Role of Insulin-like Growth Factor II Receptor in Transdifferentiation of Free Silica-induced Primary Rat Lung Fibroblasts

Objective To study the role of insulin-like growth factor II receptor in free silica-induced transdifferentiation of primary rat lung fibroblasts Methods Rat lung fibroblasts and rat alveolar macrophages were cultured. A transdifferentiation model of primary rat lung fibroblasts was induced by free silica. Levels of a-SMA protein, IGF-liR protein and mRNA were measured by immunocytochemistry, Western blot and RT-PCR, respectively. Lung fibroblasts were treated with Wortmannin. Results The expression levels of a-SMA concentration and decreased after Wortmann and IGF-IIR increased with the increasing free silica n was used. Conclusion The IGF-IIR plays an important role in free silica-induced transdifferentiation of primary rat lung fibroblasts.

Mitofusion 2 Overexpression Decreased Proliferation of Human Embryonic Lung Fibroblasts in Acute Respiratory Distress Syndrome through Inhibiting RAS-RAF-1-ERK1/2 Pathway

Acute respiratory distress syndrome(ARDS)is one of the most fatal diseases worldwide.Pulmonary fibrosis occurs early in ARDS,and its severity plays a crucial role in ARDS mortality rate.Some studies suggested that fibroproliferation is an essential mechanism in ARDS.Mitofusion2(Mfn2)overexpression plays a role in inhibiting cell proliferation.However,the role and potential mechanism of Mfn2 on the proliferation of fibroblasts is still unknown.In this study,we aimed at exploring the effect of Mfn2 on the human embryonic lung fibroblasts(HELF)and discussed its related mechanism.The HELF were treated with the Mfn2 overexpressing lentivirus(adv-Mfn2).The cell cycle was detected by flow cytometry.MTT,PCR and Western blotting were used to investigate the effect of Mfn2 on the proliferation of the HELF,collagen expression,the RAS-RAF-1-ERK1/2 pathway and the expression of cycle-related proteins(p21,p27,Rb,Raf-1,p-Raf-1,Erk1/2 and p-Erk1/2).The co-immunoprecipitation assay was used to explore the interaction between Mfn2 and Ras.The results showed that the overexpression of Mfn2 inhibited the proliferation of the HELF and induced the cell cycle arrest at the G0/G1 phase.Meanwhile,Mfn2 also inhibited the expression of collagen I,p-Erk and p-Raf-1.In addition,an interaction between Mfn2 and Ras existed in the HELF.This study suggests that the overexpression of Mfn2 can decrease the proliferation of HELF in ARDS,which was associated with the inhibition of the RAS-RAF-1-ERK1/2 pathway.The results may offer a potential therapeutic intervention for patients with ARDS.

Retinoic Aacid Diminished the Expression of MMP-2 in Hyperoxia-exposed Premature Rat Lung Fibroblasts through Regulating Mitogen-activated Protein Kinases

This study examined the effects of retinoic acid （RA）, PD98059, SP600125 and SB203580 on the hyperoxia-induced expression and regulation of matrix metalloproteinase-2 （MMP-2） and metalloproteinase-2 （TIMP-2） in premature rat lung fibroblasts （LFs）. LFs were exposed to hyperoxia or room air for 12 h in the presence of RA and the kinase inhibitors PD98059 （ERK1/2）, SP600125 （JNK1/2） and SB203580 （p38） respectively. The expression levels of MMP-2 and TIMP-2 mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. MMP-2 activity was measured by zymography. The amount of p-ERK1/2, REK1/2, p-JNK1/2, JNK1/2, p-p38 and p38 was determined by Western blotting. The results showed that： （1） PD98059, SP600125 and SB203580 significantly inhibited p-ERK1/2, p-JNK1/2 and p-p38 respectively in LFs; （2） The expression of MMP-2 mRNA in LFs exposed to hyperoxia was decreased after treatment with RA, SP600125 and SB203580 respectively （P0.01 or 0.05）, but did not change after treatment with PD98059 （P0.05）. Meanwhile, RA, PD98059, SP600125 and SB203580 had no effect on the expression of TIMP-2 mRNA in LFs exposed to room air or hyperoxia （P0.05）; （3） The expression of pro- and active MMP-2 experienced no change after treatment with RA or SP600125 in LFs exposed to room air （P0.05）, but decreased remarkably after hyperoxia （P0.01 or 0.05）. SB203580 inhibited the expression of pro- and active MMP-2 either in room air or under hyperoxia （P0.01）. PD98059 exerted no effect on the expression of pro- and active MMP-2 （P0.05）. It was suggested that RA had a protective effect on hyperoxia-induced lung injury by down-regulating the expression of MMP-2 through decreasing the JNK and p38 activation in hyperoxia.

