共查询到20条相似文献，搜索用时 46 毫秒
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Reasons for performing study: Further knowledge of equine keratinocyte physiology and keratinocyte response to various stimuli is important in developing a better understanding of disease states involving the epidermis. Objectives: To assess the inflammatory cytokine response of cultured equine keratinocytes to various pathogen‐associated molecular pattern molecules (PAMPs) from both Gram‐negative and positive bacteria likely to be present in equine sepsis. Methods: Keratinocytes were isolated from skin of 2 horses and primary cultures performed. Keratinocytes were harvested for RNA extraction after exposure to lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), bacterial DNA (CpG), flagellin or maintained in medium (controls) for 4 or 24 h. Real time‐quantitative PCR was used to quantify interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and CXCL8 mRNA concentrations. Results: Increases (P<0.05) in IL‐1β, IL‐6 and CXCL8 mRNA concentrations were induced by LPS exposure compared to controls. Increased mRNA concentrations of both IL‐6 and CXCL8 were also noted (vs. controls) upon exposure to flagellin. Overall, responses were greater at 4 h. No increases (P>0.05) in cytokine expression by keratinocytes were present after LTA, PGN or CpG exposure. Conclusions: Increased proinflammatory cytokine expression in response to LPS and flagellin indicate that equine keratinocytes have functional TLR4 and TLR5 receptor signalling. However, the lack of keratinocyte stimulation by PGN, LTA or CpG provides no evidence for functional TLR2, TLR9 or NOD receptor signalling. These results suggest that equine keratinocytes are more responsive to PAMPs usually associated with Gram‐negative sepsis and unresponsive to PAMPs most commonly associated with Gram‐positive sepsis. Potential relevance: The increased incidence of injury of epidermal structures in clinical cases of Gram‐negative (vs. Gram‐positive) sepsis in the horse may be due to a lack of functional TLR signalling for Gram‐positive PAMPs in the equine keratinocyte. 相似文献
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To investigate the effects of different doses of compound Chinese herbal medicinal polysaccharides (cCHMPS) on TLR4 and downstream MyD88 dependent signal transduction pathway components in chicken lymphocytes of different MHC B-LβⅡ genotypes,PCR-SSCP technique was applied to group layer according to different MHC B-LβⅡ genotypes.The peripheral blood lymphocytes of chicken with different MHC B-LβⅡ genotypes were collected,and added with 100,75,50 and 0 μg/mL cCHMPS (high,middle and low dose groups and control group),respectively,then co-culturing for 16,24,32 and 48 h.The expression of TLR4,MYD88 and TRAF-6 mRNA were detected using Real-time PCR method.The results showed that compared with control group,cCHMPS could significantly improve the expression levels of TLR4,MYD88 and TRAF-6 mRNA of different MHC B-Lβ Ⅱ genotypes chickens (P < 0.05);The expression levels of TLR4,MYD88 and TRAF-6 mRNA of AA genotype chicken lymphocyte in middle and low dose groups were higher than those of high dose group (except TLR4 gene cultured for 16 h);The expression of TLR4,MyD88 and TNAF-6 mRNA of BB genotype in high dose group were higher than those of other dose groups (except TLR4 gene cultured for 32 and 48 h);The expression of TLR4,MyD88 and TNAF-6 mRNA of BC genotype in low dose were higher than that of other dose groups (except TLR4 gene cultured for 16 h).There results indicated that cCHMPS played an important role in the body’s immune regulatory mechanism by binding to TLR4 in the surface of lymphocytes,activating the downstream MyD88-dependent signal transduction pathway,regulating cellular immunity,and cCHMPS optimum immunomodulatory does were different in each MHC B-Lβ Ⅱ genotype chickens. 相似文献
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In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. 相似文献
