Prognostic value of tumour‐associated macrophages in canine mammary tumours

Tumour‐associated macrophages (TAMs) have already been associated in human breast cancer to a poor prognosis. As a part of a tumoural microenvironment, TAMs have an important contribution influencing neoplastic progression. Hitherto, in canine mammary tumours (CMT) the prognostic value of TAMs has not been reported. In this study, MAC387 immunohistochemical expression was evaluated in 59 CMTs (20 benign and 39 malignant). The TAM value was significantly higher in malignant than benign CMT (P = 0.011). In malignant CMT, TAMs were associated with skin ulceration (P = 0.022), histological type (P = 0.044), nuclear grade (P = 0.031) and tubular differentiation (P = 0.042). The survival analysis revealed a significant association between tumours with higher levels of TAMs and the decrease in overall survival (P = 0.030). TAMs have proven to have a prognostic value. These findings suggest the future possibility of using TAMs as a novel therapeutic target in CMT.     

不同产地猴头茵子实体粗多糖特征及其刺激巨噬细胞产生NO的活性

提取7种不同来源的猴头菌(Hericium erinaceus)子实体粗多糖,对其理化性质和体外免疫活性进行分析.结果表明:不同产地的猴头菌子实体粗多糖含量和得率差异较大,以来源于黑龙江省牡丹江市海林县(H1)、林口县(H3)的子实体粗多糖含量、得率较高;各粗多糖提取物分子量分布相似,单糖组成均以葡萄糖为主,并含有岩藻糖和半乳糖;7种子实体粗多糖提取物均能增强小鼠巨噬细胞株RAW264.7生成NO的量,以500 μg/mL H3提取物活性最高、H1次之.     

Th1/Th2型免疫反应相关基因在巨噬细胞RAW 264.7应对细粒棘球绦虫原头蚴刺激时的表达谱研究

本研究旨在解析巨噬细胞RAW264.7在应对细粒棘球绦虫原头蚴刺激时其Th1、Th2型免疫反应相关基因的差异表达规律,为进一步揭示巨噬细胞抗细粒棘球绦虫原头蚴免疫调控机制奠定理论基础.将细粒棘球绦虫原头蚴和巨噬细胞RAW264.7共培养6、24、72 h,收集RAW264.7细胞,提取总RNA,构建cDNA文库,利用R...     

Effect of P2X7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7

AIM: To establish a cell line of stable silencing of P2X₇ receptor (P2X₇R) expression through short hairpin RNA (shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line. METHODS: Stable silencing of P2X₇R gene in the RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine P2X₇R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X₇R silencing in G418 resistant cells was verified by immunofluorescent microscopy and real-time PCR, respectively. The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation assay. The cell cycle distribution and apoptosis were evaluated by flow cytometry. RESULTS: The expression of P2X₇R at mRNA and protein levels was down-regulated by 80% in sh P2X₇R group compared with negative control (NC) plasmid transfection. In addition, P2X₇R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05). Compared with NC cells, P2X₇R silencing resulted in an increase in the phagocytosis of the cells (P<0.05). CONCLUSION: A cell line RAW264.7 of stable silencing of P2X₇R expression was successfully established. P2X₇R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macrophages.     

IL-4对肝片吸虫感染诱导的巨噬细胞中脂肪酸结合蛋白2分布的影响

为阐明小鼠感染肝片吸虫后巨噬细胞中脂肪酸结合蛋白2(FABP2)的分布,采用肝片吸虫囊蚴为感染源,经口分别感染雌性BALB/c野生型(WT)及其IL-4单抗处置小鼠,在运用特异性PCR鉴定成功感染小鼠后获取巨噬细胞,并设立人工巯基醋酸盐诱导巨噬细胞对照组进行体外培养。用荧光定量PCR检测选择性激活巨噬细胞的标记蛋白Relmα、Ym1和 Arginase1以确定其激活状态。采用特异性抗体对所获取的巨噬细胞细胞质染色后用共聚焦显微镜摄取不同时间点从不同小鼠体内获取的巨噬细胞染色后的图片。野生型BALB/c巨噬细胞中Relmα、Ym1和Arginase1均大量表达;IL-4单抗处置BALB/c小鼠和巯基醋酸盐诱导小鼠中巨噬细胞中的Relmα、Ym1和Arginase1的表达量较野生型小鼠中的极显著或显著下降。FABP2在FeMΦ中并不分布于细胞质膜下,而是呈点状广泛分布于细胞质中,其荧光强度在12~24 h间呈上升趋势,在24~48 h间却呈下降趋势。     

Infectious pancreatic necrosis virus establishes an asymptomatic carrier state in kidney leucocytes of juvenile Atlantic cod, Gadus morhua L

Juvenile Atlantic cod (10 g) were infected with infectious pancreatic necrosis virus (IPNV) by intraperitoneal injection and cohabitation. Fish showed no signs of disease but IPNV could be re-isolated from kidney tissue for up to 12 weeks. On weeks 2, 5, 8, 10, 11 and 12 following infection, kidney leucocytes were fractionated on Percoll gradients, and cells separated into plastic adherent and non-adherent cell populations after overnight incubation. IPNV was detectable in lysates of both cell populations and in supernatants by culture in CHSE-214 cells. Wells containing 10(5)-10(6) macrophages had an IPNV TCID(50) of about 10(3)/well and in serially diluted macrophages the minimum number of cells required to detect virus ranged from 10(1) to 10(4). These data indicate that about one in 10(4) macrophages were infected and the mean number of virus/infected cell was about 10. Replication of IPNV in the macrophages was low as the titre of the virus in macrophage lysates did not increase between days 1 and 3 of culturing the macrophages, but virus was released into the supernatant over this time.     

