首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 62 毫秒
1.
γδ T cell responses are induced by various viral and bacterial infections. Different γδ T cells contribute to activation and regulation of the inflammatory response and to epithelial repair. How γδ T cells respond to rotavirus infection and how the colonization of probiotics influences the γδ T cell response were unknown. In this study, we evaluated by multicolor flow cytometry the frequencies and distribution of total γδ T cells and three major subsets (CD2-CD8-, CD2+CD8- and CD2+CD8+) in ileum, spleen and blood of gnotobiotic (Gn) pigs at early (3-5 days) and late phases (28 days) after rotavirus infection. The Gn pigs were inoculated with the virulent human rotavirus Wa strain and colonized with a mixture of two strains of probiotics Lactobacillus acidophilus and Lactobacillus reuteri. In na?ve pigs, the highest frequency of total γδ T cells was found in blood, followed by spleen and ileum at the early age (8-10 days old) whereas in older pigs (32 days of age) the highest frequency of total γδ T cells was found in ileum and spleen followed by blood. Rotavirus infection significantly increased frequencies of intestinal total γδ T cells and the putatively regulatory CD2+CD8+ γδ T cell subset and decreased frequencies of the putatively proinflammatory CD8- subsets in ileum, spleen and blood at post-infection days (PID) 3 or 5. The three γδ T cell subsets distributed and responded differently after rotavirus infection and/or lactobacilli colonization. The CD2+CD8+ subset contributed the most to the expansion of total γδ T cells after rotavirus infection in ileum because more than 77% of the total γδ T cells there were CD2+CD8+ cells. There was an additive effect between lactobacilli and rotavirus in inducing total γδ T cell expansion in ileum at PID 5. The overall effect of lactobacilli colonization versus rotavirus infection on frequencies of the CD2+CD8+ γδ T cell subset in ileum was similar; however, rotavirus-infected pigs maintained significantly higher frequencies of CD8- subsets in ileum than lactobacilli-colonized pigs. The dynamic γδ T cell responses suggest that γδ T cell subsets may play important roles in different stages of immune responses after rotavirus infection and probiotic colonization. The knowledge on the kinetics and distribution patterns of γδ T cell subsets in na?ve pigs and after rotavirus infection or lactobacilli colonization provides the foundation for further mechanistic studies of their functions.  相似文献   

2.
Although swine are natural hosts for influenza A viruses, the porcine T-cell response to swine influenza A virus (FLUAVsw) infection has been poorly characterized so far. We have studied Ki-67 expression and FLUAVsw-specific production of IFN-γ, TNF-α and IL-2 in CD4+ and CD8β+ T cells isolated from piglets that had been intratracheally infected with a H1N2 FLUAVsw isolate. IFN-γ+TNF-α+IL-2+ multifunctional CD4+ T cells were present in the blood of all infected animals at one or two weeks after primary infection and their frequency increased in four out of six animals after homologous secondary infection. These cells produced higher amounts of IFN-γ, TNF-α and IL-2 than did CD4+ T cells that only produced a single cytokine. The vast majority of cytokine-producing CD4+ T cells expressed CD8α, a marker associated with activation and memory formation in porcine CD4+ T cells. Analysis of CD27 expression suggested that FLUAVsw-specific CD4+ T cells included both central memory and effector memory populations. Three out of six animals showed a strong increase of Ki-67+perforin+ CD8β+ T cells in blood one week post infection. Blood-derived FLUAVsw-specific CD8β+ T cells could be identified after an in vitro expansion phase and were multifunctional in terms of CD107a expression and co-production of IFN-γ and TNF-α. These data show that multifunctional T cells are generated in response to FLUAVsw infection of pigs, supporting the idea that T cells contribute to the efficient control of infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0182-3) contains supplementary material, which is available to authorized users.  相似文献   

3.
1. This experiment investigated growth performance and nutrient utilisation responses of broilers to partial replacement of maize and soyabean meal in broiler diets with 100 g kg?1 maize Distillers’ Dried Grain with Solubles (mDDGS) as well as responses to supplementation of an admixture of carbohydrases and protease (XAP) or phytase individually or in combination in the diets.

2. A total of 288 one-day-old broilers were allocated to 8 treatments in a randomised complete block design and a 2 × 2 × 2 factorial arrangement of treatments. The three factors were two levels each of mDDGS (0 or 100 g kg?1), phytase (0 or 1000 FTU kg?1), and XAP (0 or 500 mg kg?1).

3. Each treatment had 6 replicate cages with 6 birds per replicate cage. The control diets were formulated to meet all nutrient requirements of broilers according to National Research Council recommendations of 1994, but were marginally deficient in non-phytate P and ME.

