Background:Ischemic stroke has been regarded as a major cause of disability and death around the world due to limited effective therapies.Accumulating evidence have shown that although microglia are polarized to an an...Background:Ischemic stroke has been regarded as a major cause of disability and death around the world due to limited effective therapies.Accumulating evidence have shown that although microglia are polarized to an anti-inflammatory M2 phenotype in the early stage of ischemia,they transform progressively into a proinflammatory M1 phenotype.Bone marrow-derived mesenchymal stem cells(BMSCs)may be used to treat ischemic injury through regulating the poststroke inflammatory response.However,the mechanism by which BMSCs can treat ischemic stroke remains unclarified.Objective:This study aimed to investigate whether BMSCs shift M1-to-M2 phenotype transformation of mi-croglia/macrophages and enhance neurogenesis in a rat transient middle cerebral artery occlusion(tMCAO)model.Methods:Ninety-minute tMCAO was applied to the rats,followed by reperfusion.BMSCs were transplanted into the rats via intravenous injection at 24 h after tMCAO.After being randomly divided into the sham group,the MCAO group,and the BMSCs group,the rats’behavior was assessed at 1,3,7,and 14 days following tM-CAO.qRT-PCR,double-immunofluorescence staining,and Western blot were performed at 3 and 14 days after tMCAO to determine M1/M2 polarization of microglia/macrophages.Neurogenesis was examined by double-immunofluorescence staining at 14 days after tMCAO.Expression of brain-derived neurotrophic factor(BDNF)was measured on the protein level by immunofluorescence staining at 3 and 14 days after tMCAO.Results:We found that BMSCs treatment promoted the recovery of neurological function after tMCAO,inhibited the expression of TNF𝛼,iNOS and CD16/32,which are markers of M1 microglia/macrophage,and enhanced the expression of IL10,TGF𝛽and CD206 that are markers of M2 microglia/macrophage.Moreover,BMSCs treatment promoted neurogenesis and M2-derived BDNF expression after tMCAO.Conclusion:It is indicated by the results that BMSCs modulate neuroinflammation and enhance neurogenesis,which could be due to transforming microglia/macrophages from the M1 polarization state towards M2 in a rat tMCAO model.展开更多
目的探讨脂多糖(lipopolysaccharide,LPS)对破骨细胞生成及其骨吸收功能的作用及机制。方法取雄性C57BL/6小鼠股骨及胫骨骨髓,分离培养骨髓源巨噬细胞(bone marrow-derived macrophages,BMMs),并行流式细胞仪鉴定。取BMMs采用不同浓度LP...目的探讨脂多糖(lipopolysaccharide,LPS)对破骨细胞生成及其骨吸收功能的作用及机制。方法取雄性C57BL/6小鼠股骨及胫骨骨髓,分离培养骨髓源巨噬细胞(bone marrow-derived macrophages,BMMs),并行流式细胞仪鉴定。取BMMs采用不同浓度LPS(0、100、200、500、1 000、2 000 ng/m L)培养后,以细胞计数试剂盒8(cell counting kit 8,CCK-8)检测不同浓度LPS对细胞活性影响。为探讨LPS对破骨细胞生成的影响,取BMMs分为巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)组、M-CSF+核因子κB受体活化因子配体(receptor activator of nuclear factorκB ligand,RANKL)组、M-CSF+RANKL+50 ng/m L LPS组、MCSF+RANKL+100 ng/m L LPS组,对应培养后行抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色观察,计算破骨细胞面积百分比。为探讨LPS对Connexin43蛋白及基因表达影响,将BMMs分别分为对照组(M-CSF+RANKL)、LPS组(M-CSF+RANKL+100 ng/m L LPS)以及对照组(M-CSF+RANKL)、50 ng/m L LPS组(MCSF+RANKL+50 ng/m L LPS)、100 ng/m L LPS组(M-CSF+RANKL+100 ng/m L LPS)培养后,行Western blot以及实时荧光定量PCR检测。为探讨LPS对破骨细胞骨吸收能力的影响,将BMMs分为M-CSF组、M-CSF+RANKL组、M-CSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组对应培养后,采用骨吸收实验检测骨吸收面积百分比。结果流式细胞仪鉴定培养细胞为BMMs。CCK-8法检测显示与其他浓度相比,100 ng/m L LPS明显促进BMMs活性(P<0.05)。TRAP染色示,M-CSF组未见破骨细胞生成;与M-CSF+RANKL组相比,MCSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组破骨细胞体积更大、细胞核更多,其中后者最显著,3组破骨细胞面积百分比差异均有统计学意义(P<0.05)。Western blot检测,LPS组Connexin43蛋白相对表达量较对照组明显提高(P<0.05);实时荧光定量PCR检测示,对照组、50 ng/m L LPS组以及100 ng/m L LPS组Connexin43基因相对表达量逐渐增加,比较差异有统计学意义(P<0.05)。骨吸收实验示,M-CSF组未形成破骨细胞骨吸收;M-CSF+RANKL组、M-CSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组骨吸收面积百分比逐渐增加,比较差异均有统计学意义(P<0.05)。结论 100 ng/m L LPS能够促进Connexin43的表达,从而使破骨细胞生成增多,骨吸收功能加强。展开更多
