LIGHT of pulmonary NKT cells annihilates tissue protective alveolar macrophages in augmenting severe influenza pneumonia

CD1d-restricted natural killer T(NKT)cells are innate-like T lymphocytes with protective or pathogenic roles in the development of influenza pneumonia.Here,we show that lung-infiltrated and activated NKT cells are the major cellular source of LIGHT/TNFSF14,which determines the severity of pulmonary pneumonia by highly deteriorative influenza A virus(IAV)infection.Compared to wild-type mice,LIGHT^(-/-)mice exhibit much lower morbidity and mortality to IAV,due to alleviated lung damage and reduced apoptosis of alveolar macrophages(AMs).LIGHT preferentially promotes cell death of lymphotoxin β receptors positive(LTβR^(+))AMs but not herpesvirus entry mediator positive(HVEM^(+))AMs.Therefore,these results suggest that NKT-derived LIGHT augments cell death of the tissue protective AMs in exacerbating lung pathology and susceptibility to fatal influenza infection.Suppression of LIGHT signaling might be a viable option in the treatment of influenza-associated acute respiratory distress syndrome.

Primary Culture of Alveolar Epithelial Type Ⅱ Cells and Its Bionomic Study

To establish a better method of primary culture for alveolar epithelial type Ⅱ cells （AEC Ⅱ ） and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin （i The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A （SPA）. The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 × 10^7, and a purity of about 75%-84 %. Cells could be quickly identified with AKP staining. AEC Ⅱ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC Ⅱ, and AKP staining is simple in cell identification. AEC Ⅱ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia

The ubiquitin ligase,Itch,is required to prevent autoinflammatory disease in mice and humans.Itch-deficient mice develop lethal pulmonary inflammation characterized by the production of Th2 cytokines(for example,interleukin-4(IL-4));however,the contribution of Itch to immune defense against respiratory pathogens has not been determined.We found that Itch-deficient mice were highly susceptible to intranasal infection with the respiratory pathogen Klebsiella pneumoniae.Infected Itch-deficient mice exhibited increased immune cell infiltration,cytokine levels and bacterial burden in the respiratory tract compared with control mice.However,numbers of resident alveolar macrophages were reduced in the lungs from Itch-deficient mice both before and after infection.High levels of Th2 cytokines in the respiratory tract correlated with deceased alveolar macrophages,and genetic ablation of IL-4 restored alveolar macrophages and host defense to K.pneumoniae in Itch-deficient mice,suggesting that loss of alveolar macrophages occurred as a consequence of Th2 inflammation.Adoptive transfer of Itch−/−CD4+T cells into Rag−/−mice was sufficient to drive reduction in numbers of Itch-replete alveolar macrophages.Finally,we found that Stat6 signaling downstream of the IL-4 receptor directly reduced fitness of alveolar macrophages when these cells were exposed to the Itch−/−inflamed respiratory tract.These data suggest that Th2 inflammation directly impairs alveolar macrophage fitness in Itch−/−mice,and elucidate a previously unappreciated link between Th2 cells,alveolar macrophages and susceptibility to bacterial infection.

Anti Inflammatory Property of PDRN—An <i>in Vitro</i>Study on Cultured Macrophages

Skin aging and most age-related diseases are associated with a low-grade chronic inflammation. The nucleoside adenosine, a potent endogenous anti-inflammatory agent, is deeply involved in inflammatory diseases and, by interaction with the adenosine A2 receptor (A2AR) it immediately promotes a mechanism of defence against the inflammatory damage. The aim of our study was to investigate whether polydeoxyribonucleotide (PDRN), a mixture of deoxyribonucleotides polymers of different lengths that like adenosine, binds the A2A receptor, can reduce the inflammatory state in the macrophage cell line. RAW264.7, murine macrophage cells, were incubated with PDRN in the presence and in the absence of lipopolysaccaride (LPS), which was the major component of the outer membrane of gram-negative bacteria and which acted as a strong macrophage activator. We assessed the production of nitric oxide and the secretion of inflammatory mediators (i.e., TNF-α, IL-10, IL-12 and VEGF-A). Our data showed that PDRN produced a significant decrease of inflammation in macrophages pre-stimulated with LPS, assessed in terms of the nitric oxide content (p 2A receptor, contributed to a great extent towards reducing inflammation.

