归参汤对免疫抑制小鼠血清中IL-2、TNF-α和脾细胞Th1/Th2细胞因子的影响

为了探讨归参汤(GST)对免疫抑制小鼠血清中IL-2、TNF-α和脾细胞Th1/Th2细胞因子的影响。本试验采用环磷酰胺制造免疫抑制模型,利用双抗夹心ELISA法,观察归参汤对小鼠血清中IL-2、TNF-α的影响;RT-PCR法观察归参汤对小鼠脾细胞中INF-γ、IL-4、T-bet和GATA-3的mRNA表达的影响。结果显示:归参汤高剂量组可以显著增加免疫抑制小鼠血清中IL-2、TNF-α的含量(P<0.01),提高INF-γ的表达(P<0.05),降低IL-4的含量(P<0.05),升高T-bet(P<0.05),降低GATA-3的表达(P<0.05)。归参汤的免疫增强作用与升高血清中IL-2、TNF-α有关,且升高INF-γ/IL-4纠正免疫抑制小鼠Thl/Th2细胞因子的比例紊乱,其机制可能部分归因于对T-bet和GATA-3的mRNA表达的影响。     

Spatiotemporal heterogeneity of PPARγ expression in porcine uteroplacenta for regulating of placental angiogenesis through VEGF-mediated signalling

Non-infectious prenatal mortality severely affects the porcine industry, with pathological placentation as a likely key reason. Previous studies have demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) deficiency causes defects in the uteroplacental vasculature and induces embryonic losses in mice. However, its role in porcine placental angiogenesis remains unclear. In the present study, PPARγ expression was investigated in porcine uteroplacental tissues at gestational day (GD) 25, GD40 and GD70 via quantitative polymerase chain reaction (qPCR), Western blot and immunohistochemistry (IHC). Moreover, the roles of PPARγ in porcine placental angiogenesis were investigated using a cell model of porcine umbilical vein endothelial cells (PUVECs) to conduct proliferation, migration and tube formation assays in vitro and a mouse xenograft model to assess capillary formation in vivo. The results showed that PPARγ was mainly located in the glandular epithelium, trophoblast, amniotic chorion epithelium and vascular endothelium, as indicated by the higher expression levels at GD25 and GD40 than at GD70 in endometrium and by higher expression levels at GD40 and GD70 than at GD25 in placenta. Moreover, PPARγ expression was significantly downregulated in placenta with dead foetus. In PUVECs, knocking out PPARγ significantly inhibited proliferation, migration and tube formation in vitro and inhibited capillary formation in mouse xenografts in vivo by blocking S-phase, promoting apoptosis and downregulating the angiogenic factors of VEGF and its receptors. Overall, the spatiotemporal heterogeneity of PPARγ expression in porcine uteroplacental tissue suggests its vital role in endometrial remodelling and placental angiogenesis, and PPARγ regulates placental angiogenesis through VEGF-mediated signalling.     

Immunohistochemical expression of interferon-γ in different types of granulomatous lesions associated with bovine paratuberculosis

Animals infected with Mycobacterium avium subsp. paratuberculosis (Map) show a variety of lesions, from focal forms, seen in subclinical stages to diffuse lesions in clinical cases. The purpose of this study was to evaluate the local expression of IFN-γ by immunohistochemistry in relation with the type of lesion in naturally Map-infected cows. The number of immunolabelled cells, −the majority morphologically consistent with lymphocytes-, was higher in focal and diffuse paucibacillary forms than in diffuse multibacillary lesions, where they appeared closely related to epithelioid cells. Diffuse multibacillary lesions had the lowest numbers, but higher than controls, and positive cells were intermingled among the macrophages. The peripheral IFN-γ production was higher in all Map infected cows and a positive correlation was found with the number of immunolabelled cells in the intestine. The findings of this study show that IFN-γ would play a role in the development of the different types of lesions in paratuberculosis, and also points out the importance of adequate sampling of lymphoid tissue containing samples when studying the local immune response in which IFN-γ expression may be involved, especially in cases where focal lesions are present.     