Tumor tissues contain cancer cells,other cellular and non-cellular comp onen ts.Tumor microe nvir onments consist of cancer cells and various types of stromal cells,can cer associated fibroblasts,bone marrow-derived c...Tumor tissues contain cancer cells,other cellular and non-cellular comp onen ts.Tumor microe nvir onments consist of cancer cells and various types of stromal cells,can cer associated fibroblasts,bone marrow-derived cells,en dothelial cells,and hematopoietic cells,mainly tumor-associated macrophages and tumor-infiltrating lymphocytes.Increasing recent evidence has demonstrated that alteration of tumor microenvironments is deeply implicated in tumor progression and metastasis in gastric can cer(GC)patients.Recent in vestigati ons have provided in sights into the molecular mecha ni sms of the interaction between tumor cells and tumor microenvironments.Interactions between cancer cells and their microe nvir onment with cytok ines and microRNA in extracellular vesicles,such as the exosome,can have a substa ntial impact on tumor characteristics.Alterati ons in the tumor microe nvironment may play a crucial role in facilitating the progression of tumor cells and metastasis,as well as the activation of cell signaling pathways,which are associated with GC cell proliferati on and in vasi on by genetic or epigenetic alterations.In this review,significant molecular in sights into the tumor microenvironment,which consist of cancer associated fibroblasts,bone marrow-derived cells,tumor-associated macrophages and tumor-infiltrating lymphocytes;the interactions between cancer cells and their microenvironment;and the clinical impacts of alterations of GC microenvironments will be discussed.展开更多
基金supported by the National Natural Sci-ence Foundation of China(Grant Nos.81274113,81873028,81903949)the Natural Science Foundation of Zhejiang Province(Grant No.LQ19H290004).
文摘Background:Ischemic stroke has been regarded as a major cause of disability and death around the world due to limited effective therapies.Accumulating evidence have shown that although microglia are polarized to an anti-inflammatory M2 phenotype in the early stage of ischemia,they transform progressively into a proinflammatory M1 phenotype.Bone marrow-derived mesenchymal stem cells(BMSCs)may be used to treat ischemic injury through regulating the poststroke inflammatory response.However,the mechanism by which BMSCs can treat ischemic stroke remains unclarified.Objective:This study aimed to investigate whether BMSCs shift M1-to-M2 phenotype transformation of mi-croglia/macrophages and enhance neurogenesis in a rat transient middle cerebral artery occlusion(tMCAO)model.Methods:Ninety-minute tMCAO was applied to the rats,followed by reperfusion.BMSCs were transplanted into the rats via intravenous injection at 24 h after tMCAO.After being randomly divided into the sham group,the MCAO group,and the BMSCs group,the rats’behavior was assessed at 1,3,7,and 14 days following tM-CAO.qRT-PCR,double-immunofluorescence staining,and Western blot were performed at 3 and 14 days after tMCAO to determine M1/M2 polarization of microglia/macrophages.Neurogenesis was examined by double-immunofluorescence staining at 14 days after tMCAO.Expression of brain-derived neurotrophic factor(BDNF)was measured on the protein level by immunofluorescence staining at 3 and 14 days after tMCAO.Results:We found that BMSCs treatment promoted the recovery of neurological function after tMCAO,inhibited the expression of TNF𝛼,iNOS and CD16/32,which are markers of M1 microglia/macrophage,and enhanced the expression of IL10,TGF𝛽and CD206 that are markers of M2 microglia/macrophage.Moreover,BMSCs treatment promoted neurogenesis and M2-derived BDNF expression after tMCAO.Conclusion:It is indicated by the results that BMSCs modulate neuroinflammation and enhance neurogenesis,which could be due to transforming microglia/macrophages from the M1 polarization state towards M2 in a rat tMCAO model.
