细胞固定技术在两种细胞共同培养研究中的应用

目的探讨细胞固定对鉴别支气管上皮细胞和嗜酸粒细胞两种细胞共同培养过程中细胞代谢产物来源的作用及其机制。方法用1％多聚甲醛固定嗜酸粒细胞和支气管上皮细胞系BEAS-2B细胞；通过锥虫蓝、伊红细胞染色，观察多聚甲醛固定对细胞形态、结构、活力和与正常细胞接触培养过程中与正常细胞黏附能力的影响；用酶联免疫吸附测定（ELISA）方法定量分析固定细胞与正常细胞共同培养过程中对白细胞介素（IL）-6释放的影响。结果多聚甲醛固定后支气管上皮细胞和嗜酸粒细胞均变为死亡细胞，但其形态、结构及嗜酸粒细胞中颗粒着色无明显变化；多聚甲醛固定的BEAS-2B细胞与正常及固定的嗜酸粒细胞接触培养仅有极少量细胞发生黏附，而多聚甲醛固定的嗜酸粒细胞与正常BEAS-2B细胞接触培养则有较大数量的细胞发生黏附，但黏附细胞数量少于两种正常细胞的共同培养；固定后的嗜酸粒细胞与正常BEAS-2B细胞共同培养过程中可诱导BEAS-2B细胞释放IL-6。但与嗜酸粒细胞单独培养比较，固定后BEAS-2B细胞与正常嗜酸粒细胞共同培养对细胞培养上清液中IL-6浓度无明显影响。结论经多聚甲醛固定后，细胞死亡并丧失其代谢功能，但其表面仍保留可诱导其他细胞活化的结构成分，此技术可用于两种或多种细胞共同培养过程中代谢产物来源的鉴别。     

细胞固定在两种细胞联合培养研究中的应用

目的研究多聚甲醛固定于支气管上皮细胞和嗜酸性粒细胞,固定后细胞形态、结构,细胞与正常细胞共同培养过程中对细胞间粘附、活化,正常细胞释放细胞因子能力的影响,探讨细胞固定对鉴别两种细胞共同培养过程中细胞代谢产物来源的作用及其机制。方法用1%多聚甲醛固定嗜酸性粒细胞和支气管上皮细胞系BEAS-2B细胞;通过胎酚蓝、伊红细胞染色,观察多聚甲醛固定对细胞形态、结构、活力和与正常细胞接触培养过程中与正常细胞粘附能力的影响;用酶联免疫吸附试验(en-zyme-linkedimmunosorbentassay,ELISA)方法定量分析固定细胞与正常细胞共同培养过程中对白细胞介素-6(interlukin-6,IL-6)释放的影响。结果多聚甲醛固定后支气管上皮细胞和嗜酸性粒细胞均变为死亡细胞,但其形态、结构及嗜酸性粒细胞中颗粒着色无明显变化;多聚甲醛固定的BEAS-2B细胞与正常及固定的嗜酸性粒细胞接触培养仅有极少量细胞发生粘附,而多聚甲醛固定的嗜酸性粒细胞与正常BEAS-2B细胞接触培养则有较大数量的细胞发生粘附,但粘附细胞数量少于两种正常细胞的共同培养;固定后的嗜酸性粒细胞与正常BEAS-2B细胞共同培养过程中可诱导BEAS-2B细胞释放IL-6。但与嗜酸性粒细胞单独培养比较,固定后BEAS-2B细胞与正常嗜酸性粒细胞共同培养对细胞培养上清液中IL-6浓度无明显影响。结论经多聚甲醛固定后,细胞死亡并丧失其代谢功能,但其表面仍保留可诱导其他细胞活化的结构成份,此技术可用于两种或多种细胞共同培养过程中代谢产物来源的鉴别。     