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Zhang H Lunney JK Baker RB Opriessnig T 《Veterinary immunology and immunopathology》2011,140(1-2):152-158
The objective of this study was to determine cytokine and chemokine mRNA expression profiles in tracheobronchial lymph nodes from pigs singularly infected with porcine circovirus type 2 (PCV2), Mycoplasma hyopneumoniae (MHYO), or coinfected with both. Twenty-eight pigs were randomly assigned to one of four groups: (1) negative controls (NEG), (2) inoculated with MHYO (IMHYO), (3) inoculated with MHYO and PCV2 (CoI), and (4) inoculated with PCV2 (IPCV2). MHYO infection significantly (P<0.05) stimulated innate cytokines, IL1B and IL8. PCV2 infection significantly stimulated expression of IFNG, IL8, NOS2A and chemokines CCL2, CCL5, and CXCL10. IFNB, IL1B and IL12 were slightly increased with PCV2 infection and IFNA and IL4 were significantly downregulated. Compared to NEG pigs, coinfection resulted in a significant increase in expression of IFNG, IL1B, IL8, CCL5, CXCL10, and weak stimulation of IFNB, IL6 and IL10; IL13 and IFNA were significantly downregulated. Overall MHYO potentiated PCV2 infection by increasing IFNG and IL10 mRNA expression levels. The increase of IFNG and chemokines and decrease of IFNA in IPCV2 and CoI pigs were correlated with increased severity of lymphoid lesions and the presence of PCV2 antigen. In summary, this work provided evidence that the increased severity of lesions in PCV2 and MHYO coinfected pigs was associated mainly with the presence of PCV2 antigen and alterations of cytokine mRNA expression profiles. 相似文献
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为探究Toll样受体(TLRs)介导的信号通路在马链球菌马亚种(S.equi)感染小鼠巨噬细胞RAW264.7中的作用,收集S.equi感染后不同时间点的RAW264.7细胞,提取总RNA并反转录成cDNA,利用实时荧光定量PCR技术检测细胞Toll样受体1、2、6(TLR1、TLR2、TLR6)、接头蛋白骨髓分化蛋白88(MyD88)及细胞因子IL-1、IL-6、IL-10、IL-12、TNF-αmRNA的表达情况。结果显示,S.equi感染RAW264.7细胞后6h时,TLR1、TLR2、TLR6与MyD88mRNA水平均较对照组没有显著差异(P>0.05);感染后12h时,TLR1、TLR2和TLR6mRNA表达量未出现明显上升(P>0.05),而MyD88mRNA水平极显著升高(P<0.01);感染后24h时,TLR1、TLR2和TLR6mRNA表达水平出现极显著升高(P<0.01),MyD88mRNA表达没有显著变化(P>0.05),且IL-10和IL-12mRNA水平与对照组相比极显著升高(P<0.01),IL-1、IL-6和TNF-αmRNA水平均极显著下降(P<0.01)。结果表明,TLRs介导的信号通路参与S.equi感染RAW264.7细胞的免疫应答反应。 相似文献
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Skjolaas KA Burkey TE Dritz SS Minton JE 《Veterinary immunology and immunopathology》2006,111(3-4):199-209
The gastrointestinal epithelium represents a barrier to potentially invasive enteric pathogens, maintains a role in innate immune surveillance, and is a source of both chemokine and cytokine chemotactic mediators in response to bacterial invasion. In the current study, we evaluated cytokine and chemokine mediators known to regulate movement of macrophages (macrophage migration inhibitory factor; MIF), neutrophils (IL8), dendritic cells (CCL20), and epithelial remodeling (osteopontin; OPN) in response to invasive swine enteropathogens Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). For the in vivo experiment, weaned pigs served as uninfected controls (0 h) or were given 3 x 10(9) CFU ST orally. Pigs were sacrificed at 8, 24, 48, and 144 h after inoculation and total RNA was extracted from defined segments of proximal (PI) and distal (DI) ileum. Relative expression of MIF and OPN were not affected by ST. IL8 expression was increased numerically (P = 0.17 for the interaction term) at 24 and 144 h in the PI and these increases accounted for greater expression in the PI relative to the DI (P < 0.05). Relative expression of CCL20 was increased at 24 h after ST (P < 0.05). Next, we evaluated the time course of MIF, IL8, CCL20, and OPN mRNA expression induced by application of lipopolysaccharide (LPS), ST or SC in vitro using pig jejunal epithelial cells (IPEC-J2). Cells were grown to confluency on permeable membranes, and treated apically with LPS (10 ng/mL), ST or SC (10(8)/well). After 1 h, cells were washed to remove LPS or extracellular bacteria, and media containing gentamicin was added to kill remaining extracellular bacteria. Media