Essential Gene(s) Targeted by Peptide Nucleic Acids Kills <i>Mycobacterium smegmatis</i>in Culture and in Infected Macrophages

Background: Antisense peptide nucleic acids (PNAs) exhibit growth inhibitory effects on bacteria by inhibiting the expression of essential genes and could be promising therapeutic agents for treating bacterial infections. A study was carried out to determine the efficacy of several antisense PNAs in inhibiting extracellular and intracellular growth of Mycobacterium smegmatis. Methods: Six PNAs obtained from a commercial supplier were tested to evaluate the inhibitory effect on bacterial growth by inhibiting the expression of the following essential genes: inhA (a fatty acid elongase), rpsL (ribosomal S12 protein), gyrA (DNA gyrase), pncA (pyrazinamidase), polA (DNA polymerase I) and rpoC (RNA polymerase β subunit) of M. smegmatis. Each PNA was tested at 20 μM, 10 μM, 5 μM and 2.5 μM concentrations to determine whether they caused a dose dependent killing of M. smegmatis cultured in Middlebrook 7H9 broth or in a J774A.1 murine macrophage cell line. Results: In Middlebrook broth, the strong growth inhibitory effect against M. smegmatis was observed by PNAs targeting the inhA and rpsL genes at all four concentrations. The PNAs targeting the pncA, polA and rpoC genes were found to exhibit strong growth inhibition against M. smegmatis but only at 20 μM concentration. No growth inhibition of M. smegmatis was seen in pure culture when treated with PNAs targeting gyrA and a mismatch PNA targeting dnaG (DNA primase). All six PNAs showed killing of M. smegmatis in J774A.1 macrophage cell line that were statistically significant (p < 0.05). Conclusion: It may be concluded from this study that PNAs could be potential therapeutics for mycobacterial infections.

Essential role of monocytes and macrophages in the progression of acute pancreatitis

Acute pancreatitis(AP) is an inflammatory condition of the pancreas caused by an imbalance in factors involved in maintaining cellular homeostasis.Earliest events in AP occur within acinar cells accompanied by other principal contributors to the inflammatory response i.e.the endothelial cells,immunocytes(granulocytes,monocytes/macrophages,lymphocytes) and neutrophils.Monocytes/macrophages are important inflammatory mediators,involved in the pathophysiology of AP,known to reside in the peritoneal cavity(in the vicinity of the pancreas) and in peripancreatic tissue.Recent studies suggested that impaired clearance of injured acini by macrophages is associated with an altered cytokine reaction which may constitute a basis for progression of AP.This review focuses on the role of monocytes/macrophages in progression of AP and discusses f indings on the inflammatory process involved.

