上调miR-181a抑制香烟提取物诱导的支气管上皮细胞致炎因子生成与collagen IV、fibronectin和α-SMA表达

目的:探讨微小RNA-181a(miR-181a)对香烟提取物(cigarette smoke extract,CSE)诱导的人支气管上皮细胞(human bronchial epithelial cells, HBECs)致炎因子生成与IV型胶原蛋白(collagen IV)、纤连蛋白(fibronectin)和α-平滑肌肌动蛋白(α-SMA)表达的影响,并分析其可能的机制。方法:RT-qPCR检测CSE诱导下HBECs中miR-181a的表达情况。转染miR-181a mimic后经ELISA检测肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、IL-6和转化生长因子β1(transforming growth factor-β1,TGF-β1)的水平;Western blot检测collagen IV、fibronectin和α-SMA的表达;并进一步评估NF-κB/TGF-β1/Smad3信号通路的活性。结果:CSE可显著增加HBECs中致炎症因子IL-1β、IL-6、TNF-α和TGF-β1的生成,显著上调collagen IV、fibronectin和α-SMA的表达,同时细胞内miR-181a的表达明显降低(P0.05);转染miR-181a mimic可显著抑制CSE诱导的HBECs致炎因子生成及collagen IV、fibronectin和α-SMA表达(P0.05)。此外,Western blot的结果显示转染miR-181a mimic可抑制CSE诱导的NF-κB/TGF-β1/Smad3信号活性(P0.05)。结论:上调miR-181a表达可部分逆转CSE诱导的HBECs致炎因子的释放及collagen IV、fibronectin和α-SMA表达,其作用机制可能与抑制NF-κB/TGF-β1/Smad3信号通路的活化有关。     

大鼠肝纤维化与microRNA-181a调控肝星状细胞自噬的关系

目的:观察四氯化碳(CCl4)诱导肝纤维化(HF)大鼠肝脏结构的改变、肝组织中转化生长因子β1(TGF-β1)、微小RNA-181a(microRNA-181a)、自噬标志性蛋白LC3-II/-I和beclin-1水平及胶原沉积的变化,以及mi?croRNA-181a对TGF-β1诱导大鼠肝星状细胞(HSCs)自噬的作...     

一氧化氮对香烟烟雾提取物诱导大鼠肺泡巨噬细胞核因子кB活化的影响

目的:探讨一氧化氮(NO)对香烟烟雾提取物(CSE)诱导的大鼠肺泡巨噬细胞(AM)中核因子κB(NF—κB)活化的影响及机 制。方法:将大鼠AM与不同浓度的NO前体左旋精氨酸(L-Arg)或iNOS特异性抑制剂N6-(1-亚氨乙基)赖氨酸(L-NIL)及CSE共同培养,用免疫细胞化学染色法检测NF-κB,用Western blot检测I-κB蛋白含量,用Griess法测定培养上清液中NO的水平。结果:CSE可使NF-κB活化细胞的百分率增加,I-κB的水平下降。加入CSE和低浓度L-Arg培养的AM,NF-κB活化细胞的百分率显著高于只加入CSE的AM;而I-κB的水平显著低于只加入CSE的AM。加入CSE和高浓度L-Arg培养的AM,NF-κB活化细胞的百分率显著低于只加入CSE的AM,而I-κB的水平无显著变化。加入CSE和不同浓度的L-NIL培养的AM,NF-κB活化细胞的百分率显著低于只加入CSE的AM;而I-κB的水平则显著高于只加入CSE的AM,并呈浓度依赖(P<0.01)。结论:内源性NO对香烟烟雾所致NF-κB的活化具有双向调控作用。     

血清miR-181b和miR-27a表达与颈动脉粥样硬化程度相关

目的 分析血清微小RNA(miR)-181b、miR-27a表达与颈动脉粥样硬化(CAS)病变程度的关系.方法 选取血脂异常患者102例,健康人100名为对照组,RT-qPCR检测血清miR-181b和miR-27a的表达.对比血脂异常患者与对照组血清miR-181b、miR-27a表达差异,分析血脂异常患者发生CAS...     

