控机制。【方法】 以泰州鹅为试验对象，通过同源克隆、RACE和生物信息学分析等方法获得鹅p21基因全长序列和5′-侧翼区序列特征；构建6个不同缺失片段的启动子区双荧光素酶报告载体并分析其荧光素酶活性，进而确定p21基因核心启动子区；对核心启动子区转录因子结合位点生肌决定因子(MyoD)(+25~+36 bp)进行定点突变，并构建突变报告基因载体，在C2C12细胞系内初步鉴定鹅p21基因核心转录调控因子。【结果】 鹅p21基因cDNA全长1 943 bp，CDS区大小为453 bp，编码151个氨基酸，蛋白序列包含高度保守的CDI家族结合位点。系统进化树分析表明，鹅p21基因与鸭亲缘关系最近，与鸡和火鸡有较强的进化关系。鹅p21基因5′-侧翼区包含启动子元件，—35~+37 bp是核心启动子区，发挥正向调控作用，结合定点突变技术初步鉴定MyoD是鹅p21基因核心转录调控元件。【结论】 本研究获得了鹅p21基因完整的cDNA序列和启动子区域，MyoD是p21基因核心转录调控因子，为探究p21基因在鹅胚胎期肌肉发育过程中的调控机制提供理论依据。 相似文献
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旨在分析鹅MyoG基因启动子活性区域和转录因子,探究该基因的转录调控机制。本研究首先通过PCR扩增泰州鹅MyoG基因5'侧翼区序列1 245 bp并对其进行测序和生物信息学分析,其次,构建4个不同缺失片段的双荧光素酶报告载体,转染C2C12细胞系。进一步利用在线软件预测核心启动子区关键转录因子,对转录因子结合位点HNF4（-521～-503 bp）、USF （-379～-370 bp）和E2（-296～-281 bp）进行定点突变并构建突变报告基因载体,在C2C12细胞系内初步鉴定MyoG基因核心转录调控因子。最后,采集70日龄泰州鹅胸肌、腿肌、心、肝、脾、肺、肾和下丘脑组织样,利用荧光定量PCR检测MyoG基因和核心转录调控因子的组织表达谱。结果表明,扩增得到的鹅MyoG基因5'侧翼区序列包含启动子元件;利用双荧光素酶报告载体检测到鹅MyoG基因启动子区-624～-154 bp区域存在关键顺式调控元件;结合定点突变技术初步鉴定USF是鹅MyoG基因核心转录调控元件。组织表达谱研究进一步表明,MyoG和USF基因在鹅8个不同组织中均有表达,且在胸肌、腿肌和心组织中共同高表达（P<0.01）。鹅MyoG基因5'侧翼区具有启动子转录活性,-624～＋37 bp是核心启动子区,USF是MyoG核心转录调控因子。试验结果为探究MyoG基因在鹅肌肉发育过程的调控机制提供理论依据。 相似文献
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Infection of goats by caprine arthritis-encephalitis virus (CAEV) provides a convenient example of the infiltration of various tissues by leukocytes following a natural lentiviral infection. This event is important in determining organ susceptibility and local immunity. Caprine vascular endothelial cells are susceptible to infection by CAEV in vitro, so we have investigated the consequences of this infection on the transmigration of uninfected leukocytes in an in vitro model. After in vitro infection by CAEV or stimulation by TNFalpha, the endothelial cells allowed the passage of tenfold more leukocytes from uninfected donors than did the uninfected endothelial cells. The transmigrating leukocytes were enriched in CD8+ lymphocytes, and the leukocytes appeared to have been activated during transmigration, as demonstrated by their expression of IL2R, MHC class II antigens and gamma-delta T-lymphocyte markers. CD4+, CD8+ and B-lymphocytes all proliferated in culture after transmigration. These results suggest that any possible infection or specific stimulation of endothelia in an infected animal could profoundly influence the choice of target organs and could activate the cells involved in local mucosal immune responses. 相似文献