桔梗皂苷D对小鼠淋巴细胞和巨噬细胞免疫功能的影响

【目的】探讨桔梗皂苷D对小鼠淋巴细胞和巨噬细胞免疫功能的影响。【方法】无菌分离小鼠脾淋巴细胞和腹腔巨噬细胞,制备淋巴细胞和巨噬细胞悬液。分别培养这2类细胞,并分为空白对照组、阳性对照组(左旋咪唑处理细胞)及25,50,75和100μg/mL桔梗皂苷D组,采用MTT法检测桔梗皂苷D对淋巴细胞增殖和巨噬细胞吞噬功能的影响,采用ELISA法检测桔梗皂苷D对淋巴细胞IL-2、IL-4和巨噬细胞TNF-α、IL-12分泌的影响。【结果】桔梗皂苷D能够促进淋巴细胞增殖,增强巨噬细胞的吞噬功能,并可刺激淋巴细胞IL-2、IL-4和巨噬细胞TNF-α、IL-12的分泌,其中以50μg/mL组效果最佳。【结论】桔梗皂苷D能增强小鼠淋巴细胞和巨噬细胞的免疫调节活性。     

三氯生通过诱导巨噬细胞非mTOR途径自噬加强胞内菌杀伤

为了研究三氯生能否引起细胞自噬并探讨其可能的诱导通路,揭示三氯生作为抗菌药物的新作用机制,利用免疫荧光、菌落计数、免疫印迹等技术检测经三氯生处理过细胞的自噬水平、相关蛋白表达量的变化及杀菌能力的改变。结果表明:三氯生能引起Hela细胞和Raw264.7细胞的自噬,并且该自噬为完全自噬。三氯生诱导的小鼠巨噬细胞系Raw264.7细胞自噬依赖的是胞外信号调节激酶(Extracellular signal-regulated kinase,ERK)途径,而非经典的雷帕霉素靶蛋白(Mammalian target of rapamycin,m TOR)途径。此外,三氯生通过该作用机制加强了巨噬细胞对胞内菌的杀伤作用。研究表明,三氯生诱导自噬的现象在细胞中普遍存在,自噬在对抗病原微生物中起到重要作用,诱导自噬是三氯生作为抗菌药物的新作用机制。     

PRRSV和PCV2共感染对猪肺泡巨噬细胞CD80-CD86mRNA转录的影响

用猪生殖与呼吸综合征病毒（PRRSV）与猪圆环病毒2型（PCV2）共感染40日龄健康大白仔猪，利用实时荧光定量PCR技术对共感染仔猪肺泡巨噬细胞（PAM）共刺激分子CD80一CD86的mRNA转录水平进行了定量分析。结果表明，在感染后第3d和第7dCD80与CD86mRNA转录显著下调（P〈0．05），感染后第14dCD86mRNA转录水平仍低于未感染对照组。尽管CD80mRNA转录水平在第14d和第28d高于对照组，CD86mRNA转录水平在第28d高于对照组，两者在第42d均高于对照组，但无显著差异。证实，PRRSV和PCV2共感染可导致猪肺泡巨噬细胞的共刺激分子CD80-CD86基因转录在感染早期明显受到抑制，PAM的抗原呈递能力受到影响。     

Role of Fatty Acid Binding Protein 4 in Regulating Macrophage Autophagy Induced by BCG Infection

To investigate the role of FABP4 on regulating fatty acid metabolism and autophagy in macrophage infected with BCG, small interfering RNA for FABP4 were transfected into RAW264.7 cells alone or combined with BCG infection. The immunoblot and immunofluorescence were used to detect the expression of FABP4, the accumulation of lipid droplets, the expression of fatty acid β-oxidation and autophagy related factors. The results showed that the infection of BCG increased the expression of FABP4 and accompanied with the accumulation of lipid droplets in RAW264.7. Moreover, the knockdown of FABP4 decreased lipid droplets but promoted the expression of carnitine palmitoyltransferase 1A (CPT1A) (P <0.05) and the production of ATP (P <0.05). Meanwhile, the knockdown of FABP4 suppressed the expression of AMPK, p-ULK1, ATG5, ATG7, ATG12 and LC3B which are autophagy related factors (P<0.001). Our results indicated that the knockdown of FABP4 promoted fatty acid oxidation, decreased fatty acid level and suppressed autophagy via AMPK signal pathway in BCG infected RAW264.7 cells.     