肺泡巨噬细胞免疫代谢与慢性阻塞性肺疾病关系的研究进展

肺泡巨噬细胞（AMs）在慢性阻塞性肺疾病（COPD）的发病中起着重要作用,代谢途径和代谢产物的改变是AMs功能和相关疾病进展的关键因素之一。本文就影响AMs代谢变化的因素,以及代谢变化如何影响AMs的功能作了阐述,并进一步总结了AMs的免疫代谢变化在COPD中的研究进展,以期为COPD的防治提供新的思路和途径。     

miR-17调控自噬保护高糖环境下内皮细胞

目的:研究miR-17调控的自噬对高糖环境下内皮细胞氧化应激反应和凋亡的影响。方法:体外培养人脐静脉内皮细胞(HUVECs),并加入5.5mmol/L(对照组)和16.7mmol/L(高糖组)的葡萄糖进行干预,通过检测超氧化物歧化酶(SOD)活性,丙二醛(MDA)含量及自噬相关基因LC3、Beclin-1和SQSTM1的表达,比较各组HUVECs氧化应激和自噬水平的改变。再将HUVECs转染miR-17 mimics,并以自噬抑制剂3-甲基腺嘌呤(3-MA)干预,非转染miR-17mimics作为对照,观察miR-17对HUVECs自噬、氧化应激和凋亡的影响,并评价3-MA抑制HUVECs自噬后上述指标的改变。结果:与对照组比较,高糖组HUVECs的SOD活性下降,MDA水平增加,同时LC3II/I比例和Beclin-1上调,SQSTM1表达减少(P0.01)。在高糖环境下,转染miR-17 mimics后的HUVECs的LC3II/I比例和Beclin-1表达上调,SQSTM1水平降低,SOD活性部分恢复,MDA水平下降,细胞凋亡减少(P0.01)。经3-MA干预后,HUVECs上述效应明显减弱(P0.01)。结论:miR-17可上调内皮细胞的自噬水平,减轻高糖诱导的内皮细胞氧化应激反应,减少内皮细胞凋亡。     

miR-155/自噬/外泌体对酒精性肝病的影响

目的:研究酒精性肝病(ALD)中酒精持续激活小鼠肝脏巨噬细胞(即Kupffer细胞)并诱发肝脏炎性损伤的机制.方法:使用Gao-binge法制备慢性ALD小鼠模型,观察肝损伤,检测外泌体的浓度和大小分布,Western blot和RT-qPCR检测自噬相关蛋白和微小RNA-155(miR-155)的表达.体外实验中,对...     

敲低Wnt7a抑制卡介苗诱导的小鼠肺泡上皮细胞自噬

目的 探究无翅基因7a(Wnt7a)对牛结核分枝杆菌卡介苗(BCG)感染的肺泡上皮细胞自噬水平的调控作用。方法使用干扰Wnt7a慢病毒及BCG单独作用或共处理TC-1小鼠肺泡上皮细胞,设置小干涉RNA对照(si-NC)组、 si-NC联合BCG组、 Wnt7a小干涉RNA(si-Wnt7a)组和si-Wnt7a联合BCG组。Western blot法检测Wnt7a、微管相关蛋白1轻链3(LC3)、 P62及自噬相关基因5(ATG5)蛋白表达水平,利用免疫荧光细胞化学染色检测LC3的分布以确定细胞自噬情况。结果 与si-NC组相比,BCG感染的TC-1细胞Wnt7a、 LC3和ATG5蛋白表达均升高、 LC3绿色荧光斑点亮度与分布显著增加;与BCG组感染的TC-1细胞相比,si-Wnt7a联合BCG组Wnt7a、 LC3、 P62及ATG5蛋白表达均显著降低,LC3绿色荧光斑点亮度与分布显著降低。结论 敲低Wnt7a抑制BCG诱导的肺泡上皮细胞自噬。     

miR-7基因敲减对LPS诱导巨噬细胞炎症反应的影响

观察miR-7基因敲减(knockdown, KD)对LPS诱导的巨噬细胞炎症反应的影响,并初步探讨其机制。分别用0、50、100、200 ng/mL LPS刺激野生型(wild type, WT)小鼠骨髓来源巨噬细胞12 h,经实时荧光定量PCR(real-time fluorescence quantitative PCR, Real-time PCR)检测发现,随着LPS刺激浓度的增加,巨噬细胞中miR-7的表达水平逐渐升高,且在100 ng/mL LPS浓度刺激下miR-7表达最显著(P0.01)。用100 ng/mL LPS处理miR-7 KD小鼠和WT小鼠骨髓来源巨噬细胞12 h,镜检观察到巨噬细胞形态均由圆形变为长梭形,并伸出长长的伪足。应用流式细胞术检测巨噬细胞表面CD80和CD86分子的表达情况,结果显示,LPS作用后,miR-7 KD小鼠骨髓来源巨噬细胞表面CD80和CD86的比例较WT小鼠显著上调(P0.05)。Real-time PCR和ELISA结果显示,miR-7 KD小鼠巨噬细胞在LPS刺激后,炎性因子IL-1β、IL-6和TNF-ɑ的表达显著增加(P0.05)。Western blotting检测结果显示,LPS刺激后,与对照组相比,miR-7 KD小鼠巨噬细胞中NF-κB p65和p-NF-κB p65的蛋白水平显著升高(P0.01)。以上数据显示,miR-7基因KD可显著增强LPS诱导的巨噬细胞炎症反应,可能与NF-κB信号通路的传递增强有关,提示miR-7在巨噬细胞炎症过程中发挥重要的调控作用。     