Macrophage exosomes transfer angiotensin Ⅱ type 1 receptor to lung fibroblasts mediating bleomycin-induced pulmonary fibrosis

Background:Macrophages are involved in the pathogenesis of idiopathic pulmonary fibrosis,partially by activating lung fibroblasts.However,how macrophages communicate with lung fibroblasts is largely unexplored.Exosomes can mediate intercellular communication,whereas its role in lung fibrogenesis is unclear.Here we aim to investigate whether exosomes can mediate the crosstalk between macrophages and lung fibroblasts and subsequently induce fibrosis.Methods:In vivo,bleomycin(BLM)-induced lung fibrosis model was established and macrophages infiltration was examined.The effects of GW4869,an exosomes inhibitor,on lung fibrosis were assessed.Moreover,macrophage exosomes were injected into mice to observe its pro-fibrotic effects.In vitro,exosomes derived from angiotensin Ⅱ(Ang Ⅱ)-stimulated macrophages were collected.Then,lung fibroblasts were treated with the exosomes.Twenty-four hours later,protein levels ofα-collagen I,angiotensin Ⅱ type 1 receptor(AT1R),transforming growth factor-β(TGF-β),and phospho-Smad2/3(p-Smad2/3)in lung fibroblasts were examined.The Student's t test or analysis of variance were used for statistical analysis.Results:In vivo,BLM-treated mice showed enhanced infiltration of macrophages,increased fibrotic alterations,and higher levels of Ang Ⅱ and AT1R.GW4869 attenuated BLM-induced pulmonary fibrosis.Mice with exosomes injection showed fibrotic features with higher levels of Ang Ⅱ and AT1R,which was reversed by irbesartan.In vitro,we found that macrophages secreted a great number of exosomes.The exosomes were taken by fibroblasts and resulted in higher levels of AT1R(0.22±0.02 vs.0.07±0.02,t=8.66,P=0.001),TGF-β(0.54±0.05 vs.0.09±0.06,t=10.00,P<0.001),p-Smad2/3(0.58±0.06 vs.0.07±0.03,t=12.86,P<0.001)andα-collagen I(0.27±0.02 vs.0.16±0.01,t=7.01,P=0.002),and increased Ang Ⅱ secretion(62.27±7.32 vs.9.56±1.68,t=12.16,P<0.001).Interestingly,Ang Ⅱ increased the number of macrophage exosomes,and the protein levels of Alix(1.45±0.15 vs.1.00±0.10,t=4.32,P=0.012),AT1R(4.05±0.64 vs.1.00±0.09,t=8.17,P=0.001),and glyceraldehyde-3-phosphate dehydrogenase(2.13±0.36 vs.1.00±0.10,t=5.28,P=0.006)were increased in exosomes secreted by the same number of macrophages,indicating a positive loop between Ang Ⅱ and exosomes production.Conclusions:Exosomes mediate intercellular communication between macrophages and fibroblasts plays an important role in BLM-induced pulmonary fibrosis.

Clusterin as a serum biomarker candidate contributes to the lung fibroblasts activation in chronic obstructive pulmonary disease

Background:Fibrosis in the peripheral airways contributes to airflow limitation in patients with chronic obstructive pulmonary disease(COPD).However,the key proteins involved in its development are still poorly understood.Thus,we aimed to identify the differentially expressed proteins(DEPs)between smoker patients with and without COPD and elucidate the molecular mechanisms involved by investigating the effects of the identified biomarker candidate on lung fibroblasts.Methods:The potential DEPs were identified by isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomic analysis.The messenger RNA and protein levels of clusterin(CLU)in COPD patients and 12%cigarette smoke extract(CSE)-treated human bronchial epithelial cells were determined at the indicated time points.Furthermore,anin vitro COPD model was established via the administration of 8%CSE to normal human lung fibroblasts(NHLFs)at indicated time points.The effects of CSE treatment andCLU silencing on proliferation and activation of lung fibroblasts were analyzed.Results:A total of 144 DEPs were identified between COPD patients and normal smokers.The iTRAQ-based proteomics and bioinformatics analyses identified CLU as a serum biomarker candidate.We also discovered that CLU levels were significantly increased(P<0.0001)in Global Initiative for Obstructive Lung Disease II,III,and IV patients and correlated(P<0.0001)with forced expiratory volume in 1 s(R=-0.7705),residual volume(RV)(R=0.6281),RV/total lung capacity(R=0.5454),and computerized tomography emphysema(R=0.7878).Similarly,CLU levels were significantly increased in CSE-treated cells at indicated time points(P<0.0001).The CSE treatment significantly inhibited the proliferation,promoted the inflammatory response,differentiation of NHLFs,and collagen matrix deposition,and induced the apoptosis of NHLFs;however,these effects were partially reversed byCLU silencing.Conclusion:Our findings suggest that CLU may play significant roles during airway fibrosis in COPD by regulating lung fibroblast activation.

