African Dust Storms Reaching Puerto Rican Coast Stimulate the Secretion of IL-6 and IL-8 and Cause Cytotoxicity to Human Bronchial Epithelial Cells (BEAS-2B)

African dust storm events (ADE) travel across theAtlantic Ocean(ADEAO) and reach the Puerto Rican coast (ADEPRC), potentially impacting air quality and human health. To what extent seasonal variations in atmospheric particulate matter (PM) size fractions, composition and sources trigger respiratory-adverse effects to Puerto Ricans is still unclear. In the present study, we investigated the pro-inflammatory and cytotoxic effects of PM samples harvested during ADEAO (PM10), ADEPRC (PM2.5 and PM10) and Non-ADE (Pre-and Post-ADEAO and Non-ADEPRC), using BEAS-2B cells. Endotoxins (ENX) in PM2.5 and PM10 extracts and traces of metals (TMET) in PM2.5 extracts were also examined. IL-6 and IL-8 secretion and cytotoxicity were used as endpoints. ADEAO and ADEPRC extracts were found to be more cytotoxic than Non-ADE and ADEAO were more toxic than ADEPRC extracts. PM10 extracts from ADEAO and Post-ADEAO caused significant secretion of IL-8. IL-6 and IL-8 secretion was higher following treatment with PM10 and PM2.5 ADEPRC than with Non-ADEPRC extracts. ENX levels were found to be higher in PM10 ADEAO than in the rest of the samples tested. TMET levels were higher in PM2.5 ADEPRC than in Non-ADEPRC extracts. Deferoxamine significantly reduced cytotoxicity and IL-6 and IL-8 secretion whereas Polymyxin B did not. TMET in PM2.5 fractions is a major determinant in ADEPRC-induced toxicity and work in conjunction with ENX to cause toxicity to lung cells in vitro. ENX and TMET may be responsible, in part, for triggering PM-respiratory adverse responses in susceptible and predisposed individuals.

下调HMGB2表达对肝癌LM3细胞上皮-间质转化的抑制作用及其AKT/mTOR信号通路机制

目的:探讨下调肝癌细胞中高迁移率族框蛋白2 (HMGB2)表达对肝癌细胞生物学行为及上皮-间质转化(EMT)进程的影响,并阐明其作用机制。方法:对数生长期的人肝癌LM3细胞分为阴性对照组和HMGB2 RNA干扰组(HMGB2 siRNA组),分别以Lipofectamin 2000为载体转染无关序列的RNA寡核苷酸(RNA oligo)和敲除HMGB2序列的RNA oligo。采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测2组细胞中HMGB2 mRNA和蛋白表达水平,分别采用细胞划痕实验和Transwell小室实验检测2组细胞的迁移和侵袭能力,采用Western blotting法检测2组细胞中E-钙黏蛋白(E-cadherin)、 N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达水平。结果:与阴性对照组比较,HMGB2 siRNA组细胞中HMGB2 mRNA和蛋白表达水平均明显降低(P<0.05),HMGB2 siRNA组细胞划痕愈合率明显降低(P<0.01),侵袭细胞数明显减少(P<0.01),细胞中E-cadherin蛋白表达水平明显升高(P<0.01),N-cadherin、Vimentin、mTOR、AKT和磷酸化AKT (p-AKT)蛋白表达水平明显降低(P<0.05或P<0.01)。结论:下调HMGB2的表达可降低肝癌LM3细胞迁移和侵袭能力并抑制EMT,其作用机制可能与参与调节AKT/mTOR通路相关蛋白表达有关。

Cross-talk between microRNA-let7c and transforming growth factor-β2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

木犀草素通过与B淋巴细胞瘤-2蛋白结合抑制硅肺纤维化的作用机制

目的探究中药丹参有效成分在治疗硅肺中的作用和作用机制。方法运用网络药理学和分子对接技术筛选出中药丹参治疗硅肺的有效成分和关键靶点。热转移实验检测有效成分与关键靶点的结合稳定性,细胞划痕实验检测上皮细胞的迁移能力,蛋白质印迹法检测上皮间质转化的标志蛋白、纤维化标志蛋白以及凋亡相关蛋白的表达水平。结果中药丹参的有效成分木犀草素与硅肺关键靶点B淋巴细胞瘤-2(BCL-2)具有最低的结合能,热转移实验验证木犀草素与BCL-2蛋白具有较好的结合稳定性。细胞划痕实验显示木犀草素能够抑制转化生长因子-β1刺激的上皮细胞迁移。蛋白质印迹法证明木犀草素能够抑制BCL-2蛋白的表达,与转化生长因子-β1刺激组相比,木犀草素给药及沉默BCL-2后能够抑制上皮间质转化及胶原蛋白的形成,进而抑制纤维化的进程。木犀草素给药及沉默BCL-2后能够促进细胞凋亡。结论木犀草素能够与BCL-2结合进而抑制硅肺纤维化的进程,其作用机制可能与木犀草素能够促进细胞凋亡有关。