自噬参与香烟烟雾提取物诱导的肺动脉内皮细胞凋亡

目的:探讨自噬在香烟烟雾提取物(cigarette smoke extract,CSE)诱导人肺动脉内皮细胞(human pulmonary artery endothelial cells,HPAECs)凋亡中的作用。方法:常规培养HPAECs,分为对照组、CSE组、3-甲基腺嘌呤(3-methyladenine,3-MA)组和3-MA+CSE组,应用Hoechst 33342染色和Annexin V/PI流式细胞术检测细胞凋亡,单丹磺酰尸胺(monodansylcadaverine,MDC)染色观察细胞自噬泡形成,Western bolt测定自噬相关蛋白beclin-1、LC3与cleaved caspase-3的水平。结果:MDC染色示CSE处理可以诱导细胞产生自噬泡,Western blot结果示自噬相关蛋白LC3及beclin-1表达升高,3-MA预处理后抑制上述蛋白的表达。Hoechst 33342染色和Annexin V/PI流式结果显示CSE组细胞凋亡率较对照组明显增加,在3-MA+CSE组,细胞凋亡率较CSE组进一步升高;同时,CSE组细胞cleaved caspase-3蛋白水平较对照组明显升高(P0.05),3-MA+CSE组的caspase-3表达较CSE组进一步升高。结论:CSE能诱导HPAECs发生自噬和凋亡,抑制自噬能够进一步促进CSE对HPAECs的凋亡作用,这种作用可通过激活caspase-3实现。     

Immunosuppressive properties of surfactant in alveolar macrophage NR8383

Objective: To evaluate the anti-inflammatory effects of exogenous surfactants and surfactant phospholipid without surfactant proteins (SP-A and SP-D) on the lipopolysaccharide- (LPS) stimulated rat alveolar macrophage (AM) cell line NR8383. Methods: Exogenous surfactants (beractant, calfactant or colfosceril) and surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), standardized to phospholipid content of 25–1,000 μg/ml were incubated with LPS- (1 μg/ml) stimulated NR8383 AMs. Results: TNF-α and IL-1β secretion and nitric oxide (NO) formation following LPS stimulation were inhibited by treatment with surfactants or DPPC. Furthermore, LPS-dependent NO production and iNOS protein levels were significantly suppressed in cells pretreated for one hour with beractant compared to beractant added simultaneously with or following LPS. Additionally, LPS-stimulated oxidative burst, measured by flow cytometry, was significantly decreased by beractant. Finally, beractant inhibited the translocation of NF-κB from cytoplasmic into nuclear extract in LPS-stimulated NR8383 AMs. Conclusions: Exogenous surfactants and surfactant phospholipid inhibit secretion of proinflammatory cytokines and NO in NR8383 AMs. The inhibitory effects of beractant on oxygen radical and LPS-induced NO formation may result from unique mechanisms of decreasing cell signaling. The anti-inflammatory activity of surfactant products used in the treatment of neonatal respiratory distress syndrome (RDS) may depend upon the specific preparation or dose used. Received 28 December 2006; returned for revision 4 July 2007; accepted by S. Stimpson 29 October 2007     

