DETERMINATION OF SERUM SOLUBLE MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR LEVELS IN PATIENTS WITH HEMATOLOGICAL DISEASES

Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological diseases. Methods: The concentration of M-CSFsR was determined by ELISA. The serum M-CSFsR was identified and characterized by immunoprecipitation and Western blotting. Results: The mean serum level of M-CSFsR of 123 normal individuals was 0.48 ng/ml ± 0.41 ng/ml. Immunoprecipitation and Western blotting assay revealed a ～ 90kD band of serum M-CSFsR. The mean serum M-CSFsR level of 60 patients with acute lymphoblastic leukemia (ALL), 36 patients with acute myeloblastic leukemia (AML), 13 patients with myelodysplastic syndrome (MDS) and 42 patients with aplastic anemia (AA) .were 0.22 ng/ml±0.23 ng/ml, 0.17 ng/ml±0.16 ng/ml, 0.19 ng/ml±0.16 ng/ml and 0.23 ng/ml±0.21 ng/ml, respectively, which were significantly lower than that of normal subjects (P=0.002 ,P<0.0001,P<0.0001 andP<0.0001). The mean serum M-CSFsR level of 51 idiopathic thrombocytopenic purpura (ITP) patients was significantly higher than that of normal subjects (2.05 ng/ml±2.75 ng/ml,P<0.0001). Conclusion: The serum M-CSFsR levels of patients with ALL, AML, MDS and AA were significantly lower, while the level of patients with ITP was significantly higher than that of normal individuals. Patients with severe ITP (platelet count<30×l09/L) had the highest M-CSFsR level. It suggested that the abnormal levels of serum M-CSFsR may associate with some hematological diseases and may contribute to the pathological process.

CO-EXPRESSION OF MACROPHAGE COLONY-STIMULATING FACTOR WITH ITS RECEPTOR IN HUMAN HEPATOMA CELLS AND ITS POTENTIAL ROLES

Objective: To investigate the potential role of macrophage colony-stimulating factor (M-CSF) and macrophage colony-stimulating factor receptor (M-CSF-R) on the growth of human hepatoma cells. Methods: Specimens of different origin, including tissues of human hepatocellular carcinoma (HCC), human fetal liver (FL) and normal liver (NL), the hepatoma cell lines, as well as the peripheral blood mononuclear cells (PBMC) from patients with HCC or liver metastatic tumor (LMT), were used to detect the expression levels of M-CSF and M-CSF-R by ABC immunohistochemistry staining and reverse transcription polymerase chain reaction methods the expression levels of M-CSF and M-CSF-R. Influence of monoclonal antibody against M-CSF (B5) or M-CSF-R (RE2) on proliferation ability of hepatoma cell linesin vitro was also studied. Results: The results showed that hepatoma tissues produced elevated levels of both M-CSF and M-CSF-R compared with those of fetal liver (P<0.001). The M-CSF/M-CSF-R expression levels of PBMC from hepatoma patients were higher than those of LMT patients (P<0.01,P<0.05) and the normal people (P<0.001). The hepatoma cell lines showed strong positive for M-CSF and M-CSF-R production. Both B5 and RE2 displayed a dose-dependent inhibitory effect on the growth and proliferation of hepatoma cells. Conclusion: The study indicates a co-expression model for M-CSF-R in hepatoma cells, suggesting an involvement of M-CSF/M-CSF-R in growth signaling of those malignant cells. The M-CSF/M-CSF-R seems to function through an autonomy mechanism in human hepatoma.

The roles of macrophage migration inhibitory factor in retinal diseases

Macrophage migration inhibitory factor(MIF),a multifunctional cytokine,is secreted by various cells and participates in inflammatory reactions,including innate and adaptive immunity.There are some evidences that MIF is involved in many vitreoretinal diseases.For example,MIF can exacerbate many types of uveitis;measurements of MIF levels can be used to monitor the effectiveness of uveitis treatment.MIF also alleviates trauma-induced and glaucoma-induced optic nerve damage.Furthermore,MIF is critical for retinal/choroidal neovascularization,especially complex neovascularization.MIF exacerbates retinal degeneration;thus,anti-MIF therapy may help to mitigate retinal degeneration.MIF protects uveal melanoma from attacks by natural killer cells.The mechanism underlying the effects of MIF in these diseases has been demonstrated:it binds to cluster of differentiation 74,inhibits the c-Jun N-terminal kinase pathway,and triggers mitogen-activated protein kinases,extracellular signal-regulated kinase-1/2,and the phosphoinositide-3-kinase/Akt pathway.MIF also upregulates Toll-like receptor 4 and activates the nuclear factor kappa-B signaling pathway.This review focuses on the structure and function of MIF and its receptors,including the effects of MIF on uveal inflammation,retinal degeneration,optic neuropathy,retinal/choroidal neovascularization,and uveal melanoma.