4. Weight gain and gain:food were higher in broilers receiving diets containing mDDGS. The coefficient of apparent ileal N digestibility was lower in diets with mDDGS. Phytase increased the coefficient of apparent ileal DM digestibility in all diets.

5. Phytase improved the coefficient of the apparent total tract DM retention independently of mDDGS and tended to improve the coefficient of apparent P retention in the diets without mDDGS. The enzymes were additive in their effects in the diets with mDDGS. Overall, the results showed that adding 100 g kg?1 mDDGS to a maize–soyabean meal diet had no negative effect on growth when energy and nutrient concentrations were similar to the maize–soyabean meal diet, and that phytase or an admixture of carbohydrases and protease individually or in combination modestly improved nutrient utilisation independently of mDDGS addition. Combination of the enzymes did not produce greater benefit than the use of phytase alone.  相似文献   


4.
Larval development, mucosal mast cell (MMC) and eosinophil responses in worm-nai;ve lambs, yearling goats and goat kids were compared using two different experimental challenge regimes involving oral administration of infective Teladorsagia circumcincta L(3). Experimental challenge regimes enabled primary and secondary immune responses in the two species to be compared. Goats carried higher worm burdens than lambs and there were significant differences in the stages of development attained by the larval challenge that established in the two species. Possible physiological reasons for these differences are discussed. There were also differences in the establishment and development of larvae in individual yearlings which may indicate the development of a weak age-related immune response. Quantitative analysis of MMC and globule leukocyte (GL) recruitment and functional activity in the form of mast cell-specific proteinase (MCP) production demonstrated differences between the species with goat tissues containing significantly higher numbers of GL and lower concentrations of MCP than the lambs. Quantitative analysis of blood and tissue eosinophil responses failed to demonstrate any significant differences in either species under the two challenge regimes.  相似文献   

5.
《Veterinary microbiology》1997,57(4):337-345
Southern blots prepared with DNA from 20 Merino lambs that previously had chronic dermatophilosis (chronic) and 20 lambs that previously had mild dermatophilosis lesions (resistant) were hybridised with DNA sequences of the genes for the T-cell receptor-β (TCRβ), a TCR-associated peptide ‘cluster designation 3γ chain’ (CD3γ) and ovine Major Histocompatibility Complex class 1 (ov. MHC class 1). There was a significant difference in the incidence of an allele of CD3γ between the chronic and resistant lambs. No significant difference in the incidence of alleles of TCRβ or ov. MHC class 1 was detected.  相似文献   

6.
1. Three experiments performed to study the effects of amino acid imbalances on the growth of genetically lean (LL) and fat (FL) male chickens from 28 to 42 d of age were described by Leclercq et al. (1994). The plasma amino acid concentrations of birds on selected treatments from that paper are reported here. In experiment 1, three dietary concentrations of digestible lysine were compared (4.75, 6.75 and 7.75 g/kg). In experiment 2, two dietary concentrations of digestible arginine were compared (6.53 and 10.00 g/kg). In experiment 3, three diets were compared: a high‐protein diet (189 g CP/kg), a low‐protein diet containing added essential amino acids (144 g CP/kg), and this low‐protein diet supplemented with 40 g/kg of non‐essential amino acids (NEAA; glutamic and aspartic acids).

2. The present results are compared with two earlier reports on the same genotypes. The LL consistently had lower plasma concentrations of me‐thionine, cystine, phenlyalanine, isoleucine and valine, and higher concentrations of histidine, than the FL chickens. In 4 of 5 experiments, LL leucine concentrations were lower, and glutamic acid, tyrosine, glutamine and alanine were higher, than in the FL. The other amino acids measured; arginine, lysine, aspartic acid, glycine and serine, exhibited variable responses among the experiments.

3. When the limiting essential amino acids, lysine and arginine, were added to a deficient diet, the plasma concentration of the supplemented amino acid increased while the others remained constant or decreased.

4. When glutamic and aspartic acids were added to the low protein diet, plasma amino acid responses were similar to those of adding a limiting amino acid to a deficient diet, except that alanine exhibited a dramatic increase.

5. Although there were genotype by diet interactions for several amino acids, the interactions were caused by differences in the degree of the responses, not in their direction.

