背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
Objective To examine the effect of neuropeptide Y （NPY） on TGF-β1 production in RAW264.7 macrophages. Methods Enzyme linked immunosorbent assay （ELISA） was used to detect TGF-β1 production. Cell counting kit 8 ...Objective To examine the effect of neuropeptide Y （NPY） on TGF-β1 production in RAW264.7 macrophages. Methods Enzyme linked immunosorbent assay （ELISA） was used to detect TGF-β1 production. Cell counting kit 8 （CCK-8） was used to assay the viability of RAW264.7 cells. Western blot was used to detect the phosphorylation of PI3K p85. Results NPY treatment could promote TGF-β1 production and rapid phosphorylation of PI3K p85 in RAW264.7 cells via Y1 receptor. The elevated TGF-β 1 production induced by NPY could be abolished by wortrnannin pretreatment. Conclusion NPY may elicit TGF-β production in RAW264.7 cells via Y1 receptor, and the activated PI3K pathway may account for this effect.展开更多
Two immunomodulatory polysaccharides(Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cell...Two immunomodulatory polysaccharides(Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cells. Both the level of released nitric oxide(NO) and expression of inducible nitric oxide synthase(iNOS) m RNA were significantly enhanced in the RAW264.7 macrophages cells treated by Vp2a-Ⅱ and Vp3. Vp2a-Ⅱ(100–800 μg/m L) and Vp3(400 μg/mL) could significantly increase the phagocytic activity of RAW264.7 cells and the secretion and m RNA expression of TNF-α and IL-6 in a concentrationdependent manner through affecting mitogen-activated protein kinase(MAPK) activity and nuclear factor κB(NF-κB) nuclear translocation. Vp2a-Ⅱ might activate the MAPK signaling pathways and induce the nuclear translocation of NF-κB p65, whilst Vp3 likely activated the NF-κB and MAPK signaling pathways without influencing the p38 MAPK route.展开更多
OBJECTIVE The emerging role of chronic inflammation is the major degenerative diseases of modern society such as periodontitis,atherosclerosis,rheumatoid arthritis,Parkinson′s disease and even cancer.Eight components...OBJECTIVE The emerging role of chronic inflammation is the major degenerative diseases of modern society such as periodontitis,atherosclerosis,rheumatoid arthritis,Parkinson′s disease and even cancer.Eight components were isolated from Derris laxiflora Benth.,In this study,we found these compounds from Derris laxiflora Benth suppress lipopolysaccharide-induced inflammatory response in murine macrophage(RAW 264.7)cells.METHODS RAW 264.7cells were cultured in DMEM media supplemented with 10%(V/V)heated-inactivated FBS,penicillin 100U·mL-1 and streptomycin 100μg·mL-1.The cells were incubated at 37℃in a humidified atmosphere of 5%CO2in air.RAW264.7cells were seeded in a 24-well plate at a density of 2×105 mL-1 and then incubated with or without LPS(100ng·mL-1)in the absence or presence of compounds for 24 h.Effects of these isolates on NO production were measured indirectly by analysis of nitrite levels using the Griess reaction.Quercetin was used as a positive control.RESULTS ight components were isolated from Derris laxiflora Benth.,including three new pterocarpans 7,6′-dihydroxy-3′-methoxypterocarpan(1),derrispisatin(2),derriscoumaronochromone(3)and three new flavonoids cis-3,4′-dihydroxy-5,7-dimethoxyflavan(4),derriflavanone B(5),iso-lupinenol(6)as well as two known ones,lonchocarpol A(7)and lonchocarpol D(8).The structures of these new compounds were determined by analysis of their spectroscopic data.Raw264.7 cells were treated with the compounds from Derris laxiflora Benth for 24 h.Among them,compounds 5,7 and 8 significantly suppressed the NO production in LPS-treated RAW264.7 cells with IC50 values<10μg·mL-1.CONCLUSION In this study,we found that compounds from Derris laxiflora Benth suppresses lipopolysaccharide-induced inflammatory response in murine Raw264.7 cells.展开更多
Raw264.7 cells are monocytic cells that can differentiate to activated macrophages after lipopoly-saccharide (LPS) stimulation. Here, we analyzed the factors secreted by Raw264.7 cells in response to LPS. The culture ...Raw264.7 cells are monocytic cells that can differentiate to activated macrophages after lipopoly-saccharide (LPS) stimulation. Here, we analyzed the factors secreted by Raw264.7 cells in response to LPS. The culture media of LPS-treated Raw264.7 cells was able to stimulate growth in MEF1F2 and NIH3T3 mouse fibroblast cell lines. We identified five secreted and LPS-induced chemokines, CCL2, CCL5, CCL12, CxCL2, and CxCL10, by microarray analysis and tested their stimulatory activity. We used commercially available bacterially expressed proteins, and found only CCL12, CxCL2 and CxCL10 stimulated growth in MEF1F2 and NIH3T3 cells. The saturation density of the cells was also increased. They were not able to stimulate growth in v-Src transformed MEF1F2 or SWAP-70 transformed NIH3T3 cells. We examined signaling pathways activated by these three factors. We found that ERK and p38 MAP kinase were activated and were required for the activity to stimulate the cell growth. Other pathways including phosophatidylinositol-3 kinase (PI3K), NFκB pathways were not activated. These results suggest that Raw264.7 cells secretes growth stimulation factors for fibroblasts when differentiated to macrophages implicating that fast growth of them is related to inflamation although the reason is still unclear.展开更多
Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide ...Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.展开更多
文摘Objective To examine the effect of neuropeptide Y （NPY） on TGF-β1 production in RAW264.7 macrophages. Methods Enzyme linked immunosorbent assay （ELISA） was used to detect TGF-β1 production. Cell counting kit 8 （CCK-8） was used to assay the viability of RAW264.7 cells. Western blot was used to detect the phosphorylation of PI3K p85. Results NPY treatment could promote TGF-β1 production and rapid phosphorylation of PI3K p85 in RAW264.7 cells via Y1 receptor. The elevated TGF-β 1 production induced by NPY could be abolished by wortrnannin pretreatment. Conclusion NPY may elicit TGF-β production in RAW264.7 cells via Y1 receptor, and the activated PI3K pathway may account for this effect.