Mudskipper interleukin-34 modulates the functions of monocytes/macrophages via the colony-stimulating factor-1 receptor 1

Interleukin-34(IL-34)is a novel cytokine that plays an important role in innate immunity and inflammatory processes by binding to the colonystimulating factor-1 receptor(CSF-1R).However,information on the function of IL-34 in fish remains limited.In the present study,we identified an IL-34 homolog from mudskippers(Boleophthalmus pectinirostris).In silico analysis showed that the mudskipper IL-34(BpIL-34)was similar to other known IL-34 variants in sequence and structure and was most closely related to an orange-spotted grouper(Epinephelus coioides)homolog.BpIL-34 transcripts were constitutively expressed in various tissues,with the highest level of expression found in the brain.Edwardsiella tarda infection significantly up-regulated the mRNA expression of BpIL-34 in the mudskipper tissues.The recombinant mature BpIL-34 peptide(rBpIL-34)was purified and used to produce anti-rBpIL-34 IgG.Western blot analysis combined with PNGase F digestion revealed that native BpIL-34 in monocytes/macrophages(MOs/MФs)was N-glycosylated.In vitro,rBpIL-34 treatment enhanced the phagocytotic and bactericidal activity of mudskipper MOs/MФs,as well as the mRNA expression of pro-inflammatory cytokines like tumor necrosis factorα(BpTNF-α)and BpIL-1βin these cells.Furthermore,the knockdown of mudskipper CSF-1R1(BpCSF-1R1),but not mudskipper BpCSF-1R2,significantly inhibited the rBpIL-34-mediated enhanced effect on MO/MФfunction.In conclusion,our results indicate that mudskipper BpIL-34 modulates the functions of MOs/MФs via BpCSF-1R1.

IMMUNOHISTOCHEMICAL OBSERVATION OF MACROPHAGE COLONY STIMULATING FACTOR AND ITS RECEPTOR IN BREAST CANCER AND HEPATOMA TISSUES

Objective: To study the potential role of cellular macrophage colony-stimulating factor (cM-CSF) and cellular macrophage colony-stimulating factor receptor (cM-CSF-R) with breast cancer and hepatoma and search the way for clinical application. Methods: Frozen surgical specimens from 48 breast cancer patients, including 29 cases of histological grade II and 19 eases of grade III, and 16 hepatoma patients were investigated by Avidin Biotin Complex (ABC) immunohistochemical assay with anti-M-CSF monoclonal antibody (Mab) and anti-M-CSF-R Mab. Pathohistological examination was performed as well. Results: cM-CSF and cM-CSF-R were detected in tested specimens. The expression levels of cM-CSF and cM-CSF-R in grade III group were higher than in grade II group and more higher than control group hyperplasia of breast. Hepatoma tissues also showed higher expression level of cM-CSF and cM-CSF-R than normal adult and fetal liver. Conclusion: Breast cancer and hepatoma tissues presented higher expression levels of cM-CSF and cM-CSF-R than control and expression level might be related with tumor’s process.

Antiviral Activity of Silkworm Expressed Recombinant Human Macrophage Colony-Stimulating Factor in Vitro and Its Mechanism

Cells, pretreated with the recombinant human macrophage colony-stimulating factor (rhM-CSF) expressed in silkworm larvae, were inoculated with several viruses to observe the effect of rhM-CSF on viral replication. The results showed that in cultures of fibroblast derived from human fetal skin-muslce tissues infected with the viruses (including VSV, rhinovirus, influenza virus type A3, HSV-1, HSV-2, adenovirus type 6 and type 11), rhM-CSF inhibited the virus-induced cytopathy, which defered or relieved the cytophathy and that in the cells derived from breast feeding rabbit kidney infected with HSV-1, rhM-CSF reduced titer of the virus in a rhM-CSF dose-dependent pattern,in which rhM-CSF with the dose of 1×106 U/L made the viral titer drop dwon over 200 times. This antiviral activity of rhM-CSF could be partially neutralized by anti-IFN-βMcAb, but not by antiTNF-α, anti-IFN-α, or anti-IFA-γ McAb, indicating the mechanism of the antiviral activity of MCSF is related to the induction of IFN-β.

