鸵鸟红细胞免疫功能研究

应用红细胞Ｃ３ｂ受体花环（Ｃ３ｂＲＲ）和免疫复合物花环（ＩＣＲ）试验，证实鸵鸟红细胞表面存在补体Ｃ３ｂ受体（Ｃ３ｂｒｅｃｅｐｔｏｒ，Ｃ３ｂＲ），表明红细胞免疫系统（Ｒｃｄｃｅｌｉｍｍｕｎｅｓｙｓｔｅｍ，ＲＣＩＳ）的概念亦适用于这种动物；鸵鸟红细胞不但有传统医学观念上的功能，而且还可借助ＣＲ１（ＣｏｍｐｌｅｍｅｎｔｒｅｃｃｐｔｏｒｔｙｐｅＩ）进行免疫粘附，并以此为基础，表现出各种重要的免疫功能。     

Exendin-4的研究进展

在介绍Exendin-4的基础上,综述了目前最新的Exenatide用于治疗糖尿病的研究进展,并基于大量临床前数据,阐释了其功能、作用机制和毒理学研究。     

甘蓝自交不亲和决定因子的体外表达和相互作用的检测

 S位点受体激酶(SRK)和S位点富含半胱氨酸蛋白/S位点蛋白11(SCR/SP11)分别为甘蓝自交不亲和(self-incompatibility, SI)信号传导的雌雄决定因子。为了深入研究两者的作用机理和进行人工调控，本研究以结球甘蓝ZQ为材料，利用pET·NusA融合蛋白表达系统，将包含SRK胞外域和跨膜域的mSRK蛋白和SCR蛋白在大肠杆菌BL21中融合表达，经SDS-PAGE电泳检测表达出融合蛋白大小分别约为116 kD和74 kD。进一步将两融合蛋白进行体外相互作用的检测，结果表明mSRK与SCR蛋白能够相互结合形成稳定的复合体，这为下一步实现SRK-SCR复合体聚合与解离的人为调控提供了技术平台。     

Adenovirus-mediated mPPARγ1 gene overexpression inhibits IFN-γ-induced galectin-9 gene and protein expression in ECV304

AIM:To investigate whether adenovirus-mediated mPPARγ1 gene overexpression inhibits IFN-γ-induced galectin-9 gene and protein expression in ECV304. METHODS:A replication-deficient recombinant adenovirus expression vector of mPPARγ1 was constructed by using the AdEasy system. ECV304 were incubated for 24 h with 1×104 U/L, 5×104 U/L, 1×105 U/L and 2×105 U/L IFN-γ, respectively. ECV304 stimulated with 1×105 U/L IFN-γ were divided into 4 groups in random: P group (PPARγ1 gene overexpression), T group (treated with troglitazone 40 μmol/L in DMSO), PT group (PPARγ1 gene overexpression+troglitazone treatment) and control group. Changes of PPARγ and galectin-9 in mRNA and protein levels in different groups and subgroups were investigated by RT-PCR and immunoblotting. RESULTS: Galectin-9 expression was very few in normal ECV304. IFN-γ induced the expression of galectin-9 in ECV304. Degree of galectin-9 expression increased with the dose of IFN-γ. PPARγ1 gene overexpression inhibited IFN-γ-induced galectin-9 expression in ECV304. Galectin-9 mRNA and protein expressions from PT group and P group were inhibited in similar degree (P>0.05). However, this effect was not observed in troglitazone intervention (P>0.05). PPARγ expression was also very few in normal ECV304. PPARγ1 gene overexpression/activation had no effect on endogenous mPPARγ expression. CONCLUSION: This may partly contributed to the anti-inflammatory and immuno-regulatory effect of PPARγ1 gene overexpression by inhibiting IFN-γ-induced galectin-9 gene and protein expression in ECV304.     

