TRAIL-decoy receptor 1 plays inhibitory role in apoptosis of granulosa cells from pig ovarian follicles

Previously, we histochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovarian follicles, and demonstrated a marked reduction in the expression of TRAIL-decoy receptor-1 (DcRI) in granulosa cells of atretic follicles. In the present study, to confirm the inhibitory activity of DcR1 in granulosa cells, granulosa cells prepared from healthy follicles were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) to cleave glycophospholipid anchor of DcR1 and to remove DcR1 from the cell surface, and then incubated with TRAIL. PI-PLC treatment increased the number of apoptotic cells induced by TRAIL. The present finding indicated the possibility that TRAIL and its receptors were involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis.     

鸡肿瘤坏死因子的诱生及活性检测

通过对鸡静脉注射卡介苗 (BCG)和脂多糖 (L PS) ,应用 L92 9细胞检测鸡体外周血清、脾细胞和外周血白细胞肿瘤坏死因子 (TNF)活性 ,表明外周血清 TNF活性最高 ,其次是脾细胞 ,而外周血白细胞 TNF活性最低。本研究也表明在正常鸡的外周血液中有少量的 TNF     

鸡包涵体肝炎肿瘤坏死因子消长规律的研究

自1975年从动物体内提取肿瘤坏死因子（TNF）以来，许多科学家从哺乳动物及禽类体内提取TNF并对其理化性质、生物学活性进行了不同程度的研究和探讨。而用鸡包涵体肝炎病毒诱生TNF与该病之间相关性及产生的消长规律的研究尚属空白。本研究应用鸡包涵体肝炎病毒（FAV-HA毒株）接种SPF雏鸡，在接种后的不同日龄采血，提取TNF，并对其免疫活性（对L₉₂₉细胞的杀伤率）进行了检测，从而进一步研究其产生TNF的消长规律及与该病毒的相关性。研究结果表明，鸡包涵体肝炎病毒经口腔感染SPF雏鸡可刺激机体产生TNF，在感染的不同日龄鸡的TNF活性出现一定的消长规律。这进一步说明鸡包涵体肝炎(IBH)与TNF的产生及其活性具有密切的相关性。     

Effects of proinflammatory cytokines on canine articular chondrocytes in a three-dimensional culture

OBJECTIVE: To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system. SAMPLE POPULATION: Humeral head articular cartilage chondrocytes obtained from 6 adult dogs. PROCEDURE: Chondrocytes were cultured in a 3-D system for < or = 12 days in serum-free medium with IL 1alpha, IL-1beta, or TNF-alpha at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines. RESULTS: In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1beta (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-alpha-treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1beta (50 ng/mL) and IL-1beta (100 ng/mL) groups at days 1 and 3, in the IL-1beta (100 ng/mL) group at day 6, and in all TNF-alpha groups at days 1, 3, and 6. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that TNF-alpha more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1beta. In vitro, IL-1alpha appeared to have a minimal effect on canine chondrocytes.     

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5 ± 1.5%) and CEC-32 (RIF 7.0 ± 0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R_low. Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2 ± 0.3%) similar to that of R_low (1.1 ± 0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.     