ANTIGEN ASSOCIATION OF J6-1 CELL MEMBRANE ASSOCIATEDFACTOR RECEPTOR WITH MACROPHAGE COLONYSTIMULATING FACTOR RECEPTOR

Objective: To verify the antigen association of MAF-J6-1 receptor with M-CSFR and to further study the role of M-CSF and its receptor mediated juxtacrine in promoting leukemic cell proliferation. Methods: Monoclonal antibody (McAb) of MAF-J6-1R RE2 and polyclonal antibody (PolyAb) of rhM-CSFR were prepared. The specificity of McAb RE2 to M-CSFR was confirmed by indirect ELISA, cross-neutralizing assay with J6-1 cell colony formation and neutralization test by ELISA. Results: the reactive activity of purified RE2 to M-CSFR was over 1: 16000. The inhibitory activity of M-CSFR and MAF-J6-1R could be blocked by RE2 and anti-M-CSFR antibody. The reactivity of RE2 to M-CSFR could be reduced by M-CSFR. Conclusion: The specificity of RE2 to M-CSFR was confirmed and the antigen association of MAF-J6-1R with M-CSFR was proved. It suggests that M-CSF and its receptor mediated auto-juxtacrine stimulation could be an operative mechanism in either leukemia or nonhematological malignancies.

Granulocyte Colony-Stimulating Factor Enhances the Anticancer Effects of Cisplatin against Lung Cancer by Promoting Angiogenesis

The G-CSF is used as a therapeutic drug of the febrile neutropenia in lung cancer chemotherapy, however, there were few reports that showed the effects of combination effects of G-CSF and anticancer drugs against lung cancer. In the present study, we investigated the effects of G-CSF and the combination effects of G-CSF and cisplatin on lung cancer growth. We investigated the effect of G-CSF against the LL-2 and KLN-205 cells by MTT assay and tried to detect the G-CSF receptor by RT-PCR. Next, to analyze the G-CSF effects in vivo, we transplanted the LL-2 into C57BL/6 mice, intraperitoneally administered G-CSF (30 micro/kg/day) with or without cisplatin (5 mg/kg), measured the tumor size and analyzed pathologically by HE and immunostaining. In vitro analyses, G-CSF showed no effects in LL-2 and KLN-205 cells, and RT-PCR revealed no G-CSF receptor mRNA. In vivo analyses, G-CSF alone did not significantly suppress tumor growth. However, concurrent G-CSF administration with cisplatin significantly enhanced the tumor suppressing effect of cisplatin in early stage of tumor growth. The analysis data of vWF immunostaining indicated that the neovascularization in the peripheral region of the tumors was more enhanced in G-CSF treatment mice. ELISA assay revealed that G-CSF did not influence the serum concentration of TNF-alpha and IL-12 in tumor-bearing mice. This study suggests that concurrent (combination) administration of cisplatin with G-CSF is a safe and effective method for enhancing anticancer effects and reducing chemotherapeutic agent-induced myelosuppression.

Molecular cloning, pathologically-correlated expression and functional characterization of the colony- stimulating factor 1 receptor （CSF-1R） gene from a teleost, Plecoglossus altivelis

Colony-stimulating factor 1 receptor （CSF-1R） is an important regulator of monocytes/macrophages （MO/MФ）. Although several CSF-1R genes have been identified in teleosts, the precise role of CSF- 1R in ayu （Plecoglossus altivelis） remains unclear. In this study, we characterized the CSF-1R homologue from P. altivelis, and named it PaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that PaCSF-1R was most closely related to that of Japanese ricefish （Oryzias latipes）. Tissue distribution and expression analysis showed that the PaCSF-1R transcript was mainly expressed in the head kidney-derived MO/MФ, spleen, and head kidney, and its expression was significantly altered in various tissues upon Vibrio anguillarum infection. After PaCSF-1R neutralization for 48 h, the phagocytic activity of MO/MФ was significantly decreased, suggesting that PaCSF-1R plays a role in regulating the phagocytic function of ayu MO/M（P.