大黄鱼C型凝集素受体Clec4e的分子鉴定及凝集特性

C型凝集素受体（C-type lectin receptor，CTLR）是可以特异性结合糖类病原体相关分子模式（pathogen-associated molecular patterns，PAMPs）的模式识别受体（pattern recognition receptors，PPRs），在先天性免疫中发挥着重要作用。为了揭示硬骨鱼CTLR的生物学功能，本研究以从大黄鱼（Larimichthys crocea）转录组数据库中筛选出的一个CTLR基因—C型凝集素结构域家族4成员E基因（C-type lectin domain family 4 member E gene，Clec4e）为研究对象，研究其分子特征、表达分布和凝集特性。结果显示，LcClec4e cDNA全长1546 bp，开放阅读框（open reading frame，ORF）771 bp，编码254个氨基酸。LcClec4e的N端有一个跨膜区，无信号肽，C端含有一个糖识别结构域（carbohydrate recognition domain，CRD），其中含有糖结合位点EPN和WFD以及6个可形成二硫键的保守半胱氨酸。系统发育分析表明，LcClec4e与多种鲈形目鱼类Clec4e具有较近的亲缘关系。荧光定量PCR结果显示，LcClec4e在所检测的10种组织中呈组成型分布，且在肝脏中表达量最高；LcClec4e在来源于大黄鱼头肾组织的原代巨噬细胞、淋巴细胞和粒细胞中均有表达，且在巨噬细胞中表达量最高；经灭活溶藻弧菌刺激后，LcClec4e在3种免疫细胞中的表达均极显著上调。原核表达的重组LcClec4e胞外段（recombinant LcClec4e-extracellular domain，rLcClec4e-ex）具有Ca2+依赖性的凝集活性，可凝集小鼠、家兔的红细胞，以及嗜水气单胞菌、变形假单胞菌、溶藻弧菌和坎氏弧菌等4种水产常见的革兰氏阴性菌。D-葡萄糖、D-果糖、D-甘露糖、D-麦芽糖、α-乳糖和脂多糖均可抑制rLcClec4e-ex对大黄鱼重要病原菌变形假单胞菌的凝集作用，说明LcClec4e可能与变形假单胞菌表面的糖类物质结合。这些研究结果提示，LcClec4e可能作为一种PPR，通过结合病原菌表面的糖类PAMPs来识别病原，参与大黄鱼抗细菌感染的免疫防御。     

Bortezomib attenuates LPS-induced inflammatory response of human monocytes through regulating miR-223/NLRP3 signaling pathway

AIM To investigate the effects of bortezomib (BTZ) on microRNA-223 (miR-223)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathway and lipopolysaccharide (LPS)-induced inflammatory response of human monocytes. METHODS Monocytes were isolated and purified from peripheral blood of rheumatodid arthritis (RA) patients. The levels of interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in supernatants of the monocytes were determined by ELISA, and the optimal induction time of LPS and the optimal treatment concentration of BTZ were selected according to the levels of IL-6, IL-1β and TNF-α. The monocytes were divided into control group, LPS induced group and BTZ group. The level of miR-223 in the monocytes was measured by RT-qPCR, and the protein levels of NLRP3, caspase-1, suppressor of cytokine signaling 1 (SOCS1) and SH2 domain-containing inositol phosphatase-1 (SHIP-1) in the monocytes were determined by Western blot. RESULTS The monocytes successfully isolated and purified from the peripheral blood of RA patients were spherical, evenly distributed and regular in shape.And after LPS induction for 24 h, the cells were mostly spindle-shaped and aggregated. According to the levels of IL-6, IL-1β and TNF-α, the optimal induction time of LPS was 24 h, and the optimal concentration of BTZ was 50 nmol/L. Compared with control group, the levels of miR-223, SOCS1 and SHIP-1 in LPS induction group were significantly decreased (P<0.05), and the levels of NLRP3 and caspase-1 were significantly increased (P<0.05). Compared with LPS induction group, the levels of miR-223, SOCS1 and SHIP-1 in BTZ group were significantly increased (P< 0.05), and the levels of NLRP3 and caspase-1 were significantly lowered (P<0.05). CONCLUSION Bortezomib may block the activation of miR-223/NLRP3 signaling pathway, thus reducing the secretion of inflammatory factors in LPS-induced human monocytes.     