Cytokine profiles in peripheral blood mononuclear cells from patients with cystic echinococcosis

IntroductionCystic echinococcosis (CE) is a chronic zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus), which affects domestic and wild carnivores as the definitive host and ungulates as intermediate hosts. In intermediate hosts, both Th1 and Th2 cells are involved in the immune responses to an echinoccocal infection. This study aimed to investigate production of IL-4, IL-10, and IFN-γ cytokines in peripheral blood mononuclear cells (PBMCs) of CE patients before and after surgical treatment.MethodsTo evaluate cytokine production in response to E. granulosus antigens, we investigated IL-4, IL-10, and IFN-γ production in PBMCs of 20 CE patients in response to hydatid cyst fluid antigen (HCF-Ag) before and after surgical treatment using ELISA.ResultsThe mean IL-4 production from HCF-Ag stimulated PBMCs was significantly decreased (p < 0.05), while IFN-γ was significantly increased in HCF-Ag stimulated PBMCs in patients after surgery (p = 0.005).Furthermore, our results showed that there is no significant difference between IL-10 production in patients before and after treatment (p = 0.562).ConclusionsOur data Indicated production of IL-4 in cultured PBMCs of CE patients stimulated with HCF-Ag was decreased significantly. While, production of IFN-γ was increased significantly in responses to HCF Ag after surgery. We concluded that the evaluation of IL-4 and IFN-γ in HCF-Ag stimulated PBMCs of CE patients should be considered as a useful marker in the follow up of patients with cystic echinococcosis.     

利用米糠外添GAD制备γ-氨基丁酸的工艺条件优化

以稻米加工副产品米糠为原料,采用单因素和正交实验确定了外添谷氨酸脱羧酶(GAD)制备γ-氨基丁酸(GABA)的最佳工艺条件:GAD酶用量175u/g,酶解时间2h,pH5.5,酶解温度45℃。经验证实验,在最佳工艺条件下GABA生成量为1530.7mg/100g米糠。     

复方术苦芩提取液对腹泻小鼠iIELs及十二指肠INF-γ、IL-4 mRNA转录的影响

旨在研究复方术苦芩提取液对腹泻小鼠小肠上皮内淋巴细胞（intestinal intraepithelial lymphocyte,iIELs）及十二指肠INF-γ、IL-4 mRNA转录的影响。将72只小鼠随机分为6个组,分别为空白组、感染组、阳性药物组（黄芪多糖口服液,浓度为1.2 mg·mL^-1）和药物Ⅰ、Ⅱ、Ⅲ组（复方术苦芩提取液,生药浓度分别为0.1、1和10 mg·mL^-1）。除空白组外,小鼠连续给药7 d[灌胃0.5 mL·（次·d）^-1]后腹腔注射5×10⁷ CFU大肠杆菌,并将感染组小鼠出现腹泻时设定为试验0 h。于试验6 h和4 d时各处死6只小鼠,制作小肠病理切片并计数iIELs数量,qRT-PCR分析十二指肠段INF-γ、IL-4 mRNA水平。结果显示：阳性药物组和药物Ⅰ、Ⅱ、Ⅲ组对腹泻小鼠的保护率分别为66.7%、50.0%、66.7%、75.0%。试验6 h时,与空白组相比,感染组小鼠iIELs数量增加,十二指肠INF-γ mRNA水平降低、IL-4 mRNA水平增高,差异均极显著（P<0.01）;与感染组相比,阳性药物组和药物Ⅰ、Ⅱ、Ⅲ组小鼠iIELs数量减少,阳性药物组和药物Ⅲ组小鼠十二指肠INF-γ mRNA水平增高、IL-4 mRNA水平降低。试验4 d时,与空白组相比,感染组小鼠十二指肠和回肠的iIELs数量增加,空肠iIELs数量减少,其十二指肠INF-γ、IL-4 mRNA水平增高,差异均极显著（P<0.01）;与感染组相比,阳性药物组和药物Ⅰ、Ⅱ、Ⅲ组小鼠十二指肠和回肠的iIELs数量（Ⅰ组十二指肠除外）均减少,空肠iIELs数量增加,阳性药物组和药物Ⅲ组十二指肠段INF-γ、IL-4 mRNA水平均降低。结果表明,复方术苦芩提取液通过调控小鼠小肠iIELs数量,平衡十二指肠INF-γ/IL-4 mRNA转录,达到对小鼠腹泻的保护作用。     

乙酸浓度对奶牛乳腺上皮细胞甘油三酯含量及瘦素和过氧化物酶增殖物激活受体γ基因表达量的影响

本试验旨在研究乙酸浓度对奶牛乳腺上皮细胞甘油三酯(TAG)含量及瘦素(leptin)和过氧化物酶增殖物激活受体γ(PPARγ)基因表达量的影响。将传至第3代的奶牛乳腺上皮细胞以适宜的密度培养48 h后,随机分为6个处理,各处理分别采用乙酸浓度为0(对照组)、4、6、8、10和12 mmol/L的培养液。置于37℃5%CO2培养箱继续培养48 h。收集细胞,观测脂滴形成状况,测定TAG含量及leptin和PPARγ基因表达量。结果表明:随乙酸浓度的增加,培养的奶牛脂肪前体细胞脂滴形成增多;随乙酸浓度的增加,TAG含量和PPARγ基因表达量均呈显著的线性或二次曲线增加(P<0.05),且以乙酸浓度为10和12 mmol/L时效果较好;一定浓度的乙酸可上调leptin基因表达量,尤以浓度为8、10 mmol/L时上调作用较好。结果提示,乙酸对奶牛乳腺上皮细胞内脂滴的形成、TAG的积累、leptin和PPARγ基因的表达有显著的促进作用。本试验条件下,乙酸浓度为10～12 mmol/L时能较好地促进PPARγ基因的表达,浓度为8～10 mmol/L时能较好地促进leptin基因的表达。     