Receptor role of membrane bound macrophage colony-stimulating factor

According to the definition of cytokine, the direction of signaling should be from cytokine to receptor. The counter receptor was presented. Membrane bound macrophage colony-stimulating factor (m-M-CSF) and its receptor (M-CSF-R) were shown in human leukemic cell line J6-1 as autojuxtacrine mechanism. Soluble M-CSF receptor (sM-CSF-R), which was isolated from J6-1 cells membrane, was added into J6-1 cell culture. It was observed inhibition of J6-1 cell proliferation, decreasing of mitosis index and ratio of multinuclear cells, enlargement of cell diameter and volume, down regulation of numerous surface antigens. Dramatic change of intracellular pH was shown between several min to 20 min after treatment of sM-CSF-R. It suggested that some information was transmitted via m-M-CSF from sM-CSF-R. This counter signaling was not influenced by saccharification of m-M-CSF.

抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用

目的探讨敲低长链非编码RNA(lncRNA)牛磺酸上调基因1(TUG1)抑制核苷酸结合寡聚结构域样受体蛋白1(NLRP1)炎症小体在缓解阿尔茨海默病进展中的作用。方法选取9~10周龄遗传背景为C57/BL6的野生型小鼠(WT组,10只)或淀粉样前体蛋白(APP)/早老素1(PS1)转基因小鼠(30只)。APP/PS1转基因小鼠随机分为模型(model)组,模型+敲低lncRNA TUG1组[model+lncRNA TUG1短发夹RNA(shRNA)组]和model+shRNA非靶标(NT)组,每组10只。分别采集12周龄第1天(3月龄)和32周龄第1天(8月龄)小鼠外周血和脑皮质组织,并分离皮质中的原代小胶质细胞和原代星形胶质细胞,每个时间点每组5只小鼠。Real-time PCR分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和巨噬细胞移动抑制因子(MIF)mRNA的水平,以及原代星形胶质细胞中补体蛋白C1r和C1s mRNA的水平。ELISA法测定其外周血浆中MIF含量。对3月龄和8月龄小鼠脑皮质原代小胶质细胞和原代星形胶质细胞共培养。CCK-8法测定上述2种细胞的增殖能力。Western blotting分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织中MIF、白细胞介素1β前体(pro-IL-1β)、凋亡相关斑点样蛋白(ASC)、Caspase-1(p20)、Caspase-1(full)、NLRP1及NLRP3蛋白的表达水平。采用免疫荧光染色法测定8月龄各分组小鼠脑皮质组织中β淀粉样蛋白(Aβ)表达。结果3月龄和8月龄时,与WT组小鼠相比,model组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF相对表达水平显著上调,原代小胶质细胞和原代星形胶质细胞增殖能力增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF的相对表达水平显著降低,原代小胶质细胞和原代星形胶质细胞增殖能力降低(P<0.05)。与WT组相比,model组小鼠外周血浆中MIF含量显著升高;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)、NLRP1以及NLRP3的蛋白表达水平显著升高;Aβ免疫荧光强度明显增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠外周血浆中MIF含量显著降低;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)和NLRP1的蛋白表达水平显著降低,Aβ免疫荧光强度明显降低(P<0.05),而NLRP3蛋白质的表达水平无明显变化(P>0.05)。与model组相比,model+shRNA NT组小鼠上述所有检测指标差异均无显著性(P>0.05)。结论APP/PS1转基因小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF因子表达上调与脑皮质内NLRP1炎症小体激活成正相关,敲低lncRNA TUG1可缓解阿尔茨海默病的进展。