BTG2、ARL2在非小细胞肺癌中的表达及其病理分型的相关性

目的探讨ADP核糖基化样因子2(ARL2)、B细胞异位基因2(BTG2)蛋白在非小细胞肺癌组织中表达水平及其病理分型相关性分析。方法选择2021年5月—2022年5月本院收治的60例非小细胞肺癌患者作为研究对象,共收集非小细胞肺癌的癌组织和癌旁正常组织标本60对,其中非小细胞肺癌的癌组织作为实验组,癌旁正常组织作为对照组。应用免疫组化方法分别检测非小细胞肺癌患者的癌组织和癌旁组织中ARL2蛋白和BTG2蛋白的表达水平。分析ARL2蛋白和BTG2蛋白的表达水平与非小细胞肺癌的临床病理分型相关性。结果实验组中ARL2蛋白表达阳性率为66.7%,,明显高于对照组的25.4%(P<0.05);实验组中BTG2蛋白表达阳性率为20.6%,明显低于对照组的71.4%(P<0.05)。ARL2、BTG2蛋白表达与患者年龄、性别无相关性,差异无统计学意义(P>0.05);而与肿瘤TNM分期、淋巴结转移、肿瘤最大直径、复发率、病理学分级、复发相关性密切,差异具有统计学意义(P<0.05)。统计患者的1年生存率为47.6%(29/60),其中ARL2阳性生存率为31.0%(6/20),ARL2阴性生存率为81.0%(30/40);BTG2阳性生存率为96.1%(25/26),BTG2阴性生存率为35.0%(11/34);ARL2阴性、BTG2阳性非小细胞肺癌患者生存率显著增高(P<0.001)。结论非小细胞肺癌患者的ARL2蛋白表达明显升高,BTG2蛋白表达水平明显降低,证实了ARL2水平的高表达和BTG2的低表达,对非小细胞肺癌患者的ARL2阳性蛋白、BTG2阴性表达与病理分型有着十分密切的相关性,值得未来在预后方面进一步研究探讨。

EOTAXIN AND EOTAXIN-2 EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELL

Objective To study the role of eotaxin and eotaxin-2 expression by Th2 cytokine and analyze their relationship in normal human bronchial epithelial cell line-BEAS-2B cell. Methods Levels of eotaxin mRNA and protein expression in the bronchial epithelial cell line BEAS-2B cell were determined with RT-PCR and ELISA. We also used RT-PCR to evaluate eotaxin-2 expression under the regulation of Th2 cytokine IL-4 and IL-13 as well as proinflammatory agent-TNFα. Results Eotaxin mRNA expression was the highest at the time point of 12h under the stimulation of TNF-α. While Th2 cytokine IL-4 and IL-13 had the amplification effect on the expression. Eotaxin protein was also elevated with the combination stimulation of proinflammatory agent TNF-α and IL-4 in dose and time dependent manner（ P 〈 0. 01 ）. These results were also seen when the cells were stimulated by TNF-α and 1L- 13. Eotaxin-2 mRNA expression was the highest at the time point of 8h. The expression evaluated by semi-quantitative RT-PCR also elevated under the co-stimulation of TNF-α and IL-4 or TNF-α and IL-13 and it should significantly correlate with Eotaxin （ P 〈 0. 05 ）. Conclusion This study demonstrated that Th2 cytokine like IL-4 and IL-13 enhances eotaxin and eotaxin-2 expression when co-stimulated with proinflammatory agent TNF-α. These results showed that Th2 cytokines existence is the strong evidence for bronchial epithelial cells taking part in the allergic inflammation especially in eosinophils recruitment.