文摘目的探讨脂多糖(lipopolysaccharide,LPS)对破骨细胞生成及其骨吸收功能的作用及机制。方法取雄性C57BL/6小鼠股骨及胫骨骨髓,分离培养骨髓源巨噬细胞(bone marrow-derived macrophages,BMMs),并行流式细胞仪鉴定。取BMMs采用不同浓度LPS(0、100、200、500、1 000、2 000 ng/m L)培养后,以细胞计数试剂盒8(cell counting kit 8,CCK-8)检测不同浓度LPS对细胞活性影响。为探讨LPS对破骨细胞生成的影响,取BMMs分为巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)组、M-CSF+核因子κB受体活化因子配体(receptor activator of nuclear factorκB ligand,RANKL)组、M-CSF+RANKL+50 ng/m L LPS组、MCSF+RANKL+100 ng/m L LPS组,对应培养后行抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色观察,计算破骨细胞面积百分比。为探讨LPS对Connexin43蛋白及基因表达影响,将BMMs分别分为对照组(M-CSF+RANKL)、LPS组(M-CSF+RANKL+100 ng/m L LPS)以及对照组(M-CSF+RANKL)、50 ng/m L LPS组(MCSF+RANKL+50 ng/m L LPS)、100 ng/m L LPS组(M-CSF+RANKL+100 ng/m L LPS)培养后,行Western blot以及实时荧光定量PCR检测。为探讨LPS对破骨细胞骨吸收能力的影响,将BMMs分为M-CSF组、M-CSF+RANKL组、M-CSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组对应培养后,采用骨吸收实验检测骨吸收面积百分比。结果流式细胞仪鉴定培养细胞为BMMs。CCK-8法检测显示与其他浓度相比,100 ng/m L LPS明显促进BMMs活性(P<0.05)。TRAP染色示,M-CSF组未见破骨细胞生成;与M-CSF+RANKL组相比,MCSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组破骨细胞体积更大、细胞核更多,其中后者最显著,3组破骨细胞面积百分比差异均有统计学意义(P<0.05)。Western blot检测,LPS组Connexin43蛋白相对表达量较对照组明显提高(P<0.05);实时荧光定量PCR检测示,对照组、50 ng/m L LPS组以及100 ng/m L LPS组Connexin43基因相对表达量逐渐增加,比较差异有统计学意义(P<0.05)。骨吸收实验示,M-CSF组未形成破骨细胞骨吸收;M-CSF+RANKL组、M-CSF+RANKL+50 ng/m L LPS组、M-CSF+RANKL+100 ng/m L LPS组骨吸收面积百分比逐渐增加,比较差异均有统计学意义(P<0.05)。结论 100 ng/m L LPS能够促进Connexin43的表达,从而使破骨细胞生成增多,骨吸收功能加强。
文摘Tumor tissues contain cancer cells,other cellular and non-cellular comp onen ts.Tumor microe nvir onments consist of cancer cells and various types of stromal cells,can cer associated fibroblasts,bone marrow-derived cells,en dothelial cells,and hematopoietic cells,mainly tumor-associated macrophages and tumor-infiltrating lymphocytes.Increasing recent evidence has demonstrated that alteration of tumor microenvironments is deeply implicated in tumor progression and metastasis in gastric can cer(GC)patients.Recent in vestigati ons have provided in sights into the molecular mecha ni sms of the interaction between tumor cells and tumor microenvironments.Interactions between cancer cells and their microe nvir onment with cytok ines and microRNA in extracellular vesicles,such as the exosome,can have a substa ntial impact on tumor characteristics.Alterati ons in the tumor microe nvironment may play a crucial role in facilitating the progression of tumor cells and metastasis,as well as the activation of cell signaling pathways,which are associated with GC cell proliferati on and in vasi on by genetic or epigenetic alterations.In this review,significant molecular in sights into the tumor microenvironment,which consist of cancer associated fibroblasts,bone marrow-derived cells,tumor-associated macrophages and tumor-infiltrating lymphocytes;the interactions between cancer cells and their microenvironment;and the clinical impacts of alterations of GC microenvironments will be discussed.