PTHrP促进RANKL诱导巨噬细胞分化为破骨细胞参与中耳胆脂瘤骨破坏

目的:骨质进行性吸收破坏是中耳胆脂瘤最重要的临床特征之一,可导致一系列颅内外并发症,而目前中耳胆脂瘤骨破坏的机制尚未明确。本研究旨在探究甲状旁腺激素相关蛋白(parathyroid hormone-related protein,PTHrP)参与中耳胆脂瘤骨破坏的机制。方法:收集后天性中耳胆脂瘤患者的25例胆脂瘤标本和13例外耳道正常皮肤组织标本。采用免疫组织化学染色方法检测PTHrP、核因子κB受体活化因子配体(receptor activator for nuclear factor-kappa B ligand,RANKL)和骨保护素(osteoprotegerin,OPG)在中耳胆脂瘤和外耳道正常皮肤组织中的表达,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法检测中耳胆脂瘤和外耳道正常皮肤组织中是否存在TRAP阳性多核巨噬细胞。选取小鼠单核巨噬细胞RAW264.7细胞进行干预,分为RANKL干预组和PTHrP+RANKL共同干预组,采用TRAP染色法检测2组破骨细胞的生成情况,实时聚合酶链反应(real-time polymerase chain reaction,real-time PCR)检测干预后2组破骨细胞相关基因TRAP、组织蛋白酶K(cathepsin K,CTSK)和活化T细胞核因子1(nuclear factor of activated T cell cytoplasmic 1,NFATc1)的mRNA表达水平,骨吸收陷窝实验检测2组破骨细胞的骨吸收功能。结果:免疫组织化学染色结果显示,PTHrP和RANKL在中耳胆脂瘤组织中的表达均显著增高,OPG表达降低(均P<0.05),且PTHrP的表达与RANKL、RANKL/OPG比值均呈显著正相关,与OPG表达呈显著负相关(分别r=0.385、r=0.417、r=-0.316,均P<0.05)。同时,PTHrP、RANKL的表达水平与中耳胆脂瘤的骨破坏程度均呈显著正相关(分别r=0.413、r=0.505,均P<0.05)。TRAP染色结果显示中耳胆脂瘤上皮周围基质中有大量TRAP阳性细胞,并存在细胞核数量为3个或3个以上的TRAP阳性破骨细胞。RANKL或PTHrP+RANKL联合干预5 d后,与RANKL干预组相比,PTHrP+RANKL联合干预组的破骨细胞数量显著增加(P<0.05),且破骨细胞相关基因TRAP、CTSK和NFATc1的mRNA表达水平均升高(均P<0.05)。骨吸收陷窝扫描电镜结果显示RANKL干预组、PTHrP+RANKL联合干预组的骨片表面均形成骨吸收陷窝;与RANKL干预组相比,PTHrP+RANKL联合干预组的骨片表面骨吸收陷窝数量显著增加(P<0.05),面积也更大。结论:PTHrP可能通过促进RANKL诱导胆脂瘤组织周围基质中的巨噬细胞分化为破骨细胞,参与中耳胆脂瘤骨破坏。

Internalization and half-life of membrane-bound macrophage colony-stimulating factor

The binding of recombinant human macrophage colony-stimulating factor (M-CSF) soluble receptor (rh-M-CSF-sR) to membrane-bound macrophage colony stimulating factor (m-M-CSF) and the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes mediated by m-M-CSF were studied with a model of J6-1 cell line. The results indicated that m-M-CSF bound rh-M-CSF-sR with high affinity (Kd= 1.78×10 12 mol/L) and mediated a temperature- and energy-dependent internalization of rh-M-CSF-sR, and that internalized rh-M-CSF-sR could return to the cell surface in an m-M-CSF-bound state, suggesting that m-M-CSF may have a capability to mediate the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes. In addition, the half-lives of cell-associated M-CSF and its receptor of stimulated normal human cord blood mononuclear cells (CBMCs) and 4 leukemic cell lines were measured by indirect immunoflu-orescence and flow cytometry. The results showed that the half-lives of the various kinds of M-CSF isoforms and