Effects of transforming growth factor β2 and connective tissue growth factor on induction of epithelial mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells

AIM:To Investigate the effects of transforming growth factorβ2(TGF-β2)and connective tissue growth factor(CTGF)on transdifferentiation of human lens epithelial cells(HLECs)cultured in vitro and synthesis of extracellular matrix(ECM).METHODS:HLECs were treated with TGF-β2(0,0.5,1.0,5,10μg/L)and CTGF(0,15,30,60,100μg/L)for different times(0,24,48,72h)in vitro and the expression ofα-smooth muscle actin(α-SMA),the main component of the extracellular matrix typeⅠcollagen(Col-1)and fibronectin(Fn)were measured by using real-time polymerase chain reaction(PCR)and western-blot.RESULTS:TGF-β2 and CTGF significantly increased expression ofα-SMA mRNA and protein(P【0.05,P【0.001),Fn mRNA and protein(P【0.001),Col-1 mRNA and protein(P【0.001).TGF-β2 could induce HLECs expression of CTGF mRNA and protein in dosedependent manner(P【0.05,P【0.001).TGF-β2 and CTGF could induce HLECs to expressα-SMA,Fn and Col-1 in time-dependent manner.Each time of TGF-β2and CTGF induced HELCs expression ofα-SMA,Fn,Col-1 mRNA and protein was significant increase compared with control(P【0.05,P【0.001).CONCLUSION:TGF-β2 and CTGF could induce HLECs epithelial mesenchymal transition and ECM synthesis.

LPS-Induced Proliferation and Chemokine Secretion from BEAS-2B Cells

The surface antigen CD14 plays an important role in innate immunity, serving as a pattern recognition receptor for lipopolysaccharides (LPS). The aim of this study was to investigate the proliferation, NFκB activation, and chemokine secretion of BEAS-2B cells, a human bronchial epithelial cell line, after LPS stimulation, and some details of inVolved signaling. The presence of CD14 was investigated by flow cytometry. Cell proliferation was measured with a [3H]-thymidine incorporation assay. sCD14, RANTES, and IL-8 concentrations in cell supernatants were measured by ELISA. BEAS-2B cells express CD14 on their surface and secrete soluble CD14 into the supernatant. Cells react on LPS with increased proliferation, activation of NFκB, and the secretion of the pro-inflammatory chemotactic cytokines IL-8 and RANTES, which proves the functionality of the CD14 receptor. Neither CD14 nor sCD14 are regulated by LPS. Specific inhibitors of various intracellular signaling pathways diminish the LPS-induced proliferation and IL-8 secretion: Thus MAP-Kinases p38 and JNK, tyrosine kinases, and PI3-kinase are involved in the signaling cascade from the LPS-CD14-complex on the cell surface to the increased cell proliferation and expression of IL-8;furthermore, ERK 1/2, IRAK 1/4, and the NFκB pathway are inVolved in the latter. The data show the existence and functionality of CD14 receptors on BEAS-2B cells and elucidate the signaling pathways inVolved. LPS is able to increase cell prolife-ration, various cytokines which are dependent on endogenous CD14. Three MAPK pathways, PI3 kinase and tyrosine kinase may be involved. Also CD14 is present/involved which was controversial.

A Phase Ⅱ Clinical Trial of Celecoxib Combined with Platinum-Based Regimen as First-Line Chemotherapy for Advanced Non-Small Cell Lung Cancer Patients with Cyclooxygenase-2 Positive Expression

Objective： To evaluate the efficacy and safety of celecoxib treatment of advanced non-small cell lung cancer （NSCLC）, and combined therapy by molecular analysis. plus platinum-doublet as first-line chemotherapy in to determine the subgroup benefiting from celecoxib Methods： A total of 44 treatment-naive patients of advanced NSCLC with positive cyclooxygenase-2 （COX-2） expression confirmed by immunohistochemical （IHC） staining were designed to receive celecoxib plus platinum-doublet chemotherapy （cisplatin plus gemcitabine, novelbine or docetaxol） from February 2005 to May 2007. On 5-7 day before chemotherapy, 400 mg celecoxib was administered twice a day orally until obvious evidence of disease progression or intolerable toxicity was found. Adverse events were recorded according to NCI-CTC criteria. The primary endpoint was overall survival （OS）. The secondary endpoints included progression-free survival （PFS）, 1-year survival rate, response rate （RR） and safety. Additionally, we detected epithelial growth factor receptor （EGFR） status including EGFR gene amplification by real-time PCR and gene mutations by DHPLC followed by sequencing. Results： The response rate was 45% （20/44）, and the disease control rate （DCR） was 59% （26/44）. The median progression-free survival time and median survival time were 6 m and 18 m, respectively. The l-year survival rate was 68%. Chemotherapy cycle numbers and best response were found to be the predictive factors for PFS by COX model analysis （P=0.023 and P=0.000, respectively）. No factor was found to affect OS. The most common toxicities included neutropenia and nausea/vomit. EGFR gene amplification was an independent prognostic factor influencing OS （P=0.0002）. Patients with EGFR mutations （exon 21） had a tendency of disease progression （P=0.041）. Conclusion： Encouraging activities of celecoxib combined with platinum-doublet chemotherapy were demonstrated in treatment-naive patients with advanced NSCLC, with good tolerances. For COX-2 IHC positive patients, positive EGFR amplification and mutation might be related to poor clinical outcomes.