and RNA were collected at 1.5, 3, and 6 h after treatment. MIF mRNA was not affected by LPS or bacterial treatment. Similarly, IL8 expression was not affected by LPS, but was increased by ST and SC relative to controls at 1.5 and 3 h post exposure (P < 0.05 for all comparisons). Treatment with SC increased CCL20 mRNA relative to controls at 3 h (P < 0.05), while ST increased CCL20 at 1.5, 3, and 6h with maximal expression at 6 h (P < 0.05 for all comparisons). ST and SC increased polarized IL8 secretion. Our data demonstrate that invasive bacterial pathogens in the pig gastrointestinal tract trigger upregulation of selected cytokine and chemokine mediators, but serovars of Salmonella elicited differing patterns of activation in vitro. 相似文献
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Uddin MJ Nuro-Gyina PK Islam MA Tesfaye D Tholen E Looft C Schellander K Cinar MU 《Veterinary immunology and immunopathology》2012,147(3-4):211-222
The Toll-like receptor (TLR)4 is critical for the recognition of Gram-negative bacterial lipopolysaccharide (LPS) but in porcine peripheral blood mononuclear cells (PBMCs) it may cooperate with other TLRs and lead to the production of inflammatory cytokines. Therefore, we analyzed TLR1-10 mRNA expression in porcine PBMCs stimulated with LPS over time (1-48 h) by using quantitative real-time PCR and cytokine proteins level by ELISA in culture supernatant. TLR1-10 mRNA was detectable in porcine PBMCs. When compared with the control (non-stimulated), TLR1 mRNA were increased (p<0.05) at 3 h after challenge with 1 μg/ml LPS, whereas TLR1 and TLR2 mRNA were increased (p<0.01) at 6 h after challenge with 10 μg/ml LPS. TLR4 increased (p<0.001) at 3h after challenge with LPS and remained constant. TLR5 and TLR6 mRNA increased (p<0.05) at 9 h and 1 h after of LPS stimulation, respectively. The mRNA of CD14 and MD2 were increased (p<0.001) at 1h after LPS stimulation. Additionally, at most of the time analyzed, the mRNA expression increased with the dose of LPS. The LPS concentration had influence (p<0.05) on all the TLRs expression except TLR10; whereas time had effect (p<0.05) on all TLRs expression except TLR2, 3, 6 and 10. When compared to the control, the cytokines IL1b, IL8 and TNFα proteins were increased (p<0.001) immediately at 1 h after LPS stimulation and remained constant till 48 h. IL12b was increased (p<0.001) 12 h after challenge with 10 μg/ml of LPS. Although IL8 level was the highest, the higher (p<0.05) expression of all these inflammatory cytokines indicate that upon interacting with TLRs, LPS exerted inflammatory response in PBMCs through the production of Th1 type cytokines. The production of cytokines was influenced (p<0.001) by both the dose of LPS and the stimulation time. Hence, the porcine PBMCs are likely able to express all members of TLRs. 相似文献
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Faleiros RR Leise BS Watts M Johnson PJ Black SJ Belknap JK 《Veterinary immunology and immunopathology》2011,144(1-2):45-51
Chemokines play a vital role in leukocyte activation and emigration that reportedly plays a central role in laminar injury in equine laminitis. The purpose of this study was to evaluate the pattern of laminar chemokine expression in horses in the classical carbohydrate overload (CHO)-model of laminitis. Laminar samples were obtained 24h following water administration in the control group (CON, n=8), and at the onset of fever (≥ 102°F, 12-22 h post CHO, DEV group, n=8) and at the onset of lameness (20-48 h post CHO, LAM group, n=8) in induced horses. Real time quantitative PCR was performed on all samples in order to determine laminar mRNA concentrations of both CXC chemokines (CXCL1, CXCL6, CXCL8) and CC chemokines (CCL2 [MCP-1], CCL3 [MIP-1α], and CCL8 [MCP-2]). Data were subjected to ANOVA followed by Student-Newman-Keuls (P<0.05). Laminar mRNA concentrations for all CXC chemokines were increased (P<0.05) at both the DEV and LAM horses when compared to the control horses, whereas mRNA concentrations