Constitutive expression of interferons in swine leukocytes

Interferon (IFN)-α and IFN-γ positive cells were revealed by flow cytometry in peripheral blood mononuclear cells (PBMC) of Specific Pathogen Free (SPF) pigs. A low prevalence of IFN-γ positive cells was also detected in PBMC of some Porcine Reproductive and Respiratory Syndrome virus-infected pigs and uninfected, control pigs. IFN-α positive cells showed phenotypes of both monocytes and plasmacytoid dendritic cells. The presence of IFN-α in PBMC was also confirmed by Western blotting. By immunoprecipitation, IFN-α was detected as 32 and 55-57 kDa bands in PBMC of healthy SPF piglets. These samples were also IFN-γ positive; the cytokine was revealed as 24, 37 and 54 kDa bands. The unusual molecular mass values of intracellular interferons were probably due to oligomerization, as previously described for human IFN-α. Swine intracellular IFN-α displayed the expected antiviral activity on bovine MDBK cells. The results indicate that interferons are constitutively expressed in swine leukocytes with peculiar molecular features.     

东北虎γ-干扰素基因的克隆、生物信息学分析及其在毕赤酵母中的表达

本试验旨在通过克隆东北虎γ-干扰素(IFN-γ)基因,研究其分子特征并预测蛋白生物学功能,为后续研究干扰素抗病毒活性做前期准备。通过RT-PCR从ConA诱导过的东北虎血淋巴细胞中扩增东北虎IFN-γ基因并测序,应用生物信息学方法进行序列分析。结果表明:东北虎IFN-γ编码区由504个核苷酸组成,共编码167个氨基酸,蛋白相对分子质量为19.59 ku,等电点为9.03,所编码的蛋白为碱性亲水性蛋白,其中前23个氨基酸可能为信号肽,IFN-γ编码蛋白保守结构域为IFN-γ超家族,且存在跨膜结构,其中1-6位氨基酸为胞内区域,7-28位氨基酸为跨膜区域,29-167位氨基酸为胞外区域;IFN-γ编码蛋白二级结构主要以α-螺旋(58.08%)和无规则卷曲(33.53%)为主,存在5个潜在的B细胞抗原表位,3个潜在的N-糖基化位点;分子进化分析显示,东北虎IFN-γ与GenBank上发表的东北虎、非洲狮、金钱豹、美洲狮、猎豹、家猫、加拿大猞猁、野猪等的核苷酸相似性为80.4%~99.8%,氨基酸相似性为70.5%~100%,东北虎IFN-γ与非洲狮、金钱豹亲缘关系最近,美洲狮、猎豹、家猫、猞猁次之,野猪最远。通过合成改造后的东北虎IFN-γ基因,构建能表达IFN-γ蛋白的重组质粒pPIC9K-IFN-γ,将其导入高效表达系统-毕赤酵母中进行诱导表达,经SDS-PAGE分析,表达蛋白的分子量约17.8 ku,与预期大小相符,表明东北虎IFN-γ成功表达。     

地塞米松对猪原代脂肪细胞分化及perilipin和PPARγ mRNA表达的影响

由断奶仔猪皮下脂肪分离血管基质细胞,增殖培养至80%融合时以10-6mol/L地塞米松处理48 h,同时设地塞米松拮抗剂RU486对照组。采用油红O染色提取法测定细胞内甘油三酯含量,相对定量实时荧光PCR方法检测细胞perilipin,GR,C/EBPβ,PPARγmRNA表达。结果显示,地塞米松显著增加细胞甘油三酯含量(P0.05),添加RU486后这种作用显著降低(P0.05);地塞米松处理组细胞中perilipin,PPARγ的mRNA表达显著高于对照组(P0.05),而C/EBPβmRNA表达较对照组显著降低(P0.05);GR mRNA表达与对照组相比无显著差异。以上结果提示:地塞米松能显著促进猪原代脂肪细胞的分化,这种作用可能通过上调perilipin mRNA表达而实现,其中PPARγ基因参与了调节。