淫羊藿苷预处理增强人牙周膜干细胞对M1型巨噬细胞的影响

背景:人牙周膜干细胞对M1型巨噬细胞促炎功能有一定抑制作用,具有抗炎等药理活性的淫羊藿苷是否能增强人牙周膜干细胞对M1型巨噬细胞的抑制作用尚不明确。目的:探究淫羊藿苷预处理人牙周膜干细胞后对M1型巨噬细胞的影响。方法:分离培养原代人牙周膜干细胞,并进行鉴定。对THP-1细胞进行诱导,采用免疫荧光染色及PCR进行M1型巨噬细胞鉴定。用含浓度10^(-7),10^(-6),10^(-5),10^(-4)mol/L淫羊藿苷的α-MEM完全培养基培养人牙周膜干细胞1,3,5,7 d,采用CCK-8法筛选合适的淫羊藿苷浓度进行实验。将α-MEM完全培养基、未处理的人牙周膜干细胞α-MEM条件培养基及淫羊藿苷预处理24 h的人牙周膜干细胞α-MEM条件培养基与RPMI-1640完全培养基以1∶1的比例对M1型巨噬细胞进行条件培养,分别为对照组、未处理组及预处理组,24 h后RT-PCR法检测M1型巨噬细胞炎症因子的mRNA表达情况;ELISA法检测M1型巨噬细胞炎症因子的蛋白表达情况;Western blot检测M1/M2型巨噬细胞表面标记物及核转录因子κB通路相关蛋白的表达。结果与结论:(1)CCK-8检测结果显示,10^(-7),10^(-6),10^(-5),10^(-4)mol/L淫羊藿苷对人牙周膜干细胞均无细胞毒性,且从第5天起,各浓度都提高了细胞活力,促进细胞增殖,选择10^(-4)mol/L淫羊藿苷进行后续实验。(2)RT-PCR法及ELISA检测结果显示,与对照组相比,未处理组及预处理组均降低了M1型巨噬细胞白细胞介素1β、白细胞介素6及肿瘤坏死因子α的表达与分泌(P<0.05),且预处理组低于未处理组(P<0.05)。(3)Western blot检测结果显示,与未处理组相比,预处理组CD86的表达明显降低(P<0.05);与对照组相比,未处理组和预处理组M2型巨噬细胞表面标志物CD206的表达均升高(P<0.01),且预处理组明显高于未处理组(P<0.01);M1型巨噬细胞经24 h条件培养后,与对照组相比,核转录因子κB/P65的表达在未处理组和预处理组均有降低(P<0.01),p-IκBα的表达仅在预处理组降低(P<0.01);与未处理组相比,预处理组核转录因子κB/P65和p-IκBα的表达均显著降低(P<0.05),而IκBα在3组中的表达差异无显著性意义。(4)上述结果证实,淫羊藿苷增强了人牙周膜干细胞对M1型巨噬细胞的抑制作用,此作用可能与抑制了巨噬细胞的核转录因子κB信号通路相关。

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.

Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice

AIM: To investigate the correlation between lymphogenous metastasis and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymph nodes. METHODS: Fifty-six inbred 615-mice were equally divided into 2 groups and inoculated with Hca-F and Hca-P cells. Their lymph node metastatic rates were examined. Growth fraction of lymphocytes in host lymph nodes was detected by flow cytometry. The Hca-F and Hca-P cells were cultured with extract of lymph node, liver or spleen. The quantity of MMPs in these supernatants was examined by zymographic analysis. The expression of Fas ligand, PCNA, Bcl-2 protein of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. The apoptosis signals of macro-phages in lymph nodes were observed with in situ DNA fragmentation. RESULTS: On the 28th day post-inoculation, the lymph node metastatic rate of HcaF was 80%(16/20), whereas that of Hca-P was 25%(5/20). The growth fraction of lymphocytes was as follows: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in HcaP cells, the peak appeared on the 7th day post inoculation and then kept at a high level. With the extract of lymph node, the quantity of the MMP-9 activity increased (P【0.01) and active MMP-9 and MMP-2 were produced by both Hca-F and Hca-P tumor cells, which did not produce MMPs without the extract of lymph node or with the extracts of the liver and spleen. The expression of Fas Ligand of Hca-F cells was stronger than that of Hca-P cells (P 【0.01). The expressions of PCNA and Bcl-2 protein of Hca-F cells in the tumors of inoculated area were the same as that of Hca-P cells. In situ DNA fragmentation showed that the positive signals of macrophages were around Hca-F cells. CONCLUSION: Secretion of MMPs which was associated with metastatic ability of Hca-F and Hca-P tumor cells depends on the environment of lymph nodes. The increased expression of Fas ligand protein of Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.