EFFECTS OF CALCIUM AND CALMODULIN INHIBITORS ON THE ABNORMAL PROLIFERATION OF LUNG FIBROBLAST

The effects of alveolar macrophage (Am) conditioned media from interstitial lung disease (ILD) patients on fibroblast (FB), and the role of calcium (Ca2+) blockers and calmodulin (CaM) inhibitors on the proliferation of lung FB were studied. We found that the AM conditioned media could stimulate FB cell proliferation and this effect could be abolished by Ca2+ blockers and CaM inhibitors. The results indicated that AM was in activated state in ILD and released some kinds of cytokines to stimulate the proliferation of FB, and Ca,2+ CaM were partially responsible for these actions.

The role of calcineurin in the lung fibroblasts proliferation and collagen synthesis induced by basic fibroblast growth factor

Objective To investigate the role of calcineurin (CaN) in the lung fibroblast proliferation and collagen synthesis induced by basic fibroblast growth factor (bFGF).Methods We used Western blot and immunohistochemical methods for investigating the content and distribution of calcineurin in the lung tissue. Calcineurin activity in different tissues was measured using 32P-labelled substrate. In the primary culture of lung fibroblasts, 3H-thymidine (3H-TdR) and 3H-proline incorporation methods were used to study the effect of cyclosporin A (CsA), an inhibitor of calcineurin, on the lung fibroblast DMA and collagen synthesis stimulated by bFGF.Results We found that calcineurin was expressed in lung tissue and has phosphatase activity (7.1±2.0 pmol Pi/mg pr/min). CsA (10^(-8)-10^(-6) mol/L) inhibited lung fibroblast 3H-TdR incorporation induced by bFGF in a dose-dependent manner, with the inhibitory rates by 20% , 46% and 66% (P< 0.01). CsA (10^(-7) -10^(-6) mol/L) inhibited 3H-proline incorporation in lung fibroblasts stimulated by bFGF, with the inhibitory rates by 21% and 37% (P<0. 01). In a culture medium, CsA (10^(-8)-10^(-6) mol/L) inhibited 3H-proline secretion induced by bFGF in a dose-dependent manner, with the inhibitory rates by 19%, 29% (P<0. 05) and 56% (P<0. 01). CsA (10^(-7) mol/L) could inhibit calcineurin activity by 44% in lung fibroblasts (P<0. 01).Conclusions Calcineurin is expressed in lung tissue and has phosphatase activity. It is involved in the bFGF stimulated lung fibroblast DNA and collagen synthesis.

Effect of intestinal ischemia-reperfusion on expressions of endogenous basic fibroblast growth factor and transforming growth factor β in lung and its relation with lung repair

AIM To study the changes of endogenoustransforming growth factor β(TGFβ)and basicfibroblast growth factor(bFGF)in lung followingintestinal ischemia and reperfusion injury andtheir effects on lung injury and repair.METHODS Sixty Wistar rats were divided intofive groups,which underwent sham-operation,ischemia(45 minutes),and reperfusion(6,24and 48 hours,respectively)after ischemia(45minutes).Immunohistochemical method wasused to observe the localization and amounts ofboth growth factors.RESULTS Positive signals of both growthfactors could be found in normal lung,mainly inalveolar cells and endothelial cells of vein.Afterischemia and reperfusion insult,expressions ofboth growth factors were increased and theiramounts at 6 hours were larger than those ofnormal control or of 24 and 48 hours after insult.CONCLUSION The endogenous bFGF and TGF βexpression appears to be up-regulated in thelung following intestinal ischemia andreperfusion,suggesting that both growth factorsmay be involved in the process of lung injury andrepair.