血清MIF、MCP-1、suPAR水平与脓毒症严重程度及合并ARDS风险的关系

目的探讨血清巨噬细胞迁移抑制因子(MIF)、单核细胞趋化蛋白-1(MCP-1)、可溶性尿激酶型纤溶酶原激活物受体(suPAR)水平与脓毒症严重程度及合并急性呼吸窘迫综合征(ARDS)风险的关系。方法回顾性分析2022年2月至2023年5月太原钢铁(集团)有限公司总医院收治的86例脓毒症患者的临床资料。依据病情程度不同将患者分为脓毒症组(n=20)、严重脓毒症组(n=48)和脓毒症休克组(n=18)。入院72 h内参考ARDS诊断标准将患者分为ARDS组(n=27)和非ARDS组(n=59)。检测并比较各组脓毒症患者血清MIF、MCP-1、suPAR水平。收集ARDS组与非ARDS组患者年龄、性别、体重指数、合并症、感染类型、既往史、心率、急性生理学和慢性健康状况评价Ⅱ(APACHEⅡ)、脓毒症相关性器官衰竭评价(SOFA)评分、白细胞计数、血乳酸、天冬氨酸转移酶(AST)、丙氨酸转移酶(ALT)、总胆固醇等指标。采用多因素Logistic回归分析对影响脓毒症患者并发ARDS的危险因素进行分析。通过受试者工作特征(ROC)曲线分析血清MIF、MCP-1、suPAR水平预测脓毒症患者并发ARDS的价值。结果脓毒症休克组患者血清MIF、MCP-1、suPAR水平分别为(94.02±10.13)、(506.55±45.15)、(13.89±3.95)ng/mL,均高于脓毒症组[(76.93±7.01)、(148.38±35.74)、(6.07±2.13)ng/mL]和严重脓毒症组[(85.46±8.74)、(327.08±40.62)、(8.42±1.07)ng/mL],而严重脓毒症组患者血清MIF、MCP-1、suPAR水平均高于脓毒症组,差异均有统计学意义(P<0.05)。ARDS组与非ARDS组患者的年龄、性别构成比、体重指数、合并症、感染类型、白细胞计数、心率、吸烟史、饮酒史、血乳酸、AST、ALT、总胆固醇比较,差异均无统计学意义(P>0.05);ARDS组患者APACHEⅡ评分、SOFA评分、有急腹症和胰腺炎占比及血清MIF、MCP-1、suPAR水平均高于非ARDS组,差异均有统计学意义(P<0.05)。经多因素Logistic回归分析结果显示,急腹症、胰腺炎、APACHEⅡ评分、SOFA评分、MIF、MCP-1、suPAR是影响脓毒症患者并发ARDS的独立危险因素(P<0.05)。经ROC曲线分析结果显示,血清MIF、MCP-1、suPAR水平均能预测脓毒症患者ARDS的发生,曲线下面积分别为0.904、0.910、0.917,预测价值较好(P<0.05)。结论血清MIF、MCP-1、suPAR水平与脓毒症患者病情程度、并发ARDS密切相关,且血清MIF、MCP-1、suPAR水平对ARDS的发生有较好的预测价值。

丁酸钠经AMPK/Nrf2/HO-1信号通路调节脂多糖诱导肺泡巨噬细胞极化的作用机制

目的探讨丁酸钠(sodium butyrate,SB)对脂多糖(lipopolysaccharide,LPS)诱导肺泡巨噬细胞极化的影响及其作用机制。方法小鼠肺泡巨噬细胞(MH-S细胞)随机分为对照(Control)组、LPS组、SB组、LPS+SB(LB)组、LPS+SB+腺苷酸活化蛋白激酶(AMPK)抑制剂(Compound C)(LC)组、LPS+SB+核因子E2相关因子2(Nrf2)抑制剂(ML385)(LM)组。通过CCK8检测MH-S细胞活力,筛选出最佳的1000 ng/mL LPS、1 mmol/L SB、10μmol/L Compound C、5μmol/L ML385药物浓度进行后续实验;实时荧光定量(qRT-PCR)检测MH-S细胞白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-10(IL-10)、白细胞分化抗原86(CD86)、巨噬细胞甘露糖受体(CD206)、AMPK、Nrf2和血红素加氧酶1(HO-1)的mRNA表达水平;酶联免疫吸附试验(ELISA)检测培养基上清IL-6、TNF-α、IL-1β和IL-10蛋白含量;流式细胞术测定M1和M2型巨噬细胞相关标记物CD86和CD206的表达。各组数据通过单因素方差分析和Tukey法进行检验。结果通过CCK8选取了1000 ng/mL LPS、1 mmol/L SB、10μmol/L Compound C和5μmol/L ML385进行造模和干预。qRT-PCR和ELISA结果一致显示,与LPS组比较,LB组M1型巨噬细胞相关促炎细胞因子IL-6、TNF-α、IL-1β显著降低(均P<0.01),但M2型巨噬细胞相关抑炎细胞因子IL-10显著升高(均P<0.01)。qRT-PCR和流式细胞术结果一致显示,与Control组比较,LPS组CD86水平显著升高(均P<0.01),SB组差异无统计学意义;与LPS组比较,LB组CD86表达水平显著降低(均P<0.01),但M2型巨噬细胞标记物CD206的变化趋势与CD86相反。qRT-PCR结果显示,与LPS组比较,LB组促进AMPK/Nrf2/HO-1的表达(均P<0.05);与LB组比较,LC组降低了AMPK/Nrf2/HO-1的表达(均P<0.05),LM组降低了Nrf2/HO-1的表达(均P<0.05)。流式细胞术结果显示,与LB组比较,LC组和LM组逆转了SB对CD86水平的抑制作用(均P<0.01);M2型巨噬细胞标记物CD206的表达趋势与M1型巨噬细胞标记物CD86相反。结论SB通过激活AMPK/Nrf2/HO-1信号通路,抑制LPS诱导的M1型、促进M2型肺泡巨噬细胞极化,改善了炎症反应。