间充质干细胞移植对降植烷诱导狼疮小鼠肺泡出血的影响及可能机制

目的:探讨间充质干细胞(mesenchymal stem cells,MSCs)移植对降植烷诱导狼疮小鼠肺泡出血的作用及可能机制。方法:10周龄雌性C57BL/6(B6)小鼠20只腹腔注射0.5 mL降植烷,3只同周龄雌性B6小鼠腹腔注射PBS作为正常对照组。1周后将20只注射降植烷小鼠随机分为MSC组和PBS组,分别给予尾静脉注射1×10^(6)人脐带来源的MSCs和PBS;观察1周后,计算造模小鼠生存率并称量小鼠体重,处死所有小鼠,取出肺脏,观察肺脏大体及肺脏HE染色评估造模小鼠肺出血严重程度。酶消化法制备获取3组小鼠肺脏单细胞悬液,流式细胞术检测3组小鼠肺部CD4^(+)、CD8^(+)T淋巴细胞,CD19^(+)B淋巴细胞,CD11b^(+)Gr1^(+)中性粒细胞,F4/80^(+)肺间质巨噬细胞的绝对数以及抗炎型CD206^(+)肺间质巨噬细胞的百分率。结果:MSC组小鼠生存率高于PBS组,但差异无统计学意义(P=0.07);体重明显高于PBS组小鼠(P<0.01);MSC组小鼠完全弥漫性肺泡出血(diffuse alveolar hemorrhage,DAH)及部分DAH的发生率低于PBS组。与PBS组相比,MSC组肺脏单细胞总数显著减少(P<0.01),肺脏CD4^(+)T细胞数量有下降趋势,而CD8^(+)T细胞及CD19^(+)B细胞的数量则明显下降(P<0.05),肺脏CD11b^(+)Gr1^(+)中性粒细胞及F4/80^(+)巨噬细胞的绝对数也均显著降低(P<0.01和P<0.05);此外,肺出血小鼠CD206^(+)抗炎型巨噬细胞的百分率显著降低(P<0.01),而MSC移植显著提高CD206^(+)抗炎型巨噬细胞的百分率(P<0.01)。结论:MSC移植可显著降低狼疮小鼠肺泡出血的发生率,其机制可能是对肺脏免疫细胞的调控及诱导巨噬细胞CD206抗炎表型的产生。

高分子三维支架材料通过诱导外周巨噬细胞的极化促进成骨前体细胞分化

目的:考察硫酸软骨素-壳聚糖-羟基磷灰石(SF-CS-HA)三维支架材料对小鼠巨噬细胞极化的影响,并进一步研究骨免疫微环境调控对小鼠颅顶骨前体细胞在骨皮质外骨生成的影响。方法:制备SF-CS-HA三维支架材料,将支架材料与小鼠巨噬细胞RAW264.7共培养,流式细胞术检测巨噬细胞的M2型分化情况,RT-qPCR检测炎症因子基因的表达,Western blot检测TGF-β蛋白表达的变化。将巨噬细胞与SF-CS-HA三维支架材料共孵育后,制备条件培养基,比较完全培养基和条件培养基对小鼠颅顶骨前体细胞MC3T3-E1(3T3)增殖、迁移、ALP活性及形态变化,并通过Western blot检测骨生成相关蛋白的表达。最后,通过动物模型验证SF-CS-HA材料对于小鼠颅骨骨量增加的影响。结果:扫描电镜观察显示,SF-CS-HA三维支架材料表面和内部都具有均匀的空腔结构。流式细胞术检测结果显示,SF-CS-HA三维支架材料能够促进小鼠外周巨噬细胞向M2型极化。RT-qPCR和Western blot结果显示,SF-CS-HA三维支架材料提高了IL-10基因和TGF-β蛋白的表达量,降低了IL-1β基因的表达量。且SF-CS-HA三维支架能够促进MC3T3-E1细胞的增殖,显著提高ALP活性,增加细胞的迁移能力,提高细胞骨架蛋白Factin的生成。而条件培养基(骨免疫微环境调控下)对于MC3T3-E1细胞增殖和迁移能力的提升更为显著。此外,Western blot结果显示,骨免疫微环境调控下,骨生成蛋白OPN、COLA1、RUNX2、OCN和TGF-β显著增加。动物实验结果显示,SF-CS-HA材料能够在体内促进小鼠颅骨的新骨形成。结论:SF-CS-HA三维支架材料能够促进小鼠外周巨噬细胞的极化,且SF-CS-HA能够调控骨免疫微环境,促进骨细胞生成,在骨皮质外达到骨增量。