The change of basic fibroblast growth factor and effect of prostacyclin in lung injury

In this study,the changes in activity and mRNA transcript of basic fibroblast growth fac-tor(bFGF)in lung injury and the effect of prostacyclin on the treatment of lung injury wereinvestigated.The result of the experiment revealed that there were not any bioactive bFGF andbFGF mRNA in the lung tissue of normal dogs.bFGF activity and bFGF mRNA were detectedonly in the injured lung tissue.Prostacyclin could slightly elevate the activity of bFGF andsignificantly elevate the level of bFGF mRNA.The findings suggest that(1)the level of bFGF in-creased after lung injury.(2)Prostacyclin could influence the expression of bFGF.(3)bFGF couldplay an important role in the repair of injury lung tissue.

EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND BASIC FIBROBLAST GROWTH FACTOR IN HUMAN NON-SMALL CELL LUNG CANCER AND THEIR CLINICAL SIGNIFICANCE

Objective: To explore the expression of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) in non-small cell lung cancer(NSCLC) and theIR clinical significance. Methods: The expression of VEGF and bFGF was examined at the protein levels by immunohistochemical staining in 96 NSCLC patients, and in 36 of which at the mRNA levels by reverse transcription-PCR analysis. Results: VEGF mRNAs were expressed predominately as its secretory forms (VEGF121 and VEGF165) in NSCLC tissues. The positive ratios of VEGF121 and VEGF165 were 69.5%(25 of 36) and 41.7%(15 of 36) respectively. The positive ratio of bFGF was 52.8(19 of 36) in the same tumor specimens. The positive ratios of VEGF and bFGF at protein levels were 55.55%(20 of 36) and 58.33%(21 of 36) respectively. A significant positive correlation was observed between VEGF and bFGF expression in NSCLC tissues(P=0.002). No significant interrelationship between VEGF, bFGF expression and clinical data(age, sex, histological subtype differentiation, P-stage, metastasis and survival) was found. Conclusions: VEGF and bFGF may play an important role in angiogenesis and act in a synergistic manner in NSCLC.

血清微小RNA-499a-5p、成纤维细胞生长因子9、炎性因子与脓毒症所致急性肺损伤患者病情严重程度和预后的关系

目的探讨血清微小RNA-499a-5p(miR-499a-5p)、成纤维细胞生长因子9(FGF9)、炎性因子与脓毒症所致急性肺损伤(ALI)患者病情严重程度和预后的关系。方法选取接受诊治的151例脓毒症患者为研究对象,根据ALI的发生情况分为脓毒症并发ALI组(68例)和一般脓毒症组(83例)。根据氧合指数,将脓毒症并发ALI患者分为轻症组(23例)、中症组(26例)和重症组(19例)。以脓毒症并发ALI患者诊治28 d内的生存情况,分为预后良好组(43例)和预后不良组(25例)。另外,选取同期体检健康者151例为健康组。分析各组miR-499a-5p、FGF9及炎症因子间的差异和相关性。采用受试者工作特征(ROC)曲线分析血清miR-499a-5p、FGF9对脓毒症患者并发ALI的预测效能。结果轻症组、中症组、重症组患者的miR-499a-5p和FGF9水平表现为依次降低,而炎症因子白细胞介素-1(IL-1)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、C反应蛋白(CRP)水平依次升高,差异有统计学意义(P<0.05)。预后不良组的miR-499a-5p、FGF9低于预后良好组,IL-1、IL-6、TNF-α、CRP水平高于预后良好组,差异有统计学意义(P<0.05)。脓毒症并发ALI患者的miR-499a-5p、FGF9与IL-1、IL-6、TNF-α、CRP呈负相关(P<0.05);miR-499a-5p可正向调节FGF9表达(P<0.05)。miR-93、miR-135a联合预测脓毒症并发ALI患者预后的诊断效能高于单独预测,差异有统计学意义(P<0.05)。结论miR-499a-5p、FGF9、炎性因子水平与脓毒症所致ALI患者的病情程度和预后相关。miR-499a-5p和FGF9可能作为潜在的生物标志物用于脓毒症并发ALI的预后评估。