The preparation of anti-hnRNP A2/B1 polyclonal antibody and its potential application in non-small cell lung cancer

Objective： In order to evaluate potential application for diagnosis and prognosis of non-small cell-lung cancer （NSCLC）, as well as to determine its role in the pathogenesis of the disease, we prepared anti-human hnRNPA2/B1 potyclonal antibody. Methods： Prokaryotic expression vector of pET28a （＋）-hnRNP A2/B1 was constructed and bansformed into E.coli BL21. The recombinant protein induced by IPTG was purified and injected to rabbits for antibody preparation. Expression of hnRN P A2/B1 was examined in 45 tissues of NSCLC and 16 inflammatory pseudotumor tissues of lung by immunohistochemistry with the antibody. The commercial hnRNP A2/B1 monoclonal antibody was used as a controI.Results： （1） Polyclonal an-tibody against hnRNP A2/B1 with high title was obtained. （2） The positive staining in NSCLC tissues was 62.22%, which was substantially higher than that in normal tissues （40%, P = 0.035） or inflammatory pseudotumor tissues （31.25%, P=0.033）. （3） Expression of hnRNP A2/B1 positively correlated with age and the history of smoking, whereas it negatively correlated with differentiation staging of tumors. （4） Follow-up study showed that the survival time of patients with positive staining was significantly shorter than that of patients without hnRNP A2/B1 expression （P=0.048）. Conclusion： It is successful to make the recombinant protein and prepare the polyclonal antibody agonist human hnRNP A2/B1. It may be a valuable marker for the diagnosis and prognosis of NSCLC. Our results provide a basis for further study in clinical application.

Exposure to ephedrine attenuates Th1/Th2 imbalance underlying OVA-induced asthma through airway epithelial cell-derived exo-somal lnc-TRPM2-AS

Although various anti-inflammatory medications,such as ephedrine,are employed to manage cough-variant asthma,their underlying mechanisms are yet to be fully understood.Recent studies suggest that exosomes derived from airway epithelial cells(AECs)contain components like messenger RNAs(mRNAs),micro-RNAs(miRNAs),and long noncoding RNA(lncRNA),which play roles in the occurrence and progression of airway inflammation.This study investigates the influence of AEC-derived exosomes on the efficacy of ephedrine in treating cough-variant asthma.We established a mouse model of asthma and measured airway resist-ance and serum inflammatory cell levels.Real-time polymerase chain reaction(RT-qPCR),Western blotting,and enzyme-linked im-munosorbent assay(ELISA)analyses were used to assess gene and protein expression levels.Exosomes were isolated and character-ized.RNA immunoprecipitation(RIP)and RNA pull-down assays were conducted to examine the interaction between hnRNPA2B1 and lnc-TRPM2-AS1.In the ovalbumin(OVA)-challenged mouse model,ephedrine treatment reduced inflammatory responses,air-way resistance,and Th1/Th2 cell imbalance.Exosomes from OVA-treated AECs showed elevated levels of lnc-TRPM2-AS1,which were diminished following ephedrine treatment.The exosomal lnc-TRPM2-AS1 mediated the Th1/Th2 imbalance in CD4^(+)T cells,with its packaging into exosomes being facilitated by hnRNPA2B1.This study unveils a novel mechanism by which ephedrine ameli-orates OVA-induced CD4^(+)T cell imbalance by suppressing AEC-derived exosomal lnc-TRPM2-AS1.These findings could provide a theoretical framework for using ephedrine in asthma treatment.