IL-6通过激活JAK2/STAT3信号通路增强小鼠肺泡巨噬细胞的吞噬功能

目的 探讨白细胞介素6(IL-6)对MH-S小鼠肺泡巨噬细胞吞噬功能的影响及其相关机制。方法 脂多糖(LPS)经气道滴入激发构建小鼠急性肺损伤(ALI)模型,ELISA检测支气管肺泡灌洗液(BALF)中IL-6的含量。体外培养MH-S细胞,在信号转导子与转录激活子3 (STAT3)抑制剂Stattic(5μmol/L)存在与否的情况下,再加入IL-6(10 ng/mL~500 ng/mL)刺激6 h,加入荧光微球孵育2 h后,采用流式细胞术检测MH-S细胞吞噬荧光微球情况;Western blot法检测磷酸化的Janus激酶2(p-JAK2)、磷酸化的STAT3(p-STAT3)、肌动蛋白相关蛋白2(Arp2)、纤维型肌动蛋白(F-actin)的表达水平。结果 鼻腔滴入LPS后,小鼠BALF中IL-6含量显著升高。随着IL-6刺激剂量的增加,MH-S细胞吞噬荧光微球的作用增强,Arp2、 F-actin蛋白的表达水平升高。100 ng/mL IL-6刺激MH-S细胞后,p-JAK2和p-STAT3蛋白的表达水平升高。阻断MH-S细胞STAT3信号后,IL-6促进细胞吞噬的效应完全消失,IL-6诱导的Arp2、 F-actin蛋白表达增加被抑制。结论 IL-6通过激活JAK2/STAT3信号通路促进MH-S细胞Arp2、 F-actin蛋白的表达增强吞噬功能。

COPD细胞微环境体外模型建立的思路与方法

慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是危害公众健康的重大慢性疾病。COPD病理机制复杂,细胞微环境改变是其病理生理的重要组成部分。细胞培养技术是研究COPD病理机制及药效药理评价的重要工具。该文介绍了肺内细胞微环境的基本构成,COPD病理进程中细胞微环境变化,总结了细胞微环境体外模型在COPD机制研究中的应用,提出COPD细胞微环境体外模型建立的思路与方法。

肺泡上皮细胞分泌的外泌体调控巨噬细胞极化在急性肺损伤中的作用

外泌体作为细胞间微环境中的信使,能够将信号在细胞之间进行传递。肺泡上皮细胞通过分泌外泌体来调节机体固有免疫反应。在特定的刺激条件下,肺泡上皮细胞分泌的外泌体通过传递不同效应活性物质,靶向调节巨噬细胞极化通路中的基因表达,参与控制肺部炎症反应的巨噬细胞极化调节。本篇综述主要阐述肺泡上皮细胞源性外泌体,通过靶向调节巨噬细胞极化,调控急性肺损伤(acute lung injury,ALI)作用的最新研究进展,为相关研究提供参考。

基于Super-SILAC定量技术系统解析槲皮素调控巨噬细胞的组蛋白翻译后修饰图谱

槲皮素(quercetin,Que)是一种具有多种生物活性的黄酮类化合物,在自然界中分布广泛,具有抗炎和抗肿瘤等生物活性。研究发现,槲皮素的抗炎活性与其对组蛋白翻译后修饰的调控密切相关。然而,槲皮素调控组蛋白翻译后修饰发挥抗炎作用的精细机制尚未有详细报道。为了探讨槲皮素调控组蛋白翻译后修饰发挥抗炎活性的作用机制,我们首先评估了槲皮素对M1型巨噬细胞极化的影响,结果发现,槲皮素可以明显下调M1型巨噬细胞的白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-6(interleukin-6,IL-6)水平;其次,利用生物质谱的细胞稳定同位素标记(stable isotope labeling by amino acids in cell culture,SILAC)技术衍生的super-SILAC方法,进行了组蛋白翻译后修饰的解析,系统定量分析了槲皮素处理后的巨噬细胞中组蛋白修饰水平的全局性变化;最终,共定量到30个组蛋白修饰位点,其中包括12个发生下调的组蛋白乙酰化修饰位点和4个发生上调的甲基化修饰位点(fold change>1.2,P<0.05);进一步对部分差异变化位点进行了蛋白质免疫印迹(western blot,WB)验证,其差异变化与质谱结果一致性良好。综上,本研究系统解析了槲皮素调控巨噬细胞的组蛋白翻译后修饰图谱,为槲皮素抗炎作用机制的研究提供了可靠的数据参考和新的线索。