MicroRNA-132转染对脂多糖诱导的肺泡巨噬细胞炎症反应的作用

 目的:探讨转染微小RNA-132（miR-132）对肺泡巨噬细胞炎症反应的作用。方法: 将体外去致热源培养的大鼠肺泡巨噬细胞株NR8383分为空白对照组、阴性对照组和转染组,分别采用miR-132增敏剂、错义链和PBS作用。转染24 h后,CCK-8法检测细胞増殖;实时荧光定量PCR检测细胞中miR-132的表达;用脂多糖（LPS）作用细胞后,凝胶电泳迁移率实验（EMSA）检测细胞中NF-κB活性;Western blotting法检测细胞中肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）的表达。结果: 与空白对照组和阴性对照组相比较,转染组细胞中miR-132的表达明显升高;转染组细胞増殖被明显抑制,与空白对照组相比较,差异有统计学意义（P<0.05）;LPS作用后,转染组NF-κB、TNF-α和IL-6表达量显著下降,与空白对照组和阴性对照组相比较,差异均有统计学意义（P<0.05）。结论: 转染miR-132可抑制NR8383细胞増殖,并抑制LPS诱导的NR8383细胞的炎症反应。     

Inhibition of Ca2+ influx by surfactant in NR8383 alveolar macrophages

OBJECTIVE: Pulmonary surfactants reduce alveolar surface tension and alter inflammatory cell function. We studied the effects of surfactant preparations on Ca2+ influx regulated by protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) and cytokine secretion in the alveolar macrophage (AM) cell line NR8383.METHODS: Fura-2-loaded AMs were stimulated with zymosan (200 microg/ml), 1,2-dioctanoyl-sn-glycerol (DOG, 20 microM) or C6-ceramide (C6C, 10 microM) in the presence of exogenous surfactants (beractant, calfactant or colfosceril) or surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), at 250 microg/ml phospholipid and changes in cytosolic free Ca2+ (Delta[Ca2+]i) and cytokines were measured. RESULTS: Zymosan-induced Delta[Ca2+]i (117 +/- 5 nM) at 3 min was reduced (p <0.001) by beractant (50 +/- 6 nM), colfosceril (61 +/- 2 nM), calfactant (46 +/- 5 nM), and DPPC (52 +/- 5 nM). Beractant inhibited the Delta[Ca2+]i by PKC stimulation with DOG and all preparations reduced the MAPK-induced Ca2+ influx by C6C. Beractant and Ca2+ channel blocker SKF 96365 (10 microM) together abolished the zymosan-stimulated Delta[Ca2+]i. Zymosan-stimulated TNF-alpha and IL-1beta secretion was also inhibited by surfactant pretreatment. CONCLUSIONS: These results indicate that exogenous surfactant inhibits Ca2+ influx and cytokine secretion in zymosan-stimulated AMs. This anti-inflammatory activity may be through an interaction with downstream signaling elements or Ca2+ channels.     

白藜芦醇调控miR-34a表达对骨肉瘤MG-63细胞生长、侵袭和自噬的影响

目的:探讨白藜芦醇通过调控微小RNA-34a(miR-34a)表达对骨肉瘤MG-63细胞生长、侵袭和自噬的影响。方法:将骨肉瘤MG-63细胞分为对照组及10、20、40、60和80μmol/L白藜芦醇处理组。采用MTT法、Transwell小室和Western blot实验检测各组骨肉瘤细胞的活力、侵袭能力及自噬蛋白表达;RT-q PCR检测各组骨肉瘤细胞中miR-34a的表达。转染miR-34a模拟物(miR-34a mimic)及阴性对照(miR-34a NC)至骨肉瘤细胞,检测miR-34a mimic和miR-34a NC在白藜芦醇处理后对骨肉瘤细胞活力和侵袭能力的影响,Western blot检测自噬标志蛋白LC3-Ⅰ、LC3-Ⅱ和beclin-1的蛋白表达。结果:与对照组相比,不同浓度的白藜芦醇可抑制肿瘤细胞的活力和侵袭能力,促进自噬(P0.05)。白藜芦醇可上调肿瘤细胞中miR-34a的表达,并具有剂量依赖性(P0.05),同时促使LC3-Ⅱ/LC3-Ⅰ比值升高,beclin-1蛋白表达量上调(P0.05)。miR-34a mimic可增加白藜芦醇对骨肉瘤细胞活力和侵袭能力的抑制,并进一步促进自噬。结论:白藜芦醇可能通过上调miR-34a表达抑制骨肉瘤MG-63细胞生长并促进其自噬。     

慢性阻塞性肺疾病小鼠模型制备方法的比较研究

目的:采用香烟烟雾(cigarette smoke,CS)暴露、肺炎克雷伯杆菌(Klebsiella pneumoniae,KP)感染、聚肌胞苷酸(polyinosinic-polycytidylic acid,Poly I:C)滴鼻、CS暴露联合KP感染和CS暴露联合Poly I:C滴鼻5种方法,建立并比较慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)小鼠模型。方法:将288只雄性BALB/c小鼠随机分为正常组(normal组)、CS组、KP组、CS+KP组、Poly I:C组及CS+Poly I:C组,每组48只。第1～8周造模,分别于第4、8、16和24周末取材。观察小鼠肺组织平均肺泡数(mean alveolar number,MAN)、肺泡平均截距(mean linear intercept,MLI)、肺功能呼气峰流速(peak expiratory flow,PEF)和50%潮气量呼气流量(50%tidal volume expiratory flow,EF50)的变化,检测肺组织中肿瘤坏死因子α(tumor nec...     