鸡软骨细胞体外分离培养鉴定以及过表达miR-15a对软骨细胞的影响

为分析miR-15a在肉鸡不同组织中的表达情况,并探究过表达miR-15a对体外培养鸡软骨细胞的影响。本研究首先通过倒置显微镜观察、PCR、凝胶电泳和甲苯胺蓝染色对体外分离培养的软骨细胞进行鉴定。通过实时荧光定量PCR检测miR-15a在肢体内外翻畸形(valgus-varus deformity, VVD)组和健康组肉鸡中(各3只)各组织的表达量。之后通过CCK8和EDU方法分析软骨细胞过表达miR-15a后细胞增殖情况。软骨细胞转染miR-15a mimics后,通过qPCR检测软骨细胞的标志基因Collagen-2、Aggrecan、Collagen-10,成熟分化基因Runx2、Sox9、VEGF、MMP9,炎性因子IL-1β、IL-6、IL-8、IL-10、TNF-α、TGF-β3以及凋亡基因Fas、FasL、Bcl-2的表达量。并构建FKBP5 3'UTR的野生型载体和突变型载体,通过双荧光素酶检测报告检测miR-15a与FKBP5的靶向关系。结果表明,本研究所用的胰蛋白酶、Ⅱ型胶原酶和透明质酸酶联合消化法成功分离得到状态良好的软骨细胞。荧光定量结果显示,miR-15a在各组织中均有表达,与健康组相比,miR-15a在VVD组的肝(P < 0.01)、脾(P < 0.05)、胸腺(P < 0.01)中的表达量显著升高,在心和胸肌组织中的表达量显著降低(P < 0.01)。CCK8与EDU分析结果显示,与NC组相比,过表达miR-15a组软骨细胞增殖速度显著降低(P < 0.01),增殖细胞数量显著减少(P < 0.01)。qPCR结果显示,与mimics NC组相比,miR-15a mimics组的软骨细胞标志基因Aggrecan、成熟分化基因Sox9、Runx2表达量显著降低(P < 0.05),Fas基因表达量极显著上升(P < 0.01),FasL基因和抗凋亡基因Bcl-2极显著下降(P < 0.01)。成功构建了FKBP5 3'UTR野生型和突变型载体,双荧光素酶检测报告结果显示预测的靶基因FKBP5与miR-15a没有靶向关系。本研究成功分离并鉴定了鸡软骨细胞,过表达miR-15a抑制鸡软骨细胞增殖、成熟和分化并促进细胞凋亡。     

A monoclonal antibody against chicken thrombocytes reacts with the cells of thrombocyte lineage

A new mouse monoclonal antibody (mAb), HUKT was raised against chicken peripheral blood thrombocytes. The mAb HUKT appeared to detect a specific marker on the surface of chicken thrombocytes. Flow cytometry (FCM) analysis revealed that it did not react with cells from the normal thymus, bursa of Fabricius, six kinds of chicken cell lines, chicken erythrocytes or human platelets. In addition, HUKT(+) cells in peripheral blood leukocytes (PBL) were CD45(low), Bu-1a(-) and CD3(-) cells. Immunoblotting analysis showed that the molecule recognized by HUKT is a monomer with an apparent molecular weight of 150 kDa under non-reducing and reducing conditions. Tissue distribution studies revealed that only cells of thrombocyte lineage in bone marrow and embryonic blood cells were stained by HUKT. The HUKT mAb presented here may be useful for both ontogenetic studies of thrombocyte lineage and immunological studies in the chicken.     

Development and use of monoclonal antibodies to chicken fibronectin to show that the chicken hepatocellular carcinoma cell line, LMH, constitutively expresses fibronectin

Fibronectin (Fn) is a high molecular weight glycoprotein and acute phase reactant that contributes to a variety of cellular activities including proliferation and wound healing. Production of Fn is influenced by cytokines such as IL-1alpha, IL-6 and TNF -alpha, and in serum Fn levels can function as an indicator of sepsis and reticulo-endothelial function. Here we describe the production of a panel of mAb to chicken Fn and give evidence that a chicken hepatocellular carcinoma cell line, LMH, constitutively expresses Fn. A capture ELISA to measure chicken Fn was developed using an IgG1 mAb (AV62) as the capture Ab, and biotinylated AV63 (IgG2b) as the detecting Ab. This study identified a single commercially available mAb directed against human Fn that also recognised chicken Fn. By contrast, the anti-chicken Fn mAbs did not cross-react with either human or bovine Fn.     

白果内酯对IL-1β诱导的ATDC5软骨细胞自噬、增殖和凋亡的影响

旨在探讨白果内酯对白介素1β(IL-1β)诱导的ATDC5软骨细胞自噬、增殖和凋亡的影响。采用10 ng·mL^-1 IL-1β诱导ATDC5软骨细胞构建体外炎症模型,随机分为对照组、IL-1β组、白果内酯组(低、中、高剂量)。利用EdU检测细胞新合成的DNA,并结合细胞核标记物(Hoechest)进行双重标记检测细胞增殖速度。使用膜Annexin-V/PI通过流式细胞仪分析ATDC5软骨细胞凋亡情况。通过mRFP-GFP-LC3双荧光系统的组合测量方法检测自噬流。蛋白免疫印迹法(Western blot)和实时荧光定量PCR(qRT-PCR)方法检测各组软骨细胞中LC3-II、Beclin1、BAX、Caspase-3和Bcl2的蛋白和mRNA表达情况。结果显示,IL-1β诱导ATDC5软骨细胞后,细胞增殖减弱,下调自噬促进凋亡,而白果内酯干预能够过促进自噬和细胞增殖,抑制凋亡。白果内酯促进ATDC5软骨细胞的增殖(P<0.05),与IL-1β组相比,抑制了LC3-II、Beclin1、BAX和Caspase-3的蛋白和mRNA表达(P<0.05),促...     

Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin A.

A microculture system in conjunction with a semiautomatic multiple sample harvester (SAMSH) was used to study the in vitro properties of chicken peripheral lymphocytes. This new procedure enabled doing rapid multiple tests, using relatively few cells, and was highly reproducible. Data were presented to show many variables that are involved in studying the concanavalin A (Con A) response of chicken lymphocytes in a microculture system. Analysis indicated that the conditions for optimal Con A stimulation as measured by incorporation of 3H-TdR include: (a) use of 2 x 10(6) cells per culture in RPMI 1640 culture medium in the absence of any serum, (b) use of 0.4 mug of Con A per culture, (c) incubation at 37 degrees C for 72 hours, and (d) addition of 1 muCi of 3H-TdR to each culture 12 to 24 hours prior to termination. This technique could be used to monitor immunocompetence of the chicken.     

Molecular study on chicken tumor necrosis factor receptor-II and tumor necrosis factor receptor-associated factor-5

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the tumor necrosis factor receptor (TNFR) superfamily. In this study, we cloned and characterized two genes that encode chicken TNFR-II and TRAF5. The initial cDNA fragments were obtained by suppressive subtractive hybridization (SSH) of chicken spleen cells with or without lipopolysaccharide stimulation (Salmonella typhimurium SL1181 (RE-mutant)). The results showed that chicken TNFR-II is 1518 bp in length with an open reading frame (ORF) of 1386 bp having 31% homology with human TNFR-II. Expression analysis of chicken TNFR-II revealed that it is highly expressed in the spleen and bursa of Fabricius. The chicken cell lines IN24, MSB1 and 1104B express TNFR-II abundantly. The time course analysis of expression in spleen, bursa of Fabricius and IN24 cell line showed that TNFR-II is maximally expressed at 6 h after stimulation in bursa of Fabricius and after 8 h stimulation in the IN24 cell line. With regard to TRAF5, the complete sequence was 1936 bp in length with an ORF of 1671 bp that showed 71.3% homology with human TRAF5. Expression analysis showed that, among the tissues examined, TRAF5 was strongly expressed in spleen and bursa of Fabricius, while among the cell lines examined, it was maximally expressed in IN24. Thus, both genes were expressed in the same tissues and cell line among examined materials. These results suggest that chicken TNFR-II may interact with TRAF5 adaptor protein to complete its signal transduction pathway.     

Molecular cloning, in vitro expression and bioactivity of goose B-cell activating factor

B-cell activating factor (BAFF), belonging to the TNF family, is critical for B cell survival and maturation. cDNA of goose BAFF (gBAFF) was amplified from goose spleen by RT-PCR. The open reading frame (ORF) of gBAFF encodes a protein of 288-amino acid. The gBAFF shows 98, 92, 44 and 55% amino acid sequence identity with duck (dBAFF), chicken (cBAFF), mouse (mBAFF) and human BAFF (hBAFF), respectively. RT-PCR results showed that gBAFF mRNA is expressed in thymus and more highly expressed in the bursa of Fabricius and spleen. Recombinant soluble gBAFF (gsBAFF) expressed in Escherichia coli has molecular weight of approximately 19kDa. In vitro, purified gsBAFF was able to promote bursa B cells survival/proliferation in goose, duck and chicken. Furthermore, recombinant dsBAFF and csBAFF have a positive effect on goose, duck and chicken bursa B cells survival/proliferation. These findings indicate that gBAFF plays an important role in the survival/proliferation of goose B cells and, owing to its high evolutionary conservation, functional cross-reactivity exists between chicken, duck and goose BAFF.     