草鱼肠巨噬细胞的分离培养与鉴定

为了建立草鱼肠巨噬细胞的分离培养与鉴定方法,本研究在采用刮除法结合胶原酶IV消化法制备肠黏膜固有层单细胞悬液的基础上,使用鱼类脏器单核细胞分离试剂盒分离肠巨噬细胞,再经差异贴壁法纯化肠巨噬细胞,最后,用含10%胎牛血清和5%草鱼血清的RPMI 1640完全培养液,在28°C、5%CO2条件下原代培养肠巨噬细胞,并通过细胞形态学检查、分子标记检测和功能验证实验加以鉴定。结果显示,每尾鱼(约250 g)肠巨噬细胞的获得量约为3×107个,细胞活率为99.6%,纯度达到95%以上;倒置显微镜观察发现,原代培养4~5 d的贴壁细胞多呈圆形或多边形,体积明显增大,细胞汇合度达到95%;光镜和电镜观察到染色后的贴壁细胞具有巨噬细胞的形态结构特征,贴壁细胞经荧光定量PCR在m RNA水平检测到巨噬细胞特异性标志分子(草鱼巨噬细胞集落刺激因子受体),功能实验证实贴壁细胞具有吞噬功能,脂多糖能够显著提高其呼吸暴发活性。本研究首次成功建立了草鱼肠巨噬细胞的分离培养与鉴定方法,为开展口服疫苗诱导的草鱼肠黏膜免疫应答研究提供细胞模型。     

猪肺巨噬细胞FcγR Ⅲ受体基因的克隆与序列分析

为研究猪肺巨噬细胞FcγR Ⅲ的生物学功能,本研究应用RT-PCR技术从猪肺巨噬细胞总RNA中克隆出猪FcγR Ⅲ的cDNA序列,并对其进行了分析。结果表明,克隆到的序列长820 bp,包含有1个771 bp完整开放阅读框(ORF),与GenBank中登录的猪FcγR Ⅲ序列(AF237453)的核苷酸同源性为99.9％;与人、牛、马、绵羊、猕猴、狗、猫、小鼠氨基酸同源性分别为61.6%、62.9%、55.3%、62.2%、63.0%、59.0%、61.8%和53.2%;蛋白质分子结构预测结果表明,该分子由信号肽（20个氨基酸）、胞外区(185个氨基酸)、跨膜区(23个氨基酸)和胞内区(28个氨基酸)组成,在胞外区存在2个Ig样结构域。猪肺巨噬细胞FcγR Ⅲ基因的成功克隆,为进一步研究其结构与功能奠定基础。     

牛抵抗素对牛肺泡巨噬细胞TLR4/MyD88非依赖信号通路基因表达的影响

旨在探究牛抵抗素通过TLR4/MyD88非依赖信号通路诱发牛肺泡巨噬细胞的相关炎症反应机理。使用100 ng·mL^-1终浓度牛抵抗素诱导牛肺泡巨噬细胞,分别在0、1.5、3.0、6.0、12.0、24.0 h时采用qRT-PCR检测TLR4/MyD88非依赖信号通路相关基因(TLR4、TRAM、NF-κB)mRNA水平表达量,通过ELISA法检测下游IL-6、1L-1β、TNF-α等促炎细胞因子表达水平。结果显示,100 ng·mL^-1牛抵抗素处理牛肺泡巨噬细胞后,TLR4、TRAM、NF-κB基因表达量于6.0 h时极显著上调(P<0.01),诱导12.0 h时,相关基因表达量达到最高;IL-6、1L-1β、TNF-α等促炎细胞因子于1.5 h表达量极显著上调(P<0.01),且均存在时间依赖效应。抵抗素诱导牛肺泡巨噬细胞后,可激活TLR4/MyD88非依赖信号通路,通过信号传递,释放大量促炎细胞因子,引起肺部炎症。     

草鱼血红蛋白通过诱导头肾巨噬细胞表达的炎症因子激活免疫反应

为了探究鱼类体内过量游离血红蛋白对鱼体的影响,本实验以草鱼为研究对象,通过体内注射血红蛋白模拟体内出血,探究血红蛋白对组织和组织中巨噬细胞的免疫调控作用。体内注射血红蛋白的实验结果显示,血红蛋白的刺激导致头肾和中肾组织中出现明显的血红蛋白沉淀,同时提高了鱼体血清中的抗氧化相关酶活性,促进了头肾组织中炎症因子基因TNF-α、IL-1β和IL-10的mRNA表达水平。普鲁士蓝和间接免疫荧光实验结果显示,头肾巨噬细胞对血红蛋白表现出了吞噬活性。荧光定量PCR检测结果显示,血红蛋白的刺激显著激活了头肾巨噬细胞中CD68、CD86、CSF和MHC-Ⅱ的mRNA表达水平。进一步的细胞因子检测结果显示,血红蛋白刺激12 h后,头肾巨噬细胞中的炎症因子基因(TNF-α、IL-1β和IL-4)、趋化因子基因(CCL20和CCL4)、IFN-α和TLR4的mRNA表达水平被显著上调表达。研究表明,草鱼头肾巨噬细胞对血红蛋白具有吞噬能力,同时血红蛋白也激活了巨噬细胞中多种细胞因子的表达。本研究结果首次阐述了草鱼血红蛋白与巨噬细胞的相互作用关系,丰富了鱼类血液基础免疫学理论,同时也为鱼类健康养殖提供了新的参考...