三项血清指标在非小细胞肺癌患者中的表达及其临床意义

目的分析血清谷胱甘肽S转移酶P1(GSTP1)、纤维母细胞生长因子受体1(FGFR1)、细胞外基质金属蛋白酶诱导因子(CD147)在非小细胞肺癌(NSCLC)患者中的表达及其临床意义。方法我院收治的96例NSCLC患者(NSCLC组)和同期体检健康者60例(对照组),比较两组血清GSTP1、FGFR1、CD147水平以及不同临床病理特征患者上述血清指标水平,分析3项指标对NSCLC的诊断效能。结果NSCLC组血清GSTP1、FGFR1、CD147高于对照组(P<0.05);不同TNM分期、淋巴结转移情况的NSCLC患者血清GSTP1、FGFR1 DNA、CD147水平比较,差异有统计学意义(P<0.05);3项指标联合诊断NSCLC的AUC、特异度、灵敏度分别为0.908、91.70%、90.00%,均高于单一指标检测(P<0.05)。结论血清GSTP1、FGFR1、CD147在NSCLC患者中显著上调,且能影响疾病的发生、进展及预后,联合检测在NSCLC临床诊疗中发挥重要价值。

LncRNA FEZF1-AS1靶向调控miR-200c-3p对人肺成纤维细胞生物学行为的影响

目的探讨FEZ家族锌指1-反义RNA1(LncRNA FEZF1-AS1)靶向调控miR-200c-3p对人肺成纤维细胞HLF生物学行为的影响。方法采用转化生长因子β1(TGF-β1)诱导HLF向肌成纤维细胞转化,分为空白对照组(Blank组)和造模组(HLF+TGF-β1组),另根据转染质粒不同将细胞分为Blank组、TGF-β1+Si LncRNA FEZF1-AS1 NC组和TGF-β1+Si LncRNA FEZF1-AS1组。采用Western blot法检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(CollagenⅠ)和波形蛋白(Vimentin)蛋白的表达。采用实时荧光定量PCR(qRT-PCR)检测LncRNA FEZF1-AS1和miR-200c-3p的表达。采用CCK-8法检测细胞增殖,细胞划痕实验检测迁移能力,Transwell实验检测侵袭能力;采用双萤光素酶实验检测FEZF1-AS1与miR-200c-3p的靶向作用关系。结果与Blank组比较,HLF+TGF-β1组α-SMA、CollagenⅠ、Vimentin蛋白表达及LncRNA FEZF1-AS1表达水平升高,miR-200c-3p表达水平降低(P<0.05);与TGF-β1+Si LncRNA FEZF1-AS1 NC组比较,TGF-β1+Si LncRNA FEZF1-AS1组细胞增殖、迁移、侵袭能力下降,LncRNA FEZF1-AS1表达及α-SMA、CollagenⅠ、Vimentin蛋白表达水平降低,miR-200c-3p表达水平升高(P<0.05);FEZF1-AS1与miR-200c-3p基因序列上存在结合位点。结论LncRNA FEZF1-AS1通过抑制miR-200c-3p促进特发性肺间质纤维化的发生、发展。

还原型谷胱甘肽对大鼠肺成纤维细胞的影响

研究过量的还原型谷胱甘肽(Glutathione,GSH)对肺成纤维细胞(Lung fibroblasts,LFS)的影响,以此来探究氧化还原平衡被打破后LFS的状态和变化。提取新鲜的大鼠LFS,通过细胞活力实验、明胶酶谱法、细胞形态实验、划痕实验、细胞免疫荧光染色和蛋白质免疫印记实验,观察不同浓度的GSH对LFS的增殖、生长、迁移以及信号通路的影响。实验表明,细胞外GSH干预可以引起LFS内稳态的改变,使局部黏着斑激酶(Focal adhesion kinase,FAK)、核因子κB(Nuclear factor kappa-B,NF-κB)、磷酸化组蛋白(Phospho-histone gamma H2A.X,γ-H2A.X)表达增高,但抑制细胞分泌的基质金属蛋白酶-2/9(MMP-2/9)的活力,使用过高浓度GSH干预时,其细胞的迁移、丝裂原活化蛋白激酶(p38)的表达、抑癌基因(p53)蛋白的表达被抑制。综上所述,过高的细胞外GSH可能导致LFS出现DNA损伤以及细胞衰竭并失能,从而引起肺组织病变。另外,过多抗氧化剂的使用会打破细胞内稳态平衡,并可能会对机体造成不可逆的损伤。