不同条件下小鼠肺泡巨噬细胞体外培养的实验研究

目的:应用文献报道的肺泡巨噬细胞(AMs)体外培养方法,利用GM-CSF、TGF-β和PPAR-γ激动剂罗格列酮(简称GTR)培养体系,分析不同年龄小鼠、不同来源前体细胞产生AM样细胞或AMs的特征。方法:收取不同周龄小鼠支气管肺泡灌洗液(BALF)、骨髓单个核细胞以及胚胎15~17 d小鼠肝脏单个核细胞,种入12孔板,置于GTR体系中培养。流式细胞术检测产出细胞AM表面标志分子F4/80、CD11b、CD170和CD11c表达。结果:培养后,光镜下BALF来源细胞基本呈圆形,骨髓来源细胞近半呈圆形,胎肝来源细胞85%左右呈圆形。4周龄小鼠BALF-AMs比例接近90%,比例和数量均随小鼠年龄增加而增加,12周龄小鼠BALF-AMs数量接近1×106个。4周龄小鼠骨髓源AM样细胞比例不到20%,8周龄和12周龄小鼠比例为40%~45%,数量随小鼠年龄增加而增加,12周龄小鼠骨髓源AM样细胞数量约为0.8×106个/孔。胎肝源AM样细胞比例约为84%,1个孔产出的AM样细胞约为4×106个。结论:不同年龄小鼠、不同来源前体细胞产生AM样细胞或AMs的纯度和数量有较大差别,应根据具体研究要求选择合适方法。

肺泡巨噬细胞和肺泡上皮细胞在急性呼吸窘迫综合征中的研究进展

急性肺损伤常导致急性呼吸窘迫综合征(ARDS)危重患者死亡,免疫细胞浸润被视为ARDS的重要标志,其中肺泡巨噬细胞(AMs)是ARDS发病过程中发挥作用的免疫细胞,AMs主要通过与肺泡上皮细胞(AECs)相互作用来参与疾病的发展。在ARDS早期阶段,激活的AMs分泌促炎细胞因子,以清除可能破坏AECs细胞结构并引起细胞死亡的病原体。在ARDS晚期,交替激活的巨噬细胞分泌抗炎细胞因子,抑制炎症反应,促进上皮再生和肺泡结构重塑。本文主要回顾AMs和AECs在ARDS中的功能和特点,以及描述AMs和AECs之间的相互作用。

骨髓间充质干细胞源外泌体对急性肺损伤大鼠肺泡巨噬细胞M1/M2极化的影响及其机制

目的:探讨大鼠骨髓间充质干细胞来源的外泌体(bone marrow mesenchymal stem cells derived exosomes,BMSCs-Exos)对脂多糖(LPS)诱导的急性肺损伤大鼠肺泡巨噬细胞NR8383 M1/M2极化的影响及其潜在的机制。方法:分离SD大鼠骨髓间充质干细胞并制备BMSCs-Exos。分别用0.1、1 mg/mL BMSCs-Exos预处理NR8383细胞1 h,再用1μg/mL LPS诱导48 h,采用ELISA和蛋白免疫印迹分别检测细胞上清液中炎性因子含量以及细胞内极化标志物蛋白表达。将NR8383细胞单独培养或与BMSCs共培养,经20μmol/L外泌体抑制剂GW4869处理并予以1μg/mL LPS诱导48 h,采用实时荧光定量PCR和蛋白免疫印迹分别检测细胞内miR-212-5p以及IL-4Rα和p-STAT6蛋白相对表达。生物信息学预测miR-212-5p与IL-4RαmRNA结合位点并通过荧光素酶实验验证。结果:成功制备BMSCs-Exos。ELISA和免疫印迹结果表明,1μg/mL LPS诱导48 h可促进NR8383细胞分泌炎性因子(TNF-α,IL-1β和IL-10)及M1极化,BMSCs-Exos预处理可明显降低LPS诱导的NR8383细胞培养上清液中炎性因子TNF-α和IL-1β含量,增加抗炎因子IL-10含量,同时抑制细胞M1极化并促进其M2极化,且1 mg/mL BMSCs-Exos明显强于0.1 mg/mL BMSCs-Exos。细胞共培养实验结果表明,1μg/mL LPS诱导48 h可增加NR8383细胞中miR-212-5p相对表达量以及IL-4Rα和p-STAT6蛋白表达,与BMSCs共培养可有效抑制LPS对上述指标的影响,但BMSCs的作用可被GW4869阻断。荧光素酶实验结果显示,miR-212-5p可与IL-4RαmRNA 3′UTR结合并促进其蛋白表达。结论:BMSCs-Exos可能通过miR-212-5p/IL-4Rα/STAT6通路抑制LPS诱导的急性肺损伤大鼠肺泡巨噬细胞NR8383的M1极化并促进其M2极化。