Effects of tissue inhibitor of metalloproteinases on canine chondrocytes cultured in vitro with tumor necrosis factor-alpha

OBJECTIVE: To elucidate tissue inhibitor of metalloproteinase (TIMP)-mediated effects on chondrocytes. SAMPLE POPULATION: Articular cartilage from humeral heads of 6 dogs. PROCEDURE: Chondrocytes from harvested specimens were cultured in 3-dimensional (3-D) agarose at 10(6) cells/mL. We prepared 3-D constructs exposed to only tumor necrosis factor (TNF)-alpha (50 ng/mL). Recombinant human TIMP-1 (255nM), -2 (285nM), or -3 (250nM) was added to liquid media bathing 3-D constructs cultured with TNF-alpha. Chondrocytes cultured without TIMP or TNF-alpha served as control samples. Samples of liquid media were collected on days 6, 9, 15, and 21 of culture for evaluation of glycosaminoglycan (GAG) and nitric oxide concentrations. The 3-D constructs were collected on days 9, 15, and 21 for evaluation of GAG, hydroxyproline (HP), and DNA contents. RESULTS: GAG content in control samples increased significantly during the study, whereas GAG content in 3-D constructs cultured with TNF-alpha or TNF-alpha plus TIMP did not increase. On day 9, GAG release from 3-D constructs cultured with TNF-alpha was significantly higher than that in other constructs. The HP content in control samples increased during the study and was significantly higher than that in all other constructs on day 21. Concentrations of nitric oxide were significantly lower in control samples on day 6, compared with concentrations for all other constructs. CONCLUSIONS AND CLINICAL RELEVANCE: Addition of TIMPs did not counteract suppression of GAG and HP accumulation in 3-D constructs exposed to TNF-alpha. Apparently, adverse effects on chondrocytes exposed to TNF-alpha cannot be prevented by addition of TIMP alone.     

miRNA-200a通过靶向ZEB1参与调节鸡胸肌细胞增殖分化

ZEB1在细胞增殖分化中发挥关键作用,然而关于ZEB1在鸡胸肌细胞增殖分化过程中的功能及其与miRNA互作的研究极少。为探索miRNA如何通过靶向ZEB1参与调节鸡胸肌细胞增殖分化,实验检测了ZEB1在55周龄和20周龄鸡胸肌组织中的表达,使用Target Scan及miRDB在线软件预测鸡ZEB1基因的靶向miRNA,构建ZEB1野生型、突变型双荧光报告载体,并在DF1细胞中验证了ZEB1的靶向miRNA,双荧光素酶报告实验结果说明miR-200a通过特异性结合ZEB1 3'非编码区种子序列直接靶向并抑制ZEB1基因的表达。结果表明:由于miR-200a在55周龄鸡胸肌表达下调,对ZEB1的抑制作用减弱,导致ZEB1在55周龄固始鸡胸肌组织表达较20周龄显著升高(P0.01)。本研究首次在鸡上证明miR-200a是ZEB1的靶向miRNA,且miR-200a可能通过靶向ZEB1参与调节鸡胸肌细胞的增殖分化,为深入理解miRNAs在家禽及其他鸟类中的分子调节机制提供了基础与依据。     

Involvement of the ERK1/2 MAPK pathway in insulin-induced S6K1 activation in avian cells

In mammals, insulin regulates S6K1, a key enzyme involved in the control of protein synthesis, via the well-documented phosphoinositide-3'kinase (PI3K) pathway. Conversely, S6K1 is activated by insulin in avian muscle despite the relative insulin insensitivity of the PI3K pathway in this tissue. Mitogen-activated protein kinase (MAPK) cascade is another insulin sensitive pathway. The aim of this study was to explore the potential involvement of the ERK1/2 MAPK pathway in the control of p70 S6 kinase (S6K1) in avian species. Firstly, we characterized ERK1/2 MAPK in various chicken tissues. ERK2 was the only isoform detected in avian species whatever the tissue studied. We also showed that ERK2 is activated in vivo by insulin in chicken muscle. The regulation and the role of ERK2 in insulin signaling were next investigated in chicken hepatoma cells (LMH) and primary myoblasts. Insulin stimulation led to ERK2 and S6K1 phosphorylation, and concomitantly increased kinase activity. U0126, an inhibitor of the ERK MAPK pathway, completely abolished insulin-induced S6K1 phosphorylation and activity in chicken myoblasts, whereas its effect was only partial in LMH cells. In conclusion, these results show that ERK1/2 MAPK is involved in the control of S6K1 by insulin in chicken cells, particularly myoblasts.