基于Notch与Wnt/β-catenin通路探讨黄芪甲苷对人成纤维细胞间质转化的作用

目的基于Notch与Wnt/β-catenin通路,探讨黄芪甲苷(astragalosideⅣ,AS-Ⅳ)对类风湿关节炎相关间质性肺病(rheumatoid arthritis associated interstitial lung disease,RA-ILD)体外模型的作用机制。方法用Luminex检测法对RA-ILD模型鼠支气管肺泡灌洗液中细胞因子进行检测,筛选出关键细胞因子;共培养人髓系白血病单核细胞(human myeloid leukemia mononuclear cells,THP-1)与正常人肺成纤维细胞(normal human lung fibroblast,NHLF)两种细胞系,加入关键细胞因子模拟RA-ILD体外模型,给予不同干预措施。通过RT-PCR、Western blot、COIP、EdU、ELISA以及双荧光素酶报告基因活性检测方法观察Snail、ZEB-1及E-Cadherin的启动子活性及mRNA表达、Notch通路中的关键蛋白表达、β-catenin的核转位水平、NHLF细胞增殖速率、ColⅠ、ColⅢ及Fibronectin的分泌水平以及β-catenin与NICD、β-catenin与LEF、NICD与CSL的相互作用。结果使用细胞因子处理NHLF细胞后,Snail、ZEB-1的启动子活性及mRNA表达明显增高,Notch通路中的关键蛋白表达水平显著上调,β-catenin的核转位明显增加,EdU阳性细胞明显增加;细胞上清中ColⅠ、ColⅢ及Fibronectin分泌水平显著上调,同时β-catenin与NICD、β-catenin与LEF、NICD与CSL的蛋白结合明显增加;使用Notch抑制剂DAPT和Jagged1 shRNA时结果与上述结论相反;AS-Ⅳ能够通过抑制Jagged1进而下调Notch及β-catenin信号通路的过度活化,抑制β-catenin与NICD、β-catenin与LEF、NICD与CSL的蛋白结合,能够剂量依赖性地抑制Snail及ZEB-1并上调E-Cadherin的启动子活性、mRNA和蛋白表达水平,抑制NHLF细胞的过度增殖及Col I、ColⅢ、Fibronectin的分泌。结论AS-Ⅳ可能通过抑制Notch和Wnt/β-catenin信号通路,抑制NHLF细胞增殖,减少ECM过度沉积,从而减缓RA-ILD肺间质纤维化进展。

非小细胞肺癌组织中FGF9、SMAD2表达与临床病理特征及预后的关系

目的 分析非小细胞肺癌(NSCLC)患者癌组织中成纤维细胞生长因子9(FGF9)、SMAD2表达与临床病理特征以及预后的相关性。方法 选取本院2018年1月-2020年12月收治的75例NSCLC患者作为研究对象,收集患者手术切除癌组织与癌旁组织,免疫组化法比较癌组织与癌旁组织FGF9、SMAD2蛋白表达情况,根据患者癌组织FGF9、SMAD2表达情况将患者分为FGF9阴性表达组、FGF9阳性表达组,SMAD2阴性表达组、SMAD2阳性表达组,比较组间临床病理特征差异;采用Spearman法分析FGF9与SMAD2表达相关性;构建KM曲线,分析患者FGF9、SMAD2表达与生存期的关联;通过COX法分析影响NSCLC患者预后不良的危险因素。结果 与癌旁组织相比,NSCLC组织中FGF9表达阳性率、SMAD2表达阳性率升高(P<0.05);是否发生淋巴结转移、TNM I+II期与TNM III期NSCLC患者FGF9阳性表达率比较差异有统计学意义(P<0.05);是否淋巴结转移、TNM I+II期与TNM III期、中高分化与低分化NSCLC患者SMAD2阳性表达率比较差异有统计学意义(P<0.05);NSCLC组织中蛋白FGF9、SMAD2表达呈正相关(P<0.05);随访期间FGF9阳性表达组患者累积生存率(20.8%)低于阴性表达组(77.3%),SMAD2阳性表达组患者累积生存率(25.9%)低于阴性表达组(76.5%),组间比较差异有统计学意义(P<0.05);FGF9阳性表达组患者无进展生存率(9.4%)低于阴性表达组(63.6%),SMAD2阳性表达组患者无进展生存率(15.5%)低于阴性表达组(58.8%),组间比较差异有统计学意义(P<0.05)。结论 NSCLC组织FGF9、SMAD2阳性表达升高,均与患者临床病理特征及预后相关,FGF9、SMAD2可能共同参与NSCLC的癌进展。