Cytologic evaluation of benign and malignant hemophagocytic disorders in canine bone marrow

Abstract: Canine hemophagocytic disorders were studied to better understand the cytologic features that differentiate benign and malignant disease. Of 286 canine clinical bone marrow reports evaluated retrospectively, 13 (4.5%) noted at least 3% hemophagocytic macrophages. Macrophages comprised between 6% and 44% of nucleated bone marrow cells. Clinical diagnoses for dogs with hemophagocytic disorders included malignant histiocytosis (n = 2), myelodysplastic syndromes (n = 4), round cell neoplasia (n = 2), immune-mediated disorders (n = 2), and idiopathic hemophagocytic syndrome (n = 3). Differentiation of benign and malignant forms of histiocytosis was problematic. Two dogs with a diagnosis of hemophagocytic syndrome had macrophages with atypical features similar to those described for malignant histiocytosis. Furthermore, only 2 of 11 dogs with presumably benign hemophagocytic disorders had exclusively mature macrophages in bone marrow. Other dogs had variable numbers of large reticular-type cells characterized by lacy chromatin, anisocytosis, anisokaryosis, and prominent and/or multiple nucleoli. On the basis of these results, cytomorphologic evaluation of bone marrow alone may not be adequate to consistently differentiate benign and malignant forms of hemophagocytic disorders.     

Properties of macrophages and lymphocytes appearing in rat renal fibrosis followed by repeated injection of cisplatin

Properties of macrophages and lymphocytes appearing in renal fibrosis remains to be investigated. F344 rats were injected once a week with cisplatin (2 mg/kg body weight) for 8 weeks and examined at post-final injection weeks 1, 3, 6, 9, and 12. Rats developed progressive renal fibrosis at weeks 1 to 6 as fibrosis-progress phase, and subsequent amelioration at weeks 9 and 12. CD68⁺ M1-macrophages and major histocompatibility complex (MHC) class II⁺ macrophages remarkably increased persistently, whereas CD163⁺ M2-macrophages slightly increased. MHC class II⁺/CD68⁺ and MHC class II⁺/CD163⁺ macrophages were present, indicating that MHC class II⁺ macrophages might have both functions of M1- and M2-macrophages. In the fibrosis-progress phase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ for M1-factors, and transforming growth factor (TGF)-β1 and IL-10 for M2-factors tended to increase; tissue injury by M1 and fibrosis by M2 might have occurred simultaneously. Lots of CD4⁺ and CD8⁺ T cells appeared in close relation with MHC class II⁺ macrophages, and mainly CD4⁺ T cells formed aggregations. In the lymphocyte aggregates collected by laser microdissection, expression of IL-17A (for Th17 cells) and forkhead box P3 (FoxP3) (for Treg) significantly increased at weeks 1 and 6, respectively; presumably, Th17 cells might be involved in tissue injury, whereas Treg might be related to fibrosis amelioration. These results suggested that macrophages and T cells may contribute interrelatedly to renal fibrosis.     

Immunohistochemical study of matrix metalloproteinases‐2 and ‐9, macrophage inflammatory protein‐2 and tissue inhibitors of matrix metalloproteinases‐1 and ‐2 in normal,purulonecrotic and fungal infected equine corneas

Objective Determine the effects of matrix metalloproteinases (MMPs)‐2, ‐9, macrophage inflammatory protein‐2 (MIP‐2), tissue inhibitors of matrix metalloproteinase (TIMP)‐1 and ‐2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Procedure Paraffin‐embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP‐2, ‐9, MIP‐2, TIMP‐1 and ‐2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 proteins. Results Matrix metalloproteinases‐2, ‐9, MIP‐2, TIMP‐1 and ‐2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP‐2 and MIP‐2, MMP‐9 and MIP‐2, and MMP‐9 and TIMP‐1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP‐9 and MIP‐2, MMP‐9 and TIMP‐2, MIP‐2 and TIMP‐1, and MIP‐2 and TIMP‐2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. Conclusion Increased immunoreactivity of MMP‐2 and ‐9 in FA and PN samples is indirectly related to MIP‐2 through its role in neutrophil chemo‐attraction. Tissue inhibitors of matrix metalloproteinase‐1 and TIMP‐2 are up‐regulated in equine purulonecrotic and fungal keratitis secondary to MMP‐2 and MMP‐9 expression. The correlation between MMPs ‐2 and ‐9, MIP‐2, TIMPs ‐1 and ‐2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.     