LncRNA FEZF1-AS1通过调控EZH2对肺间质细胞增殖、迁移及侵袭的作用

目的研究长链非编码RNA FEZ家族锌指1-反义RNA 1(lncRNA FEZF1-AS1)调控zeste同源物增强子2(EZH2)对肺间质细胞增殖、迁移、侵袭能力及上皮细胞-间质转化(EMT)的影响及其作用机制。方法将人肺腺癌细胞系A549分为对照组(control)和模型组[model,用转化生长因子β1(TGF-β1)20 ng/mL作用48 h,诱导成为肺间质细胞]。用Western blot检测细胞中E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)及波形蛋白(vimentin)的蛋白表达。RT-qPCR检测细胞中lncRNA FEZF1-AS1和EZH2基因表达。转染组细胞分为转染si NC组、si lncRNA FEZF1-AS1+OE vector组和si lncRNA FEZF1-AS1+OE EZH2组。CCK-8法检测细胞增殖、细胞划痕检测细胞迁移、Transwell小室法检测细胞侵袭;用Western blot检测细胞中E-cadherin、N-cadherin、vimentin及EZH2的蛋白表达,用RNA免疫沉淀(RIP)测定FEZF1-AS1与EZH2的直接结合作用。结果与对照组比较,模型组E-cadherin的蛋白表达水平减少(P<0.05);N-cadherin及vimentin的蛋白表达水平升高(P<0.05);与对照组比较,模型组lncRNA FEZF1-AS1与EZH2基因的表达水平明显升高(P<0.05);与si NC组相比,si lncRNA FEZF1-AS1+OE vector组细胞增殖、迁移、侵袭能力降低,E-cadherin蛋白表达升高,N-cadherin、vimentin、EZH2蛋白表达降低(P<0.05);与si lncRNA FEZF1-AS1+OE vector组比较,si lncRNA FEZF1-AS1+OE EZHZ组细胞增殖、侵袭、迁移能力升高,E-cadherin蛋白表达降低,N-cadherin、vimentin、EZH2蛋白表达升高(P<0.05);RIP实验进一步证实了lncRNA FEZF1-AS1与EZH2具有结合作用。结论LncRNA FEZF1-AS1通过调控EZH2促进肺间质细胞增殖、侵袭、转移和EMT过程。

胰岛素样生长因子1对人RPE细胞分泌TGF-β2、MMP-2的影响及机制研究

目的研究胰岛素样生长因子1(IGF-1)对人视网膜色素上皮细胞(ARPE-19)表达转化生长因子β2(TGF-β2)、基质金属蛋白酶2(MMP-2)的影响,并探索其作用机制。方法ARPE-19细胞分别按不同浓度IGF-1和不同浓度LY294002培养6 h、12 h、24 h、48 h,采用CCK-8法检测细胞活力,确定IGF-1、LY294002的最佳作用浓度与时间。细胞划痕法检测细胞迁移活性。ELISA法检测细胞培养上清液中TGF-β2浓度。将ARPE-19细胞分为对照组、IGF-1组(80μg·L^(-1) IGF-1)、IGF-1+LY294002组(80μg·L^(-1) IGF-1+30 mmol·L^(-1) LY294002)、LY294002组(30 mmol·L^(-1) LY294002),使用无血清DMEM/F12培养基培养,对照组不做任何处理,分别采用RT-PCR、Western blot检测细胞中TGF-β2、MMP-2、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(AKT)的mRNA和蛋白表达量。结果与0μg·L^(-1) IGF-1比较,80μg·L^(-1) IGF-1的细胞活力24 h变化显著(P<0.05),故确定其为IGF-1最佳作用浓度和时间。与0 mmol·L^(-1) LY294002比较,24 h的30 mmol·L^(-1) LY294002接近半数抑制浓度,故确定其为LY294002最佳作用时间和浓度。细胞划痕法检测结果显示,0μg·L^(-1) IGF-1组、40μg·L^(-1) IGF-1组、80μg·L^(-1) IGF-1组细胞迁移率整体比较及两两比较差异均有统计学意义(均为P<0.05)。ELISA检测结果显示,0μg·L^(-1) IGF-1组、40μg·L^(-1) IGF-1组、80μg·L^(-1) IGF-1组细胞上清液中TGF-β2浓度整体比较及两两比较差异均有统计学意义(均为P<0.05)。RT-PCR、Western blot检测结果显示,IGF-1、LY294002培养24 h,与对照组比较,IGF-1组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均升高,而LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05);与IGF-1组比较,IGF-1+LY294002组细胞中TGF-β2、MMP-2、PI3K、AKT的mRNA与蛋白表达水平均下降(均为P<0.05)。结论IGF-1能促进ARPE-19细胞增殖、迁移;IGF-1可能通过PI3K/AKT信号通路上调ARPE-19细胞中TGF-β2、MMP-2的表达,参与近视的发生与发展。