6. These results suggest that the FL and LL genotypes do not utilise various amino acids with the same efficiency and, as a consequence, the ideal profile of dietary amino acids should not be the same for both lines. The results support the hypothesis that selection for fatness and leanness changed the amino acid requirements independently of the: effects of food intake.  相似文献   


7.
Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10-21 months of age from a MAP infected herd with addition of either recombinant bovine IL-12 or anti-bovine IL-10 antibody with IFN-γ production in sample day samples. IFN-γ responses of sample day samples showed high correlation with responses to some antigens in day-old samples with addition of IL-12 or anti-IL-10 antibodies to cultures, indicating that day-old protocols can be applied as an alternative to the conventional IFN-γ protocol. Immunogenicity of the novel antigens was generally low for day-old samples. The most promising antigen using the day-old protocol with addition of IL-12 was latency protein LATP-2 as correlations, immunogenicity and diagnostic specificity collectively was high. The latency protein LATP-1 was the most promising antigen in the day-old protocol with addition of anti-IL-10 antibodies.  相似文献   

8.
Dendritic cells (DCs) are antigen presenting cells that potently modulate immune responses with varying outcomes depending on the DC sub-population involved. To understand how DC sub-types arise, it is necessary to determine which factors influence their differentiation. At least three major sub-populations of DCs have been described in mice: CD4+/CD8- "myeloid" DCs, CD4-/CD8+ "lymphoid" DCs and Langerhans cell-derived DCs. Whilst somewhat comparable populations have been described in man, in most other species very little is known. The identification of cytokines which stimulate proliferation of DC precursors, and the observation that the cytokine environment influences the phenotype and the function of the DCs that subsequently develop, has provided a useful tool for evaluating these rare cells. We describe the influence of cytokines on the phenotype of DCs generated in the rat. Using bone marrow cells as the source of precursors we generated "myeloid-type" DCs from the adherent population using granulocyte-macrophage colony stimulating factor (GM-CSF), IL-4 and Flt-3L or "lymphoid-type" DCs from the non-adherent population using cytokines which included IL-7, IL-3, SCF and TNFalpha. In order to facilitate similar approaches to the study of equine DCs we have identified the nucleotide sequence encoding GM-CSF from the m-RNA of equine PBMC stimulated with Concanavalin A, amplified the cDNA by PCR and cloned it in eukaryotic and prokaryotic expression vectors. We report on the structure and function of this molecule.  相似文献   

9.
Measures of behavioral responses and cardiovascular parameters to evaluate and assess animal well-being are well established. A major aspect of companion animal well-being seems to originate from direct human–animal interaction. For pet dogs, the manner in which they obtain and respond to petting and hugs could interfere with the development of a pleasant human–dog companionship. Therefore, the purpose of this study was to evaluate cardiovascular responses by dogs to physical human–dog contact and to assess these physiological responses in relation to the dogs' behavioral responses. Noninvasive measurements of privately owned dogs' (N = 28) cardiovascular parameters and behavioral responses were carried out during 9 physical human–dog interactions (e.g., petting the dog on its back, holding a forepaw of the dog). The behavioral responses were grouped in categories, for example, redirected behavior, displacement activity, and appeasement gesture. The mean heart rate (HR) and 2 cardiac activity parameters, standard deviation of normal-to-normal R–R intervals (SDNN) and root mean square of successive heartbeat interval differences/SDNN (RMSSD/SDNN) ratio, differed significantly among the human–dog interactions. Petting and holding the dog around the head was associated with an increased SDNN. An increased vagal tone was the dogs' responses to being petted at the chest. Displacement activities correlated negatively with all cardiovascular parameters (HR, SDNN, RMSSD, and RMSSD/SDNN ratio). Appeasement gestures were positively correlated with HR and occurred less under an increased vagal tone. The behavioral strategies, that is, freezing (standing motionless with all legs on the floor) and withdrawal (moving backward without any agonistic display) were negatively associated with the cardiac activity parameters, RMSSD and RMSSD/SDNN ratio. The dogs' behavioral and physiological responses suggest that some common physical human–dog interactions perceived as unpleasant by dogs. Emphasis on human signaling in human–dog interactions encourages development of recommendations for pleasant and safe human–dog contact to enhance dogs' well-being and the human–dog relationship.  相似文献   