Increased expression of HLA-DR antigen on alveolar macrophages in pulmonary diseases

Summary The expression of HLA-DR antigen on alveolar macrophages obtained by bronchoalveolar lavage in healthy controls and patients with different diseases was investigated, using cytofluorographic analysis and phykoerythrin conjugated monoclonal mouse anti-human HLA-DR antibody. Alveolar macrophages in patients with extrinsic allergic alveolitis (n=4), idiopathic lung fibrosis (n=4), sarcoidosis (n=6), rheumatoid lung disease (n=6) and pulmonary infection (n=5) showed increased density of HLA-DR antigen expression compared to healthy control subjects (n=5). The increased expression of HLA-DR antigen on alveolar macrophages confirms the importance of these cells for recognition of antigens and immunological responses in different pulmonary diseases.Abbreviations BAL bronchoalveolar lavage - PBS phosphate buffered saline     

miR-19a和miR-92a在多发性骨髓瘤中的功能及其信号通路分析

目的:利用微小RNA(microR,miRNA)种子序列的反义寡核苷酸研究miR-19a和miR-92a簇在多发性骨髓瘤中的功能及其信号通路分析。方法:实验分为miR-19a种子序列反义寡核苷酸(t-antimiR-19a)组、miR-92a种子序列反义寡核苷酸(t-antimiR-92a)组、随机对照组和空白对照组,通过MTT法检测细胞活力;使用细胞集落形成实验观察集落形成情况;Transwell小室法检测反义寡核苷酸转染细胞的侵袭能力;生物信息学软件miRFocus用来分析miR-19a和miR-92a的靶基因和其参与的信号通路。结果:MTT结果表明t-antimiR-19a和t-antimiR-92a显著抑制RPMI-8266细胞的生长,t-antimiR-19a和t-antimiR-92a的最佳浓度是0.5μmol/L,最佳作用时间是转染后48 h。细胞集落形成实验显示,相比于随机对照组,t-antimiR-19a组和t-antimiR-92a组的细胞形成集落的能力减弱,集落形成抑制率显著降低。Transwell小室实验显示,相比于随机对照组,t-antimiR-19a组和t-antimiR-92a组的侵袭能力减弱,具有侵袭能力的细胞数显著降低。miRFocus软件分析miR-19a和miR-92a参与多条与肿瘤有关的重要的信号通路。结论:miR-19a和miR-92a有可能作为治疗多发性骨髓瘤的潜在靶点。     

Antigen expression of alveolar macrophages in smokers and patients with lung diseases

Alveolar macrophage function was studied immunocytochemically using three monoclonal antibodies—macrophage CD 68 KP 1 (M), protein CD 11C (P), and anti-elastin (EL)—and three polyclonal antibodies—lysozyme (LZ), alpha-1-antitrypsin (AAT), and alpha-1-antichymotrypsin (AACT). the material for study was smears obtained from bronchial washings from 15 healthy persons and 60 patients with respiratory infections or primary or secondary malignant lung infiltration. Eight of the healthy group and 40 of the patient group were smokers (SM). the percentage of cells obtained from the washings which were macrophages was also measured. The intensity of staining reactions for each of the six antigens was noted and in general more intense staining was noted in smokers than in non-smokers. More intense staining was observed in patients with pulmonary infections (group II PI) and metastatic pulmonary infiltrations (group IV MP Ca) than in controls (group IC), while patients with primary lung cancer (group III PP Ca) had highly reduced staining reactions. the number of macrophages was similarly increased in all groups in comparison with the IC group for non-smokers and in all groups except III PP Ca for smokers. It is concluded that smoking, pulmonary infections, and metastatic infiltration of the lung are associated with an increase in the number and activity of alveolar macrophages, while patients with primary lung cancer have an increase in the number of macrophages which are functionally incompetent.