Bioactive materials from berberine-treated human bone marrow mesenchymal stem cells promote alveolar bone regeneration by regulating macrophage polarization

Alveolar bone regeneration has been strongly linked to macrophage polarization.M1 macrophages aggravate alveolar bone loss,whereas M2 macrophages reverse this process.Berberine(BBR),a natural alkaloid isolated and refined from Chinese medicinal plants,has shown therapeutic effects in treating metabolic disorders.In this study,we first discovered that culture supernatant(CS)collected from BBR-treated human bone marrow mesenchymal stem cells(HBMSCs)ameliorated periodontal alveolar bone loss.CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro.To clarify the underlying mechanism,the bioactive materials were applied to different animal models.We discovered macrophage colony-stimulating factor(M-CSF),which regulates macrophage polarization and promotes bone formation,a key macromolecule in the CS.Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats.Colony-stimulating factor 1 receptor(CSF1R)inhibitor or anti-human M-CSF(M-CSF neutralizing antibody,Nab)abolished the therapeutic effects of the CS of BBR-treated HBMSCs.Moreover,AKT phosphorylation in macrophages was activated by the CS,and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab.These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis.Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets.Overall,our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.

巨噬细胞集落刺激因子-1及其受体在牙周炎中的研究进展

牙周炎是一种影响牙齿周围支持组织的慢性炎症性疾病。牙周组织损伤主要由宿主的免疫反应介导,而目前临床治疗牙周炎主要以局部机械清除菌斑和结石为主,免疫疗法在牙周临床中应用很少。因此,对牙周炎免疫治疗的研究就显得极其重要。巨噬细胞参与宿主免疫反应的关键环节,通过识别、吞噬和清除外来病原体和异物在牙周炎的发生、发展过程中发挥重要作用。研究表明,这一过程是通过巨噬细胞集落刺激因子-1(colony stimulation factor-1,CSF-1)/巨噬细胞集落刺激因子-1受体(colony stimulation factor-1 receptor, CSF-lR)信号轴介导的巨噬细胞炎性调控、破骨细胞骨吸收调控进行的。本综述将CSF-1/CSF-1R信号轴在牙周炎中的作用及其作用机制进行总结,以期为后续研究及牙周炎的免疫治疗提供依据。

基于巨噬细胞-肌成纤维细胞转分化的肾间质纤维化病理机制及药物干预作用

在肾间质纤维化过程中,骨髓中募集的巨噬细胞可在损伤的肾脏中直接转化为肌成纤维细胞。巨噬细胞-肌成纤维细胞转分化(MMT)是肾间质纤维化的重要病理机制之一。在MMT过程中,巨噬细胞的活化受转化生长因子-β1、Janus激酶3/信号转导及转录活化因子6、Wnt等信号通路调控,阻断这些信号通路可抑制MMT,改善肾间质纤维化。在临床及体内外实验中,针对防治肾纤维化的药物干预研究较活跃,其中某些药物的干预作用可能与巨噬细胞及MMT相关。因此,深入研究肾脏MMT的病理机制及药物干预作用可以为相关疾病的治疗提供新思路。