10.
Coliform mastitis may be severe in periparturient cows due to enhanced expression of pro-inflammatory cytokines that contribute to disease pathogenesis. Tumor necrosis factor (TNF)-α is implicated with the severity of coliform mastitis by provoking inflammatory responses in affected tissues. The endothelium is an integral organ in regulating inflammatory responses and loss of endothelial integrity may be fatal. Studies in humans suggest that endothelial cell apoptosis may be a consequence of TNF-α exposure and contributes to the development of sepsis, however, its impact on bovine mammary endothelial cells (BMEC) is unknown. We sought to determine the inflammatory and apoptotic responses of primary BMEC exposed to TNF-α in vitro. Stimulation of endothelial monolayers with TNF-α resulted in significant increase of toll-like receptor 4, interleukin-6 and -8, and intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 gene expression in a time-dependent manner. Caspase-8 and caspase-3 mRNA expression, as well as caspase enzyme activity, also increased significantly following TNF-α stimulation. Cell viability assessed by ATP activity and BMEC apoptosis determined by flow cytometry revealed no significant changes across time with TNF-α stimulation. Results suggest that TNF-α stimulation, at the dose used in this study, can elicit a pro-inflammatory response in BMEC, but not induce apoptosis. The impact of TNF-α on mammary vascular function and the subsequent impact on the pathophysiology of severe coliform mastitis warrant further investigation.  相似文献   

11.
An experiment was conducted to evaluate the effects of diets containing low-, moderate-, or high-oil dried distillers dried grains with solubles (DDGS) included at conventional- or increased-inclusion rates fed to 1,500 Ross × Ross 708 male broilers that were assigned to 60 floor pens from 1 to 49 d of age. Three sources of DDGS had ether extract composition of 6.06, 8.80, or 11.59%, on dry matter (DM) basis, representing low-oil, moderate-oil, or high-oil DDGS, respectively. Diets were formulated to contain corn, soybean meal, animal protein meal as the primary ingredients, and 1 of the 3 DDGS sources at either 5, 7, 9, or 11% (conventional-inclusion rate) and 8, 10, 12, or 14% (increased-inclusion rate) in the starter (1 to 14 d), grower (15 to 24 d), finisher 1 (25 to 34 d), and finisher 2 (35 to 49 d) periods, respectively. Apparent MEn (low-oil:1,975, moderate-oil: 2,644, and high-oil: 3,137 kcal/kg) and digestible amino acid (AA) values of the 3 DDGS sources were determined from previous research. No differences were detected for cumulative BW gain and feed conversion 1 to 49 d of age or meat yields at 50 d of age. Feeding broilers diets containing the low-oil DDGS source increased feed cost per BW gain and breast meat weight of $0.025/kg and $0.004/kg compared with birds fed diets containing high-oil DDGS or moderate-oil and high-oil DDGS sources, respectively. These data indicated that DDGS source and inclusion rate did not affect cumulative growth and carcass characteristics of broilers from 1 to 50 d of age but demonstrate differences in feed cost/BW gain and feed cost/breast meat weight.  相似文献   

12.
This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.  相似文献   

13.
The daily requirement for calcium and phosphorus by giraffes to sustain the growth and maintenance of their skeletons is large. The source of sufficient calcium is browse. The source of necessary phosphorus is obscure, but it could be osteophagia, a frequently observed behaviour in giraffes. We have assessed whether bone ingested as a result of osteophagia can be digested in the rumen. Bone samples from cancellous (cervical vertebrae) and dense bones (metacarpal shaft) were immersed in the rumens of five sheep, for a period of up to 30 days, and the effect compared to immersion in distilled water and in artificial saliva for 30 days. Distilled water had no effect on the bones. Dense bone samples were softened by exposure to the saliva and rumen fluid, but did not lose either calcium or phosphorus. In saliva and rumen fluid the cancellous bone samples also softened, and their mass and volume decreased as a result of exposure to saliva, but in neither fluid did they lose significant amounts of calcium and phosphorus. We conclude that although saliva and rumen fluid can soften ingested bones, there is an insignificant digestion of bones in the rumen.  相似文献   

14.
15.
Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infections are characterized by prolonged viremia and viral shedding consistent with incomplete immunity. Type I interferons (IFN) are essential for mounting efficient antiviral innate and adaptive immune responses, but in a recent study, North American PRRSV genotype 2 isolates did not induce, or even strongly inhibited, IFN-α in plasmacytoid dendritic cells (pDC), representing “professional IFN-α-producing cells”. Since inhibition of IFN-α expression might initiate PRRSV pathogenesis, we further characterized PRRSV effects and host modifying factors on IFN-α responses of pDC. Surprisingly, a variety of type 1 and type 2 PRRSV directly stimulated IFN-α secretion by pDC. The effect did not require live virus and was mediated through the TLR7 pathway. Furthermore, both IFN-γ and IL-4 significantly enhanced the pDC production of IFN-α in response to PRRSV exposure. PRRSV inhibition of IFN-α responses from enriched pDC stimulated by CpG oligodeoxynucleotides was weak or absent. VR-2332, the prototype genotype 2 PRRSV, only suppressed the responses by 34%, and the highest level of suppression (51%) was induced by a Chinese highly pathogenic PRRSV isolate. Taken together, these findings demonstrate that pDC respond to PRRSV and suggest that suppressive activities on pDC, if any, are moderate and strain-dependent. Thus, pDC may be a source of systemic IFN-α responses reported in PRRSV-infected animals, further contributing to the puzzling immunopathogenesis of PRRS.  相似文献   