基于JAK2/STAT3通路探讨阿奇霉素对脂多糖诱导肺泡上皮细胞的保护作用

目的探究阿奇霉素(AZI)对脂多糖(LPS)诱导的人肺泡上皮细胞增殖生长、迁移的作用及相关机制。方法体外培养人肺泡上皮细胞A549,分为对照组(不做干预)、LPS组(10μg/ml LPS处理24 h)、低/中/高剂量实验组(10μg/ml LPS+2、4、8μg/ml AZI)、AZI组(10μg/ml LPS+4μg/ml AZI)、抑制剂组(10μg/ml LPS+50μmol/L JAK2/STAT3通路抑制剂AG490)、AZI+抑制剂组(10μg/ml LPS+4μg/ml AZI+50μmol/L AG490)、AZI+激活剂组(10μg/ml LPS+4μg/ml AZI+0.5μmol/L JAK2/STAT3通路激活剂Colivelin)。干预24 h后,采用细胞计数试剂盒-8(CCK-8)、酶联免疫吸附试验(ELISA)、倒置显微镜、划痕法、蛋白免疫印迹(WB)法检测炎症因子肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和IL-6表达水平、细胞生长、迁移率、上皮间质转化(EMT)及JAK2/STAT3信号通路相关蛋白表达水平。结果LPS组细胞活力较对照组下降(P<0.05)。中/高剂量实验组细胞活力较LPS组上升(P<0.05)。因此选择有显著差异的较低浓度(4μg/ml AZI)作为AZI组进行后续实验。与对照组相比,LPS组细胞生长受抑制,TNF-α、IL-1β、IL-6、E-钙黏蛋白(E-cadherin)表达降低(P<0.05),细胞迁移率、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、纤维粘连蛋白(FN)、p-JAK2、p-STAT3蛋白表达升高(P<0.05)。与LPS组相比,AZI组和抑制剂组显著扭转了上述指标的变化(P<0.05)。与AZI组相比,AZI+抑制剂组细胞生长状态较好,E-cadherin蛋白表达进一步升高(P<0.05),细胞迁移率、TNF-α、IL-1β、IL-6、N-cadherin、Vimentin、FN、p-JAK2、p-STAT3蛋白表达进一步降低(P<0.05),AZI+激活剂组则显著逆转了上述指标的变化(P<0.05)。结论阿奇霉素能够通过抑制JAK2/STAT3信号通路减轻对A549细胞的炎症损伤,促进细胞生长,并抑制其迁移与上皮间质转化(EMT)进程。

壁虎多肽混合物对人非小细胞肺癌H1299细胞增殖和凋亡的影响

目的探讨壁虎多肽混合物(GPM)对人非小细胞肺癌H1299细胞增殖和凋亡的影响。方法实验设正常对照组(等体积基础培养液)和GPM高、低剂量组(20,10 mg/mL),采用CCK-8法检测细胞增殖情况,采用流式细胞术检测细胞凋亡情况,采用实时荧光定量聚合酶链反应(RT-qPCR)法和免疫印迹(Western blot)法检测B-细胞淋巴瘤因子2(Bcl-2)、Bcl-2关联X蛋白(Bax)、磷脂酰肌醇-3-激酶(PI3K)mRNA及蛋白表达水平。结果与正常对照组比较,GPM高、低剂量组H1299细胞存活率均显著降低(P<0.01),细胞凋亡率均显著升高(P<0.01),PI3K和Bax mRNA及蛋白表达水平均显著降低(P<0.01),Bcl-2 mRNA及蛋白表达水平均显著升高(P<0.05)。结论GPM能抑制H1299细胞的增殖,其作用机制可能与调控PI3K,Bax,Bcl-2 mRNA及蛋白的表达相关。