of CCL2 and CCL8 were only increased in the LAM horses when compared to controls and the DEV horses. When taken in context with our previous studies, CXCL1, CXCL6 and CXCL8 increases precede peak laminar leukocyte accumulation. Additionally, CCL2 and CCL8 expression corroborate previous reports of monocyte/macrophage accumulation in affected laminae. Compared with previous studies, our findings demonstrate that increased laminar CXC chemokine expression consistently precedes peak leukocyte accumulation and onset of lameness in CHO laminitis models. Chemokine antagonists may be considered as possible therapeutic targets to decrease the influx of leukocytes that occurs during the development of equine laminitis. 相似文献
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Islam MA Cinar MU Uddin MJ Tholen E Tesfaye D Looft C Schellander K 《Veterinary immunology and immunopathology》2012,146(1):62-73
The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24 h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P<0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P<0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P<0.05). The level of cytokine protein IL6 and TNFα in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1 h after LPS stimulation and remained significantly higher up to 48 h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFα in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range. 相似文献
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为探究马链球菌马亚种(Streptococcus equi subsp.equi,S.equi)对小鼠骨髓源树突状细胞(bone marrow derived dendritic cells,BMDCs)TLR1、TLR2、TLR6、MyD88、IL-1、IL-6、IL-10、IL-12、TNF-αmRNA表达的影响,用GM-CSF、IL-4细胞因子刺激小鼠骨髓细胞使其诱导分化成未成熟的BMDCs,感染S.equi后16、20、26h,采用SYBR GreenⅠ实时荧光定量PCR检测感染组及未感染组细胞TLRs、MyD88和细胞因子mRNA的表达情况。结果显示,S.equi感染BMDCs后16、20、26h,TLR1、TLR2、TLR6、MyD88mRNA的表达量均显著或极显著的高于未感染组(P0.05或P0.01)。S.equi感染DCs后16、26h,IL-1的分泌量较未感染组没有变化(P0.05),IL-10、TNF-α的分泌量显著或极显著增加(P0.05或P0.01)。IL-6在S.equi感染DCs后16h的分泌量显著增加(P0.05),但感染后26h无显著变化(P0.05)。IL-12在S.equi感染DCs后16h的表达没有变化(P0.05),但感染后26h分泌量有所增加(P0.05)。说明S.equi具有调节DCs TLRs、MyD88和细胞因子表达的能力,从而介导机体的免疫应答反应。 相似文献
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M. Parys V. Yuzbasiyan‐Gurkan J.M. Kruger 《Journal of veterinary internal medicine / American College of Veterinary Internal Medicine》2018,32(1):274-279
Background
Feline idiopathic cystitis (FIC) is a common lower urinary tract disorder of domestic cats that resembles interstitial cystitis/painful bladder syndrome (IC/PBS) in humans. Diagnosis of FIC is based on clinical signs and exclusion of other disorders because of a lack of specific pathologic findings or other objective biomarkers. Cytokines are potential noninvasive biomarkers to define the presence, severity, and progression of disease, and response to treatment.Objectives
The objective of this pilot study was to determine concentrations of selected cytokines in serum from healthy cats and cats with acute FIC.Animals
Serum samples from 13 healthy cats and from 12 cats with nonobstructive acute FIC were utilized.Methods
Multiplex analysis of 19 cytokines (CCL2, CCL5, CXCL1, CXCL12, CXCL8, Flt3L, GM‐CSF, IFN‐γ, IL‐12 (p40), IL‐13, IL‐18, IL‐1β, IL‐2, IL‐4, IL‐6, PDGF‐BB, SCF, sFas, and TNF‐α) was performed with a commercially available feline‐specific multiplex bead‐based assay.Results
Mean serum concentrations of IL‐12 (p40; P < 0.0001), CXCL12 (P = 0.002), IL‐18 (P = 0.032), and Flt3L (P = 0.0024) were significantly increased in FIC cats compared to healthy cats. GM‐CSF, IL‐1b, IL‐2, and PDGF‐BB were undetectable or detected in an insufficient number of cats to allow meaningful comparisons.Conclusions and Clinical Importance
We have identified increased serum concentrations of pro‐inflammatory cytokines and chemokines CXCL12, IL‐12, IL‐18, and Flt3L in FIC‐affected cats. These findings suggest potential candidates for noninvasive biomarkers for diagnosis, staging, and therapeutic outcome monitoring of affected cats and provide additional insight into the etiopathogenesis of FIC. 相似文献14.