基因芯片用于活化支气管上皮细胞基因表达的研究

目的探讨基因芯片筛选肿瘤坏死因子(TNF)-α活化后支气管上皮细胞基因表达的变化。方法提取支气管上皮细胞系(BEAS-2B细胞)总RNA,用基因芯片检测正常培养和经TNF-α活化的BEAS-2B细胞基因表达,对上调表达的基因用逆转录聚合酶链反应(RT-PCR)确证,用流式细胞微珠方法(CBA)检测TNF-α活化的BEAS-2B细胞培养液中相关细胞因子浓度。结果TNF-α活化后BEAS-2B细胞中细胞间黏附分子(ICAM)-1、白细胞介素(IL)-8、单核细胞趋化蛋白(MCP)-1、TNF-α、IL-6基因表达显著上调(分别上调6·5、8·2、3·1、4·9和3·5倍),表达上调的基因用RT-PCR确证结果基本一致。TNF-α活化后BEAS-2B细胞培养液中细胞因子的分泌(BEAS-2B细胞培养过程中有、无TNF-α存在,IL-6、IL-8和MCP-1的分泌分别为:(117·83±18·20)ng/L和(1771·33±312·67)ng/L,P<0·001;(277·97±50·76)ng/L和(7579·20±797·15)ng/L,P<0·001;(741·53±129·91)ng/L和(12228·57±1897·58)ng/L,P<0·001),与相应基因的表达一致。结论用基因芯片方法筛选活化后支气管上皮细胞基因表达对研究支气管上皮在气道性疾病中的作用具有重要意义。     

卡介苗体外诱导鼻咽癌CNE-2Z细胞ICAM-1表达

摘要：目的：探讨卡介苗（bacillus CalmetteGuerin,BCG）对鼻咽癌（nasopharyngeal carcinoma,NPC）CNE-2Z细胞及细胞间黏附分子1（intercellular adhesion molecule1,ICAM-1）表达的影响及可能的调控机制。 方法：显微镜观察细胞形态学变化和记录细胞被胰蛋白酶完全消化所用时间;流式细胞术检测细胞表面ICAM-1表达;免疫细胞化学检测细胞NF-κB表达,western blot检测ICAM-1、NF-κB表达。 结果：BCG作用24、48和72 h细胞对培养介质的粘附力增加,且被胰蛋白酶完全消化所用的时间明显长于对照组［(5.8±0.4)、(7.8±0.4)、(8.8±0.4) min vs (5.0±0.5) min,F=22.01,P<0.01］;流式细胞术检测24、48和72 h组ICAM-1表达的平均荧光强度（MnX）明显高于对照组［(25.2±0.4)、(28.7±1.1)、(37.2±1.6) vs(23.4±0.9), F=112.34,P<0.01］;免疫细胞化学结果表明,BCG作用72 h组NF-κB表达水平高于对照组。western blot结果表明,各组NF-κB和ICAM-1表达均明显高于对照组。 结论：BCG体外诱导鼻咽癌CNE-2Z细胞ICAM-1表达,且该过程可能与NF-κB信号途径有关。     

细胞间黏附分子-1、巨噬细胞炎性蛋白-2在吸烟诱导的支气管炎大鼠模型中的变化及其意义

目的:制备吸烟诱导的慢性支气管炎大鼠模型,探讨细胞间黏附分子-1(ICAM-1),巨噬细胞炎性蛋白-2(MIP-2)在慢性支气管炎大鼠模型气道炎症中的作用.方法:雄性Wistar大鼠随机分为两组,对照组、模型组.采用单纯吸烟的方法建立慢性支气管炎大鼠模型.光镜下观察支气管肺组织病理形态学改变.分析支气管肺泡灌洗液(BALF)细胞计数和分类;用ELISA法测定肺组织匀浆MIP-2的含量;用免疫组化方法检测ICAM-1在各组支气管肺组织中的表达.结果:模型组病理形态学改变符合人类慢性支气管炎的特点.模型组支气管上皮细胞、肺泡上皮细胞及肺毛细血管内皮细胞ICAM-1表达强度与BALF中白细胞总数、中性粒细胞数成显著正相关(分别为r=0.882及r=0.843;均P＜0.01);肺组织匀浆MIP-2与BALF中白细胞总数、中性粒细胞数成显著正相关(分别为r=0.706及r=0.819;均P＜0.01).与对照组比较,模型组支气管上皮细胞,肺泡上皮细胞及肺毛细血管内皮细胞ICAM-1表达强度明显增强(P＜0.01);肺组织匀浆MIP-2含量明显升高(P＜0.01).结论:ICAM-1表达上调及MIP-2的特异性趋化作用可能参与了吸烟诱导的慢性支气管炎大鼠模型的气道炎症过程.     