Erastin通过MAPK介导的氧化应激信号通路诱导肺成纤维细胞铁死亡

目的探究Erastin对肺成纤维细胞铁死亡的作用机制。方法向小鼠肺成纤维细胞(C57/B6-L)中加入不同浓度的铁死亡诱导剂Erastin,通过细胞计数试剂(CCK-8)检测细胞活性、荧光显微镜观察氧化应激水平,蛋白质印迹法(Western blot)检测丝裂原活化蛋白激酶(MAPK)信号通路相关蛋白表达,之后分别加入p38和人细胞外信号调节激酶(ERK)的抑制剂SB203580和U0126,进一步验证Erastin诱导肺成纤维细胞铁死亡的作用机制。结果Erastin在100μmol/L药物浓度时,能够明显诱导肺成纤维细胞铁死亡,同时伴有氧化应激表达增强,此外MAPK通路中的p38和ERK蛋白磷酸化水平升高(P<0.05)。而在加入SB203580、U0126抑制剂后,肺成纤维细胞氧化应激水平明显下降,同时细胞的活性明显升高(P<0.05)。结论Erastin能够诱导肺成纤维细胞铁死亡,其作用机制可能与MAPK信号通路介导的氧化应激有关。

肺癌患者bFGF、miR-1233、D-二聚体及Fib水平变化与肺栓塞发生的关系

目的分析碱性成纤维细胞生长因子(bFGF)、微小核糖核酸-1233(miR-1233)、D-二聚体(D-D)、纤维蛋白原(Fib)水平变化与肺癌患者发生肺栓塞的相关性。方法选取2018年6月—2021年6月华北理工大学附属医院呼吸内科确诊的肺癌合并肺栓塞患者110例作为栓塞组,另外选取医院同期收治的肺癌但未合并肺栓塞患者110例作为非栓塞组,比较2组患者入院时的血清bFGF、miR-1233、血浆D-D、Fib水平,凝血功能指标、合并疾病、肺癌病理学指标及其他相关资料;受试者工作特征曲线(ROC)分析bFGF、miR-1233、D-D、Fib预测患者发生肺栓塞的价值;Logistic回归模型分析bFGF、miR-1233、D-D、Fib与肺癌患者发生肺栓塞的关系。结果栓塞组患者bFGF、miR-1233、D-D、Fib水平均高于非栓塞组(t/P=4.749/<0.001、9.244/<0.001、16.846/<0.001、9.389/<0.001);栓塞组患者的PT、APTT、INR测定值低于非栓塞组(t/P=8.131/<0.001、7.875/<0.001、6.379/<0.001);bFGF、miR-1233、D-D、Fib预测肺癌患者发生肺栓塞的ROC曲线下面积(AUC)分别为0.687、0.821、0.947、0.766,以D-D的AUC最大(Z/P=5.102/<0.001、4.910/<0.001、3.776/<0.001);Logistic回归模型分析显示,bFGF、miR-1233、D-D、Fib均升高,合并房颤是肺癌患者发生肺部栓塞的独立危险因素[OR(95%CI)=1.486(1.059~2.086)、1.672(1.128~2.479)、2.018(1.246~3.268)、1.912(1.066~3.428)、2.164(1.298~3.609)],PT、APTT、INR升高为独立保护因素[OR(95%CI)=0.618(0.404~0.946)、0.640(0.428~0.956)、0.501(0.280~0.894)]。结论血清bFGF、miR-1233、血浆D-D、Fib升高会增大肺癌患者发生肺栓塞的风险,检测上述指标,尤其是D-D水平,对于预测患者发生肺栓塞具有重要的价值。