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研究苜蓿黄酮对脂多糖(LPS)诱导下奶牛乳腺上皮细胞凋亡的影响。将奶牛乳腺上皮细胞分成4个组,即基础培养基、基础培养基中加入1 μg·mL-1的LPS、基础培养基中加入1 μg·mL-1的LPS和75 μg·mL-1苜蓿黄酮、基础培养基中加入75 μg·mL-1苜蓿黄酮。细胞在37 ℃, 5% CO2的培养箱中培养。结果表明:1)LPS刺激12 h后奶牛乳腺上皮细胞活性下降,而添加苜蓿黄酮能够极显著抑制LPS诱导下细胞活性的下降(P<0.01)。2)在LPS刺激下,细胞内的活性氧(ROS)浓度升高,而添加苜蓿黄酮能够显著降低其浓度(P<0.05)。3)LPS显著上调细胞的IL-1β、IL-6、TNF-α、TLR2、TLR4和MyD88表达(P<0.01),而苜蓿黄酮能够显著下调细胞的IL-1β、IL-6、TNF-α和TLR2表达(P<0.01或P<0.05)。4)在LPS刺激下,p53、Caspase3、p38和P-p38蛋白的表达显著升高(P<0.01或P<0.05),而添加苜蓿黄酮能够显著降低p53和p38蛋白的表达(P<0.05)。在LPS诱导下,苜蓿黄酮能够通过降低ROS浓度,抑制细胞凋亡,提高细胞活性;可能通过抑制TLR2/MyD88信号通路来降低细胞炎症因子的表达,从而保护细胞免受炎性损伤。 相似文献
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为探讨产肠毒素大肠杆菌（ETEC） K88感染仔猪发生炎症反应的分子机制,试验用ETEC K88灌服断奶仔猪,ELISA法检测攻毒后仔猪血清中白细胞介素8（IL-8）含量,实时荧光定量PCR方法检测淋巴结中Toll样受体2（TLR2）、Toll样受体4（TLR4）及其信号通路相关基因（髓样分化因子88（MyD88）、Toll相互作用蛋白（Tollip）、B细胞淋巴瘤因子3（Bcl3））的mRNA相对表达水平。结果发现,仔猪攻毒ETEC K88后6和24 h血清IL-8含量和淋巴结TLR2/4的表达水平均极显著或显著高于对照组（P<0.01;P<0.05）,且感染后24 h显著低于感染后6 h（P<0.05）;仔猪感染ETEC K88后24 h淋巴结中MyD88、Tollip和Bcl3的表达水平均极显著高于对照组（P<0.01）,但是感染后6 h时与对照组相比均无显著差异（P>0.05）。综上所述,ETEC K88感染仔猪可能是通过TLR2/4-MyD88信号通路产生炎症因子IL-8,促使仔猪出现炎症反应,且该炎症反应可能受Tollip和Bcl3蛋白的调控而被减弱。 相似文献
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LI Hai-hua ZHU Qi WANG Shi-qiong YANG Chun-lei ZHAO Xiang-hua QIAO Jia-yun WANG Wen-jie 《中国畜牧兽医》2017,44(1):262-267
To investigate the molecular mechanism of the inflammatory response in the piglets infected with enterotoxigenic E. coli (ETEC) K88, piglets were infected with ETEC K88,the IL-8 content in serum of piglets were assayed by ELISA,and the mRNA relative expression levels of TLR2/4 and its signal transduction pathway related genes (MyD88,Tollip and Bcl3) in mesenteric lymph nodes were detected by quantitative Real-time PCR. The results showed that compared with control group,the content of IL-8 in serum and the expressions of TLR2/4 in lymph nodes were all extremely significantly or significantly increased at 6 and 24 h after infection (P<0.01;P<0.05),and the IL-8 content and TLR2/4 mRNA expression at 24 h after infection were all significantly lower than those at 6 h after infection (P<0.05).In addition,the expressions of MyD88,Tollip and Bcl3 in lymph nodes were all extremely significantly increased at 24 h after infection compared with control group (P<0.01), but there was no significant difference between experimental group and control group at 6 h after infection (P>0.05). In conclusion,ETEC K88 infected piglets might produce inflammatory cytokines IL-8 through the TLR2/4-MyD88 signaling pathway,which could promote the inflammatory reaction in piglets. This inflammatory response might be regulated by Tollip and Bcl3,which could weak the inflammatory intensity in piglets. 相似文献