16.
Feline immunodeficiency virus (FIV) infection of cats is an animal model for the pathogenesis of CD4+ lymphopenia and thymus dysfunction in HIV-infected humans. Recently, a monoclonal antibody (755) was reported to recognize the feline homologue to CD45RA, allowing the enumeration of na?ve T cells in cats. We tested the hypothesis that pediatric FIV infection would be associated with a selective loss of na?ve CD4+ lymphocytes by inoculating newborn cats with a pathogenic clone of FIV (JSY3) or a related clone with an inactive ORF-A gene (JSY3-DeltaORFA), and compared the data to age-matched uninfected control cats. Both FIV inocula were associated with a reduction in the CD4-CD8 ratio (p=0.01), which was attributable to a disproportionate loss of na?ve CD4+ cells (p=0.01) vs. na?ve CD8+ cells. Therefore, the reduced CD4:CD8 ratio in FIV-infected juvenile cats is associated with a selective depletion of na?ve CD4+ cells from the blood.  相似文献   

17.
A live attenuated Salmonella enterica serovar Montevideo (SM) mutant JOL1599 was constructed by deletion of virulence-associated genes. The protective efficacy and immune response profiles of chickens immunized with JOL1599 were investigated. Immunized chickens demonstrated significant increases in plasma IgG and lymphocyte proliferative responses (P  0.05). Increased levels of IL-6, INF-γ, and IL-12 were also observed. Immunized birds strongly responded to infection by rapid stimulation of a CD4+ subset of T cells. Organ bacterial recovery assay revealed a significant reduction in the challenge strain among immunized birds. Multiple doses of JOL1599 enhanced the immune responses of the birds as revealed by ascending trends of the immunological profiles. These findings indicate that immunization of chickens with JOL1599 may provide protection against Salmonella Montevideo infection via induction of IL-6, INF-γ, and IL-12 protective cytokines, which in turn triggers conducive humoral and cell-mediated immune responses.  相似文献   

18.
Salmonella spp. is one of the major causes of food-borne illness in humans, and Salmonella enteritidis (SE) infection in commercial poultry is a world-wide problem. Here we have investigated the in vitro immune-modulating effects of β 1-4 mannobiose (MNB), which was previously found to prevent SE infection in vivo in chickens, using chicken macrophage (MQ-MCSU) cells. Treatment of MQ-NCSU cells with MNB dose-dependently increased both phagocytic activity and Salmonella-killing activity of macrophages, with the highest reduction in SE viability observed at a concentration of 40 μg/ml at 48 h post-infection. Likewise, both hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) production were increased in a dose-dependent manner by MNB. Gene expression analysis of MNB-treated macrophages revealed significant increases in the expression of iNOS, NOX-1, IFN-γ, NRAMP1, and LITAF, genes critical for host defense and antimicrobial activity, when compared to untreated cells. This data confirms that MNB possesses potent innate immune-modulating activities and can up-regulate antibacterial defenses in chicken macrophages.  相似文献   

19.
1. Three experiments were performed to study the effects of amino acid imbalance on the growth of genetically lean (LL) or fat (FL) male chickens from 28 to 42 d of age. In experiment 1, five concentrations of digestible lysine were compared (4.75, 6.75, 7.75, 8.75 and 9.75 g/kg). In experiment 2, four concentrations of digestible arginine were compared (6.53, 7.69, 8.84 and 10.0 g/kg). In experiment 3, three diets were compared: a high‐protein diet (189 g CP/kg), a low‐protein diet containing added essential amino acids (EAA) (144 g CP/kg) and this low‐protein diet supplemented with 40 g/kg of non‐essential amino acids (NEAA) (glutamic acid + aspartic acid).

2. LL birds exhibited a lower growth rate than the FL when the diet was deficient in either lysine or arginine. Plotting weight gain against lysine or arginine intake suggested that most of this effect was caused by variations in food intake.

3. When protein gains (body or total proteins) were plotted against lysine or arginine intake, LL chickens appeared more efficient than FL chickens.

4. Similar growth rates, although slightly lower in FL, were obtained with low‐ and high‐protein diets. However, NEAA supplementation of the low‐protein diet reduced adiposity of LL and did not modify that of FL. Increasing crude protein content (all amino acids) was more effective than NEAA supplementation in decreasing the adiposity of both lines.  相似文献   


20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号