非洲猪瘟病毒感染后猪外周血淋巴细胞Toll样受体信号通路的lncRNA-mRNA分析

【目的】丰富非洲猪瘟病毒（African swine fever virus,ASFV）感染后猪外周血淋巴细胞长链非编码RNA（long non-coding RNA,lncRNA）表达谱,并进一步挖掘影响Toll样受体信号通路的调控网络。【方法】试验动物感染ASFV,于第7天采集外周血并分离得到外周血淋巴细胞,运用Illumina高通量组学测序对外周血淋巴细胞中lncRNA进行测序,原始数据经处理后筛选获得差异表达的lncRNA,并进行靶基因预测,利用生物信息学方法对靶基因进行GO功能和KEGG信号通路富集分析,初步绘制与Toll样受体信号通路相关的lncRNA-mRNA调控网络,并对lncRNA-ENSSSCG00000041959在内的4个lncRNAs进行实时荧光定量RT-PCR验证。【结果】共筛选到73个差异表达的lncRNAs,其中上调表达lncRNAs 38个,下调表达lncRNAs 35个。GO功能分析结果显示,靶基因显著富集在调节免疫系统过程、防御反应、生物刺激、病毒反应和先天性免疫;KEGG信号通路富集分析显示,大部分靶基因与细胞循环、疾病及免疫应答有关,其中免疫相关信号通路有Toll样受体信号通路、TNF信号通路、产生IgA的肠道免疫网络等。进一步挖掘出lncRNA-ENSSSCG00000041959-RIPK1和lncRNA-ENSSSCG00000041959-IRAK1可能是影响Toll样受体信号通路的重要调控网络,实时荧光定量RT-PCR与测序结果一致。【结论】本研究初步鉴定出lncRNA-ENSSSCG00000041959-RIPK1和lncRNA-ENSSSCG00000041959-IRAK1可能是影响Toll样受体信号通路的lncRNA-mRNA调控网络,为进一步探索lncRNA调控ASFV感染机体免疫反应奠定了理论基础。     

The consequence of low mannose-binding lectin plasma concentration in relation to susceptibility to Salmonella Infantis in chickens

Mannose-binding lectin (MBL) is a key protein in innate immunity. MBL binds to carbohydrates on the surface of pathogens, where it initiates complement activation via the lectin-dependent pathway or facilitates opsonophagocytosis. In vitro studies have shown that human MBL is able to bind to Salmonella, but knowledge in relation to chicken MBL and Salmonella is lacking. In order to study this relation day-old chickens from two selected lines L10H and L10L, differing in MBL serum concentration, were either orally infected with S. Infantis (S.123443) or kept as non-infected controls. The differences between healthy L10H and L10L chicken sublines were more profound than differences caused by the S. Infantis infection. The average daily body weight was higher for L10H than for L10L, regardless of infection, indicating beneficial effects of MBL selection on growth. Salmonella was detected in cloacal swabs and the number of Salmonella positive chickens during the experiment was significantly higher in L10L than L10H, indicating that MBL may affect the magnitude of Salmonella colonisation in day-old chickens. MBL expression was determined in ceca tissue by real-time RT-PCR. L10H chickens showed a significantly higher relative expression than L10L at days 1 and 41 pi, regardless of infection. Finally, flow cytometric analysis of whole blood from infected chickens showed that L10H had a significantly higher count of all assessed leucocyte subsets on day 5 pi, and also a higher count of monocytes on day 12 pi than L10L. No difference was observed between infected and non-infected L10L chicken.     

Characterization of an intravenous lipopolysaccharide inflammation model in calves with respect to the acute-phase response

Our objective was to develop a lipopolysaccharide (LPS) inflammation model in calves to evaluate the acute-phase response with respect to the release of pro-inflammatory cytokines and acute-phase proteins, fever development and sickness behaviour. Fourteen 4-week-old male Holstein Friesian calves were included and randomly assigned to a negative control group (n = 3) and an LPS-challenged group (n = 11). The latter received an intravenous bolus injection of 0.5 μg of LPS/kg body weight. Blood collection and clinical scoring were performed at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 18, 24, 28, 32, 48, 54 and 72 h post LPS administration (p.a.). In the LPS group, the following clinical signs were observed successively: tachypnoea (on average 18 min p.a.), decubitus (29 min p.a.), general depression (1.75 h p.a.), fever (5 h p.a.) and tachycardia (5 h p.a.). Subsequent to the recovery from respiratory distress, general depression was prominent, which deteriorated when fever increased. One animal did not survive LPS administration, whereas the other animals recovered on average within 6.1 h p.a. Moreover, the challenge significantly increased plasma concentrations of tumour necrosis factor-α, interleukin 6, serum amyloid A and haptoglobin, with peaking levels at 1, 3.5, 24 and 18 h p.a., respectively. The present LPS model was practical and reproducible, caused obvious clinical signs related to endotoxemia and a marked change in the studied inflammatory mediators, making it a suitable model to study the immunomodulatory properties of drugs in future research.