M2型巨噬细胞抑制CD8^(+)T细胞促进食管癌细胞恶性生物行为

目的探讨M2型巨噬细胞通过抑制CD8^(+)T细胞的抗肿瘤能力参与食管癌的恶性生物学行为的作用。方法利用佛波酯(PMA)联合白细胞介素4(IL-4)/IL-13将人单核细胞白血病细胞(THP-1)诱导为M2型巨噬细胞并用实时定量PCR检测相关炎性因子。运用磁珠分选法分选健康志愿者外周血CD8^(+)T细胞,用流式细胞术验证分选纯度。构建CD8^(+)T细胞与食管鳞癌细胞的非接触性共培养体系(CD8^(+)T细胞),和构建M2型巨噬细胞、CD8^(+)T细胞与食管鳞癌细胞的非接触性共培养体系(M2型巨噬细胞联合CD8^(+)T细胞)。平板克隆实验和CCK-8细胞毒性实验检测各组肿瘤细胞增殖能力。Transwell^(TM)实验检测各组肿瘤细胞侵袭和迁移能力。流式细胞术检测并分析各组肿瘤细胞的凋亡情况。运用GraphPadPrism9.5软件进行统计学分析和制图。结果诱导后巨噬细胞IL-10和精氨酸酶1(Arg1)表达上调,IL-12和肿瘤坏死因子α(TNF-α)表达下降,具有M2表型巨噬细胞特征。磁珠分选法成功的分选出CD8^(+)T细胞,其阳性率>90%。食管鳞癌细胞与CD8^(+)T细胞非接触性共培养组的增殖、侵袭、迁移和抗凋亡能力显著低于单独癌细胞组;而食管鳞癌细胞与M2型巨噬细胞条件培养基预处理CD8^(+)T细胞共培养后,其增殖、侵袭、迁移和抗凋亡能力与食管鳞癌和CD8^(+)T细胞共培养组相比显著增强。结论M2型巨噬细胞通过抑制CD8^(+)T细胞的抗肿瘤能力促进食管鳞癌细胞的增殖、侵袭、迁移和抗凋亡。

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting

Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1 s)and type 2 cells(AEC2s)are the key cells in the repair of injured lungs as well as fetal lung development.Therefore,the purification and culture of AECls and AEC2s play an important role in the research of repair and regeneration of lung tissue.Methods:Sprague-Dawley rats(3-4 weeks,120-150 g)were purchased for experiment.Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells,and then magnetic bead cell sorting was performed to isolate Tla positive cells as AECls from the single-cell suspension by using polyclonal rabbit anti-Tla(a specific AECls membrane protein)antibodies combined with anti-rabbit IgG microbeads.Afterwards,alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining Tla-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads.Cell purity was identified by immunofluorescence staining and flow cytometry.Resii沾••The purity of AECls and AEC2s was 88.3%±3.8%and 92.6%±2.7%,respectively.The cell growth was observed as follows:AECls stretched within the 12-16 h,but the cells proliferated slowly;while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.Conclusion:AECls and AEC2s sorted by this method have high purity and good viability.Therefore,our method provides a new approach for the isolation and culture of AECls and AEC2s as well as a new strategy for the research of lung repair and regeneration.