依托咪酯通过下调含WW结构域的E3泛素蛋白连接酶2表达抑制非小细胞肺癌A549细胞增殖并诱导其凋亡的实验研究

目的探讨依托咪酯(ETO)对非小细胞肺癌(NSCLC)A549细胞增殖和凋亡的影响及含WW结构域的E3泛素蛋白连接酶2(WWP2)在其中发挥的作用机制。方法分别采用0、1、2和3 mg/L的ETO处理人正常肺上皮细胞系BESA-2B和人NSCLC细胞系A549,细胞计数试剂盒法检测细胞活力;将A549细胞随机分为对照组、ETO(3 mg/L)组、ETO+NC组和ETO+WWP2-OE组,后2组分别于ETO处理后转染空载体pcDNA3.1-NC或WWP2过表达载体pcDNA3.1-WWP2,集落形成试验检测细胞增殖能力;TUNEL染色检测细胞凋亡;实时荧光定量聚合酶链反应法检测各组细胞中WWP2的mRNA表达;STITCH数据库选择与ETO直接相互作用的蛋白质;蛋白质印迹法检测各组细胞中WWP2、增殖、凋亡和磷酸酶与张力蛋白同源物(PTEN)/磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)通路相关蛋白表达。结果ETO以剂量依赖性方式显著降低A549细胞活力(F=147.923,P<0.001);而对正常肺上皮BESA-2B细胞活力无影响(F=1.427,P=0.126)。不同浓度(0、1、2、3 mg/L)ETO处理A549细胞后,集落形成数量逐渐减少[0、1、2、3 mg/L ETO组分别为(898±38)、(785±48)、(635±36)、(388±20)个],细胞凋亡率逐渐升高[0、1、2、3 mg/L ETO组分别为(2.23±0.65)%、(7.63±0.35)%、(13.24±0.47)%、(18.93±0.36)%],呈剂量依赖性(F=218.732、352.786,均P<0.001)。STITCH数据库预测ETO可通过上调WWP2蛋白表达和下调PTEN蛋白表达与WWP2和PTEN相互作用。与对照组相比,ETO组细胞中WWP2的mRNA和蛋白表达降低,集落形成数量减少,细胞凋亡率升高,增殖细胞核抗原(PCNA)、细胞增殖抗原Ki67、B淋巴细胞瘤基因2(Bcl-2)和PTEN蛋白表达降低,Bcl-2相关X蛋白(Bax)和含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)蛋白表达升高,磷酸化PI3K(p-PI3K)/PI3K和磷酸化Akt(p-Akt)/Akt比值降低(均P<0.01);与ETO+NC组比较,ETO+WWP2-OE组细胞中WWP2的mRNA和蛋白表达升高,集落形成数量增多,细胞凋亡率降低,PCNA、Ki67、Bcl-2和PTEN蛋白表达增多,Bax和Cleaved caspase-3蛋白表达减少,p-PI3K/PI3K和p-Akt/Akt比值升高(均P<0.01)。结论ETO可抑制A549细胞增殖并促进细胞凋亡,其作用机制可能与调控WWP2的下调和PTEN/PI3K/Akt通路的激活有关。

Survivin、Bcl-2、HPV 16/18在宫颈上皮内瘤变及宫颈癌中的表达及临床意义

目的:研究Survivin、Bcl-2、HPV16/18在宫颈上皮内瘤变(CIN)及宫颈癌中的表达,探讨三者与宫颈癌的相关性。方法:采用原位杂交法检测正常宫颈组织(对照组)、CIN(CIN组)和宫颈癌(宫颈癌组)中Survivin mRNA及HPV16/18DNA的表达。采用免疫组织化学法检测Bcl-2蛋白在各组中的表达。结果:①Bcl-2蛋白、HPV16/18DNA、Survivin mRNA的阳性率在对照组、CIN组、宫颈癌组中逐渐升高,差异有高度统计学意义(P=0.000)。②Bcl-2蛋白和Survivin mRNA在宫颈癌的组织分化程度中,低分化组的表达高于高、中分化组(P<0.01),ⅡB～Ⅲ期显著高于Ⅰ～ⅡA期(P<0.05);Survivin mRNA在淋巴结转移组中高于无淋巴结转移组(P<0.01);Survivin mRNA及Bcl-2与组织类型及肿块类型无关(P>0.05);HPV16/18感染与宫颈癌组织类型、组织分化程度、临床分期、肿块类型均无关(P>0.05)。③Survivin与Bcl-2及HPV16/18在宫颈癌中的表达均呈正相关。结论:Survivin、Bcl-2及HPV16/18在宫颈癌中有异常表达。三者可能与宫颈癌的发生发展有密切关系。