子宫内膜异位症患者血清VEGFR-1和MIP-3α水平表达对术后复发的预测价值研究

目的探究血清血管内皮生长因子受体1(vascular endothelial growth factor receptor-1,VEGFR-1)和巨噬细胞炎性蛋白-3α(macrophage inflammatory protein-3α,MIP-3α)在子宫内膜异位症(endometriosis,EMs)患者中的表达以及两者联合检测对EMs患者术后复发的预测价值。方法选取2019年4月~2021年6月秦皇岛市妇幼保健院行腹腔镜手术治疗的114例EMs患者作为观察组,同期选择在该院体检的孕龄期女性114例健康体检者作为对照组。采用酶联免疫吸附法(ELISA)测定患者血清中VEGFR-1和MIP-3α水平;根据术后二年复发情况,将其分为复发组(n=78)和未复发组(n=36)。采用多因素Logistic回归分析EMs患者术后复发的影响因素;采用受试者工作特征(receiver operating characteristic,ROC)曲线分析血清VEGFR-1与MIP-3α联合检测对EMs患者术后复发的预测价值。结果与对照组相比,观察组VEGFR-1(116.25±48.57pg/ml vs 92.43±25.37pg/ml)及MIP-3α(19.25±5.24pg/ml vs 13.67±4.28pg/ml)水平升高,差异具有统计学意义(t=4.641,8.806,均P<0.05)。轻度、中度、重度EMs患者VEGFR-1水平(104.22±5.78pg/ml,118.60±6.56pg/ml,138.55±7.85pg/ml)和MIP-3α水平(15.37±1.15pg/ml,19.28±2.12pg/ml,25.42±2.56pg/ml)依次升高,差异具有统计学意义(F=147.757,133.654,均P<0.001)。复发组中后穹隆存在触痛结节及r-AFS分期(Ⅲ~Ⅳ期)占比显著大于未复发组(χ^(2)=15.139,10.310,均P<0.05);复发组术后用药6个月及以上占比显著低于未复发组(χ^(2)=15.016,P<0.001),差异具有统计学意义。多因素Logistic回归分析显示,血清VEGFR-1,MIP-3α,后穹隆存在触痛结节及r-AFS分期为EMs术后复发的危险因素(均P<0.05),术后用药6个月及以上为保护因素(P<0.05)。ROC曲线显示,血清VEGFR-1与MIP-3α联合预测EMs术后复发的曲线下面积(area under the curve,AUC)最大(0.929),其敏感度和特异度分别为85.90%和86.11%。结论VEGFR-1及MIP-3α在EMs患者血清中表达升高,且二者联合检测在预测EMs术后复发的效能更佳。

曲妥珠单抗对乳腺癌巨噬细胞相关信号因子及预后的影响

目的:探讨曲妥珠单抗对人类表皮生长因子受体2(HER2)阳性乳腺癌巨噬细胞相关信号因子及预后的影响。方法:选择2013年1月至2020年12月间在本院接受手术治疗的155名原发性非转移性HER2^(+)乳腺癌患者。通过免疫组织化学染色分析肿瘤组织中CD8^(+)、CD4^(+)、FOXP3^(+)肿瘤浸润淋巴细胞(TIL)和CD68^(+)、CD163^(+)肿瘤相关巨噬细胞(TAM)的水平。通过Spearman相关分析评估TIL和TAM之间的相关性。采用单变量和多变量Cox回归分析评估接受曲妥珠单抗辅助治疗患者的预后因素。结果:有32名(20.6%)患者在随访期间报告死亡。所有患者的中位随访时间为53.57个月(33.30~72.37)。FOXP3^(+)TIL计数与CD4^(+)TIL计数、CD8^(+)TIL计数呈正相关(P<0.001)。CD4^(+)TIL计数、CD8^(+)TIL计数、FOXP3^(+)TIL计数分别与CD163^(+)TAM、CD68^(+)TAM计数呈正相关(P<0.001)。Cox回归分析结果显示,淋巴血管侵犯(OR=2.631,95%CI=1.218~5.685),CD68^(+)TAM(OR=1.002,95%CI=1.001~1.003)、CD8^(+)TIL(OR=1.002,95%CI=1.001~1.004)、Ki-67增殖指数(OR=0.998,95%CI=0.997~1.000)是接受曲妥珠单抗辅助治疗患者预后的独立影响因素(P<0.05)。ROC分析显示,当CD68^(+)TAM的截止值为235.33时,其预测患者死亡的AUC为0.933(95%CI=0.896~0.970),敏感度为93.8%,特异度为83.7%。CD8^(+)TIL的截止值为53.67时,其预测患者死亡的AUC为0.645(95%CI=0.536~0.754),敏感度为56.3%,特异度为65.0%。结论:高CD68^(+)TAM计数、CD8^(+)TIL计数与接受曲妥珠单抗治疗的HER2^(+)乳腺癌患者的不良预后相关,并且CD68^(+)TAM计数在预测不良预后方面具有较高的预测价值。