肿瘤相关成纤维细胞外泌体对乏氧条件下肺腺癌细胞自噬的影响及其机制研究

目的 探讨肿瘤相关成纤维细胞外泌体对乏氧条件下肺腺癌A549细胞自噬的影响及其机制。方法 选取手术的肺癌标本及癌旁组织标本,进行肿瘤相关成纤维细胞(CAFs)和成纤维细胞(NFs)的原代分离和鉴定;收集CAFs和NFs细胞培养上清,利用差速离心法从细胞培养上清液中分离出外泌体(exo),并通过电子显微镜和Western Blot对分离出的外泌体进行鉴定;采用CoCL_(2)体外构建乏氧微环境,将A549细胞复苏并传代后,把外泌体与A549细胞共培养,共分为4组:常氧A549组、乏氧A549组、乏氧A549+CAFs-exo组、乏氧A549+NFs-exo组;利用Western印迹法检测不同细胞组中LC3Ⅱ、LC3Ⅰ和LC3Ⅱ/LC3Ⅰ的表达变化,并通过Western Blot确定各组中AMPK、p-AMPK、mTOR和p-mTOR的表达水平;采用CCK8法、划痕实验、Transwell法,研究外泌体在不同环境中与细胞共同培养后对细胞增殖、迁移和侵袭能力的影响。结果 Western Blot显示CAFs中FSP-1、FAP、a-SMA蛋白高表达,透射电镜观察到提取的外泌体具备典型的结构特征。与常氧A549组相比,乏氧A549各组细胞增殖、迁移、侵袭能力显著降低(P<0.001),LC3Ⅱ/LC3Ⅰ,p-AMPK/AMPK比值上升,p-mTOR/mTOR比值下降(P<0.001);与乏氧A549组相比,乏氧A549+NFs-exo组细胞增殖、迁移、侵袭能力无明显变化,LC3Ⅱ/LC3Ⅰ、p-AMPK/AMPK、p-mTOR/mTOR比值均无明显变化;与乏氧A549组相比,乏氧A549+CAFs-exo组细胞增殖、迁移、侵袭能力明显强于乏氧A549组且LC3Ⅱ/LC3Ⅰ,p-AMPK/AMPK比值上升,p-mTOR/mTOR比值下降。结论 在乏氧微环境下,肿瘤相关成纤维细胞外泌体基于AMPK/mTOR信号通路,增强A549细胞自噬,可能是A549细胞抵抗乏氧环境、维持细胞增殖的机制之一。

普列克底物蛋白2/miR-196a信号轴介导肿瘤微环境中肺癌细胞的通讯机制研究

背景与目的:阐明介导肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)与肿瘤细胞之间通讯的信号分子仍然是一个巨大的挑战,这些信号分子对癌症转移至关重要。本研究旨在探讨普列克底物蛋白2(pleckstrin-2,PLEK2)/miR-196a信号轴介导肿瘤微环境中肺癌细胞的通讯机制。方法:选择人肺腺癌细胞系H1299和人胚胎肺细胞MRC-5作为研究对象。用表达PLEK2的慢病毒(PLEK2)和载体对照(Vector)转染H1299细胞,并在转染24 h后分离外泌体(Vector_exo、PLEK2_exo)。采用miR-196a模拟物或抑制剂转染MRC-5细胞。通过蛋白质印迹法(Western blot)分析PLEK2和上皮-间充质转化(epithelial-mesenchymal transition,EMT)相关蛋白水平,采用聚合酶链反应(polymerase chain reaction,PCR)分析miR-196a表达,采用transwell实验测定细胞转移和侵袭能力。将6只雌性BALB/c-nu小鼠随机分为Vector组和PLEK2组,每组3只。通过尾静脉向各组小鼠注射转染Vector或PLEK2的H1299细胞。4周后,取出肺组织进行H-E染色和免疫组织化学染色分析α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达。所有动物实验均经长沙市第一医院(中南大学湘雅医学院附属长沙医院)伦理委员会批准(伦理编号为EI-2021-103)。结果:与Vector组相比,PLEK2组小鼠肺结节数和转移灶中α-SMA表达显著增加(P<0.001)。与Vector组相比,PLEK2组H1229细胞中miR-196a表达水平显著增加(P<0.05),并且PLEK2_exo中miR-196a表达水平显著高于Vector_exo(P<0.05)。与Vector_exo组相比,PLEK2_exo组MRC-5细胞中miR-196a、α-SMA和成纤维细胞活化蛋白(fibroblast activation protein,FAP)表达水平显著增加(P<0.05)。与阴性对照(negative control,NC)相比,miR-196a转染的MRC-5细胞中α-SMA和FAP表达水平显著增加(P<0.05)。相反,通过miR-196a抑制剂(si-miR-196a#1、si-miR-196a#)转染,α-SMA和FAP表达水平被显著抑制(P<0.05)。与NC-CM组相比,miR-196a-CM组H1299细胞的转移、侵袭细胞数和波形蛋白(vimentin)表达均显著增加(P<0.001),E-钙黏蛋白(E-cadherin)表达显著降低(P<0.001)。此外,与Vector_exo-CM组相比,PLEK2_exo-CM组H1299细胞的转移、侵袭细胞数和vimentin表达均显著增加(P<0.01),E-cadherin表达显著降低(P<0.001)。结论:PLEK2上调能够增强肺癌细胞来源的外泌体miR-196a水平,从而促进CAFs激活。激活的CAFs能够进一步增强肺癌细胞的侵袭能力。