VEGF、b-FGF、NOS_2和NOS_3在非小细胞肺癌的表达及其临床意义

目的 :研究血管内皮生长因子 (VEGF)、碱性成纤维细胞生长因子 (b -FGF)、一氧化氮合酶 2 (诱生型 ,NOS2 )、一氧化氮合酶 3(内皮型 ,NOS3)在非小细胞肺癌 (NSCLC)中的表达及其与肿瘤血管形成和淋巴结转移的关系。方法 :用免疫组化 (S -P法 )染色技术对 95例NSCLC石腊组织标本的VEGF、b -FGF、NOS2 和NOS3及肿瘤内微血管密度 (IMVD)进行检测和分析。结果 :VEGF、b FGF表达与TNM分期、IMVD及淋巴结转移有关 (P<0 .0 5) ,两者共表达与IMVD有关 (P <0 .0 1 )。NOS3与组织学分型、IMVD和淋巴结转移有关 (P <0 .0 5) ;NOS2与IMVD有关 (P <0 .0 5)。相关分析显示促血管形成因子 (VEGF、b FGF)与一氧化氮合酶 (NOS2 、NOS3)的表达呈正相关 (r=0 .30 1 8,P <0 .0 5)。结论 :VEGF和b FGF在促进血管形成和促进肿瘤转移方面可能起到协同作用 ,并且有NO的参与 ;VEGF、b FGF、NOS2

番泻苷B抑制STAT3和ERK1/2磷酸化对A549细胞生长侵袭及裸鼠成瘤的影响

目的探讨番泻苷B对A549细胞生长、侵袭及裸鼠成瘤的影响及机制。方法采用0、5、10、20μM番泻苷B处理非小细胞肺癌A549细胞,将细胞随机分为4组进行后续实验,Brdu染色检测各组细胞增殖;Hoechst染色检测细胞凋亡;划痕实验检测细胞迁移;Transwell实验检测细胞侵袭;蛋白免疫印迹检测ki67、PCNA、cl-caspase-3、cl-caspase-9、VEGF、N-cadherin和E-cadherin蛋白表达水平,STAT3和ERK1/2的磷酸化情况;建立荷瘤小鼠模型,检测肿瘤重量,免疫组化检测Ki67和VEGF表达。结果与Control组相比较,各番泻苷B剂量组Brdu阳性细胞数量、侵袭细胞数明显减少(P<0.05),细胞凋亡率明显上升(P<0.05),细胞划痕愈合率降低(P<0.05),ki67、PCNA、VEGF、N-cadherin蛋白水平明显降低(P<0.05),cl-caspase-3、cl-caspase-9、E-cadherin蛋白水平明显升高(P<0.05),STAT3和ERK1/2的磷酸化水平均明显降低(P<0.05),降低荷瘤小鼠肿瘤重量与Ki67和VEGF表达水平(P<0.05)。结论番泻苷B抑制STAT3、ERK1/2磷酸化对A549细胞体内外生长有抑制作用。

过表达BTG2增强人肺腺癌细胞的辐射敏感性

在肺癌类型中,肺腺癌占大多数,其总体生存率差,BTG2是抗增殖基因家族的一员,属于BTG/TOB家族。许多研究表明,B细胞易位基因2(BTG2)多种类型的肿瘤中存在异常表达,但其在肺腺癌放疗敏感性中发挥的调控作用尚不明确。本研究通过肺腺癌组织样本及在线数据库,探究BTG2在肺腺癌患者中的表达水平及其表达与临床预后之间的相关性,结果提示在具有放疗抗性的肺腺癌患者中,BTG2的表达水平显著降低,且在肺腺癌细胞系中,BTG2能对放疗产生响应,其在肺腺癌患者中的低表达状态与不良的预后相关(P<0.05);在人肺腺癌A549和H1299细胞系中转染过表达BTG2(OE-BTG2)慢病毒,通过克隆形成检测过表达BTG2对肺腺癌细胞系的辐射敏感性的影响,实验证实过表达BTG2可显著增强A549和H1299细胞系的辐射敏感性(P<0.05);并进一步通过WB、免疫组化检测BTG2及凋亡相关蛋白BAX的表达水平,结果证实:过表达BTG2可显著增加A549和H1299细胞辐射后的凋亡水平。最后通过裸鼠成瘤试验检测BTG2在活体中对肺腺癌辐射敏感性的影响,结果提示:在动物实验中,过表达BTG2可显著增强肺腺癌的辐射敏感性(P<0.05)及增加辐射后BAX的表达水平。综上所述,BTG2在肺腺癌组织中处于低表达状态,并且与不良的临床预后紧密相关,过表达BTG2可促进凋亡过程,增加人肺腺癌细胞系的辐射敏感性,能为克服肺腺癌的辐射抗性提供新的靶点。