基金supported by Research on Precision Nutrition and Health Food,Department of Science and Technology of Henan Province(CXJD2021006)。
文摘Two immunomodulatory polysaccharides(Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cells. Both the level of released nitric oxide(NO) and expression of inducible nitric oxide synthase(iNOS) m RNA were significantly enhanced in the RAW264.7 macrophages cells treated by Vp2a-Ⅱ and Vp3. Vp2a-Ⅱ(100–800 μg/m L) and Vp3(400 μg/mL) could significantly increase the phagocytic activity of RAW264.7 cells and the secretion and m RNA expression of TNF-α and IL-6 in a concentrationdependent manner through affecting mitogen-activated protein kinase(MAPK) activity and nuclear factor κB(NF-κB) nuclear translocation. Vp2a-Ⅱ might activate the MAPK signaling pathways and induce the nuclear translocation of NF-κB p65, whilst Vp3 likely activated the NF-κB and MAPK signaling pathways without influencing the p38 MAPK route.
基金The project supported by Department of Industrial Technology,Ministry of Economic Affairs,Chinese TaipeiMedical and Pharmaceutical Industry Technology and Development Center
文摘OBJECTIVE The emerging role of chronic inflammation is the major degenerative diseases of modern society such as periodontitis,atherosclerosis,rheumatoid arthritis,Parkinson′s disease and even cancer.Eight components were isolated from Derris laxiflora Benth.,In this study,we found these compounds from Derris laxiflora Benth suppress lipopolysaccharide-induced inflammatory response in murine macrophage(RAW 264.7)cells.METHODS RAW 264.7cells were cultured in DMEM media supplemented with 10%(V/V)heated-inactivated FBS,penicillin 100U·mL-1 and streptomycin 100μg·mL-1.The cells were incubated at 37℃in a humidified atmosphere of 5%CO2in air.RAW264.7cells were seeded in a 24-well plate at a density of 2×105 mL-1 and then incubated with or without LPS(100ng·mL-1)in the absence or presence of compounds for 24 h.Effects of these isolates on NO production were measured indirectly by analysis of nitrite levels using the Griess reaction.Quercetin was used as a positive control.RESULTS ight components were isolated from Derris laxiflora Benth.,including three new pterocarpans 7,6′-dihydroxy-3′-methoxypterocarpan(1),derrispisatin(2),derriscoumaronochromone(3)and three new flavonoids cis-3,4′-dihydroxy-5,7-dimethoxyflavan(4),derriflavanone B(5),iso-lupinenol(6)as well as two known ones,lonchocarpol A(7)and lonchocarpol D(8).The structures of these new compounds were determined by analysis of their spectroscopic data.Raw264.7 cells were treated with the compounds from Derris laxiflora Benth for 24 h.Among them,compounds 5,7 and 8 significantly suppressed the NO production in LPS-treated RAW264.7 cells with IC50 values<10μg·mL-1.CONCLUSION In this study,we found that compounds from Derris laxiflora Benth suppresses lipopolysaccharide-induced inflammatory response in murine Raw264.7 cells.
文摘Raw264.7 cells are monocytic cells that can differentiate to activated macrophages after lipopoly-saccharide (LPS) stimulation. Here, we analyzed the factors secreted by Raw264.7 cells in response to LPS. The culture media of LPS-treated Raw264.7 cells was able to stimulate growth in MEF1F2 and NIH3T3 mouse fibroblast cell lines. We identified five secreted and LPS-induced chemokines, CCL2, CCL5, CCL12, CxCL2, and CxCL10, by microarray analysis and tested their stimulatory activity. We used commercially available bacterially expressed proteins, and found only CCL12, CxCL2 and CxCL10 stimulated growth in MEF1F2 and NIH3T3 cells. The saturation density of the cells was also increased. They were not able to stimulate growth in v-Src transformed MEF1F2 or SWAP-70 transformed NIH3T3 cells. We examined signaling pathways activated by these three factors. We found that ERK and p38 MAP kinase were activated and were required for the activity to stimulate the cell growth. Other pathways including phosophatidylinositol-3 kinase (PI3K), NFκB pathways were not activated. These results suggest that Raw264.7 cells secretes growth stimulation factors for fibroblasts when differentiated to macrophages implicating that fast growth of them is related to inflamation although the reason is still unclear.
基金supported by the National Natural Science Foundation of China,No.32371048(to YK)the Peking University People’s Hospital Research and Development Funds,No.RDX2021-01(to YK)the Natural Science Foundation of Beijing,No.7222198(to NH)。
文摘Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.
