单核—巨噬细胞和ECV304内皮细胞表达死亡相关蛋白

为研究单核—巨噬细胞和ECV30 4细胞是否表达死亡相关蛋白以及影响死亡相关蛋白表达的因素。分别用氧化型低密度脂蛋白 (10 0mg L)孵育THP 1细胞 ,H2O2 (1mmol L)孵育ECV30 4细胞 ,用逆转录聚合酶链反应检测死亡相关蛋白mRNA的表达。结果发现 ,THP 1细胞及ECV30 4细胞均表达死亡相关蛋白 ,氧化型低密度脂蛋白和H2O2能分别诱导THP 1细胞和ECV30 4细胞死亡相关蛋白mRNA表达增高。实验结果提示死亡相关蛋白可能参与调节氧化应激诱导的单核—巨噬细胞和内皮细胞的凋亡。

杨树皮类脂对小白鼠单核—巨噬细胞吞噬功能的影响

本试验以作为免疫增强剂及佐剂的杨树皮类脂采用碳颗粒廓清法给小白鼠灌胃来研究杨树皮类脂对小白鼠单核—巨噬细胞吞噬功能的影响,试验给16只小白鼠注射刚果红染料和灌食5、10、20倍稀释的杨树皮类脂溶液,通过对小鼠单核-巨噬细胞吞噬刚果红染料的吞噬速率进行t检验。结果表明,5倍稀释的杨树皮类脂抑制小白鼠单核巨噬细胞的吞噬功能和生理盐水组间差异不显著(P>0.050);10、20倍稀释的杨树皮类脂溶液和生理盐水组间差异极显著(P<0.01)地提高巨噬细胞的吞噬功能;试验证明杨树皮类脂在促进小白鼠单核—巨噬细胞吞噬功能方面有较显著的作用,从而为杨树皮类脂用于免疫增强剂提供了一定的理论依据。

Adult endothelial progenitor cells from human peripheral blood maintain monocyte/macrophage function throughout in vitro culture

Mononuclear cells （MNCs） isolated from peripheral blood by density gradient centrifugation were plated on human fibronectin-coated culture plates and cultured in EGM-2 medium. Attached spindle-shaped cells, reported as endothelial progenitor cells （EPCs） by some investigators, had elongated from adherent round cells, but had not proliferated from a small number of cells as supposed previously. The growth curve of the primary EPCs showed that the cells had little proliferative capacity. Flow cytometry analysis showed that the cells could express some of the endothelial lineage markers, while they could also express CD 14, which is considered a marker of monocyte/macrophage lineages throughout culture. In endothelial function assays, the cells demonstrated a lower level of expression of eNOS than mature endothelial cells in the reverse transcription-polymerase chain reaction and did not show an ability to develop tube-like structures in angiogenesis assay in vitro. In this study, we identified the monocytoid function of EPCs by the combined Dillabeled acetylated low-density lipoprotein （Dil-Ac-LDL） and Indian ink uptake tests. All the cells were double positive for Dil- Ac-LDL and Indian ink uptake at days 4, 14 and 28 of culture, which means the EPCs maintained monocytoid function throughout the culture. Therefore, although adult EPCs from peripheral MNCs have some endothelial lineage properties, they maintain typical monocytic function and have little proliferative capacity.

Lysine-specific Demethylase 1 Represses THP-1 Monocyte-to-macrophage Differentiation

Objective To investigate the role of lysine-specific demethylase 1 （LSD1） in the process of THP-1 monocyte-to-macrophage differentiation. Methods Quantitative reverse transcription-polymerase chain reaction （qRT-PCR） and Western blotting were performed to analyze the expression of LSD1 and interleukin-6 （IL-6） in THP-1 monocytes and THP-l-derived macrophages. Chromatin immunoprecipitation （ChiP） assay was applied to detect the occupancy of LSD1 and H3K4 methylation at IL-6 promoter during THP-1 monocyte-to-macrophage differentiation. IL-6 mRNA level and H3K4 methylation at IL-6 promoter were analyzed using qRT-PCR and ChiP assay in LSD 1 -knockdown THP- 1 cells treated with 12-O-tetradecanoylphorbol- 13-acetate （TPA） for 0 4, 8, 12, and 24 hours. Fluorescence activated flow cytometry was performed to reveal the percentage of macrophages differentiated from THP- 1 monocytes. Results The expression of LSD1 reduced during THP-1 monocyte-to-macrophage differentiation （P〈0.01）. LSD1 occupancy decreased and H3K4 methylation increased at IL-6 promoter during the differentiation. With knockdown of LSD1, H3K4 methylation at IL-6 promoter was found increased after TPA treatment at different times points （all P〈0.05, except 24 hours）. The percentage of macrophages increased significantly in theTHP-I cells with LSD1 knockdown （P〈0.05）. Conclusions LSD1 is repressed during the monocyte-to-macrophage differentiation of THP-1 cells. Suppression of LSD 1-mediated H3K4 demethylation may be required for THP-1 monocyte-to-macrophage differentiation.

Profiling of differentially expressed chemotactic-related genes in MCP-1 treated macrophage cell line using human cDNA arrays

AIM: To study the global gene expression of chemotactic genes in macrophage line U937 treated with human monocyte chemoattractant protein-1 (MCP-1) through the use of ExpreeChipTMO2 cDNA array. METHODS: Total RNA was extracted from MCP-1 treated macrophage line U937 and normal U937 cells, reversely transcribed to cDNA, and then screened in parallel with HO2 human cDNA array chip. The scanned result was additionally validated using RT-PCR. RESULTS: The result of cDNA array showed that one chemotactic-related gene was up-regulated more than two-fold (RANTES) and seven chemotactic-related genes were down-regulated more than two-fold (CCR1, CCR5, ccll6, GROβ, GROγ,IL-8 and granulocyte chemotactic protein 2) in MCP-1 treated U937 cells at mRNA level. RT-PCR analysis of four of these differentially expressed genes gave results consistent with cDNA array findings. CONCLUSION: MCP-1 could influence some chemokine and receptor expressions in macrophages in vitro. MCP-1 mainly down-regulates the expression of chemotactic genes influencing neutrophilic granulocyte expression (GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2), and the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.

Inhibitory effect of Gardenblue blueberry (Vaccinium ashei Reade) anthocyanin extracts on lipopolysaccharide-stimulated inflammatory response in RAW 264.7 cells

Blueberries are a rich source of anthocyanins, which are associated with health benefits contributing to a reduced risk for many diseases. The present study identified the functional Gardenblue blueberry （Vaccinium ashei Reade） anthocyanin extracts （GBBAEs） and evaluated their capacity and underlying mechanisms in protecting murine RAW 264.7 cells from lipopolysaccharide （LPS）-stimulated inflammation in vitro. Enzyme-linked immunosorbent assay （ELISA） kit results showed that GBBAEs significantly inhibited the production of nitric oxide （NO）, prostaglandin E2 （PGE2）, interleukin-6 （IL-6）, IL-113, and interferon-y （INF-y）. Real-time polymerase chain reaction （PCR） analysis in- dicated that the mRNA expression levels of IL-6, IL-113, tumor necrosis factor-α （TNF-α）, monocyte chemoattractant protein-1 （MCP-1）, and cyclooxygenase 2 （COX-2） were suppressed in LPS-stimulated RAW 264.7 cells. Additionally, Western blot analysis was used to evaluate the relative protein expression levels of COX-2 and nuclear factor-κB p65 （NF-κBp65）. All these results suggested the potential use of GBBAEs as a functional food for the treatment of in- flammatory diseases.