猪圆环病毒2型诱导3D4/2细胞氧化应激模型的建立

通过研究不同浓度猪圆环病毒2型(PCV2)对体外感染3D4/2细胞产生氧化应激水平的影响,来确定建立PCV2体外诱导3D4/2细胞氧化胁迫模型的条件。以5个不同浓度PCV2感染组(100、10-1、10-2、10-3、10-4)作用于3D4/2细胞2h,弃去病毒液,加入含100mL/L胎牛血清1640培养液后继续培养,于4、8、12、24、48h分别收集细胞上清液或细胞,测定NO、ROS、GSH、GSSG、XOD、MPO和iNOS等指标。10-1PCV2感染3D4/2细胞24h、48h后显著升高细胞NO水平,感染4h~24h显著升高细胞ROS水平,降低细胞GSH水平和GSH/GSSG比值;10-1 PCV2感染3D4/2细胞4h、8h显著升高细胞GSSG水平;10-1PCV2感染3D4/2细胞4、8、12、24h均能显著升高细胞XOD、MPO、iNOS活力,提示10-1PCV2感染3D4/2细胞一定程度上改变了细胞氧化还原状态,诱导了细胞产生氧化应激,而病毒感染后48h未检测到PCV2核酸。表明PCV2感染后4h~24h能诱发3D4/2细胞氧化应激,选择10-1PCV2作为氧化应激模型的感染剂量。     

PYY对LPS诱导小鼠腹腔巨噬细胞TNF-α、IL-6分泌的影响

为明确PYY对巨噬细胞炎性细胞因子分泌的调节作用,本试验分离培养健康小鼠腹腔巨噬细胞,不同浓度PYY预处理后,以LPS刺激。ELISA方法检测细胞培养上清中TNF-α、IL-6含量,半定量PCR方法检测细胞中TNF-α、IL-6mRNA表达变化。结果显示：高浓度的PYY1-36（10-9-10-7 mol/L）和PYY3-36（10-8-10-7 mol/L）对LPS诱导小鼠腹腔巨噬细胞TNF-α分泌具有显著抑制作用（P〈0.05）;PYY1-36对LPS诱导小鼠腹腔巨噬细胞IL-6分泌无明显作用（P〉0.05）;不同浓度PYY3-36（10-11-10-7 mol/L）对LPS诱导小鼠腹腔巨噬细胞IL-6分泌均具有显著抑制作用（P〈0.05）。表明PYY对LPS诱导小鼠腹腔巨噬细胞炎性细胞因子TNF-α及IL-6的分泌具有一定的抑制作用,提示PYY可能通过抑制炎性细胞因子的分泌而抑制炎症性疾病的发生发展。     

感染旋毛虫小鼠腹腔巨噬细胞TLR4及细胞因子的变化

为研究旋毛虫感染对小鼠腹腔巨噬细胞Toll样受体4(TLR4)及细胞因子的影响,本研究分别取感染前、后不同时期小鼠腹腔巨噬细胞,采用半定量PCR和流式细胞术检测巨噬细胞TLR4的表达量,western blot检测TLR信号传导相关蛋白MyD88和NF-κB相对表达量,并对巨噬细胞培养上清液细胞因子浓度进行测定.结果显示,巨噬细胞TLR4在基因和蛋白水平上表达量变化趋势基本一致,呈双峰状,峰值分别在感染后4d和21 d:旋毛虫感染初期4d左右TLR4表达升高,之后降低,14d左右至最低点,感染后期21 d左右TLR4表达重新升高,然后降低并趋于稳定.MyD88/NF-κB的相对表达量及炎性因子含量变化趋势与巨噬细胞TLR4表达量变化趋势相似.由此表明,小鼠腹腔巨噬细胞TLR4的表达与旋毛虫生活史的不同阶段及抗原密切相关,在旋毛虫不同时期抗原刺激下,经由TLR4/MyD88/NF-κB传导途径活化细胞,继而引起炎性因子分泌发生变化.