哮喘豚鼠支气管肺组织IL-5及GM-CSFmRNA表达

目的 :探讨白介素 5 (IL 5 )及粒细胞 巨噬细胞集落刺激因子 (GM CSF)在哮喘豚鼠支气管肺组织中的表达。方法 :实验分为哮喘组和对照组 ,每组各 15只豚鼠。采用原位杂交方法检测两组豚鼠支气管肺组织的IL 5和GM CSFmRNA表达。结果 :哮喘组支气管肺组织IL 5和GM CSFmRNA表达强度 (细胞数 /HP)分别为 36 0 0± 8 0 0和 32 0 0± 9 0 0 ,对照组分别为 14 0 0± 5 0 0和 18 0 0± 7 0 0 ,哮喘组IL 5和GM CSFmRNA表达强度显著高于对照组 (P <0 0 1)。哮喘组肺组织嗜酸细胞 (EOS)浸润密度 (细胞数 /HP)为 48 0 0± 2 6 0 0 ,对照组为 19 0 0± 6 0 0 ,哮喘组显著高于对照组 (P <0 0 1)。结论 :哮喘豚鼠支气管肺组织IL 5和GM CSFmRNA高表达可能在气道炎症中发挥重要作用。     

Del-1对特异性过敏患儿嗜酸性粒细胞表面ICAM-1表达的影响

目的研究发育内皮基因1(Del-1)对特异性过敏患儿嗜酸性粒细胞上细胞间黏附分子1(ICAM-1)表达的影响,并初步探讨其意义。方法收集35例特异性过敏患儿外周血标本,利用磁珠分选(MACS)方法分选嗜酸性粒细胞,用Del-1、抗Del-1抗体或白细胞介素17(IL-17)处理,用逆转录聚合酶链反应(PCR)检测ICAM-1的表达,并进行统计学分析。结果与对照组比较,Del-1处理组ICAM-1表达水平下降(P0.05),抗Del-1抗体处理组ICAM-1表达水平上升(P0.05);Del-1与IL-17共同处理组ICAM-1表达水平较单独IL-17处理组下降(P0.05);但在非嗜酸性粒细胞中,Del-1或抗Del-1抗体处理组ICAM-1表达水平较对照组[用磷酸盐缓冲液(PBS)处理]差异无统计学意义(P0.05)。结论 Del-1可调节嗜酸性粒细胞表面的ICAM-1表达,提示Del-1可能通过抑制嗜酸性粒细胞的黏附作用抑制特异性过敏炎症。     

PM2.5对支气管上皮细胞色素上皮衍生因子表达的影响

目的探索不同浓度PM2.5对支气管上皮细胞(BEAS-2B)色素上皮衍生因子(PEDF)蛋白表达的影响。方法 BEAS-2B细胞传代培养,加入低、中、高浓度PM2.5悬液(25μg/ml、50μg/ml、100μg/ml)刺激24 h。采用酶联免疫吸附试验(ELISA)检测细胞培养上清液中PEDF水平;Western blotting检测BEAS-2B细胞中PEDF蛋白表达水平。结果与对照组相比,PM2.5浓度为25μg/ml时,细胞内和上清液中PEDF蛋白水平均有增加趋势,但无统计学意义(t=-0.730,t=-1.840,P0.05);PM2.5浓度为50μg/ml和100μg/ml时,细胞内和上清液中PEDF蛋白表达水平明显增高(t5.798,P0.01)。结论中、高浓度PM2.5能够增加支气管上皮细胞中PEDF蛋白水平,并呈浓度依赖性。     

辐射对肝癌细胞HepG2多药耐药基因MDR1表达影响的实验研究

目的辐射对肝癌细胞多药耐药性的影响。方法取肝癌细胞株HepG2及HepG2/ADM分别进行X线照射(10Gy),于照射前后分别采用流式细胞仪检测各组细胞表面Pgp变化,采用RT-PCR法检测MDR1表达变化。结果照射前后HepG2细胞表面P-gp阳性表达率分别为:(11.3±1.2)%(、27.4±3.9)%;HepG2/ADM组为:(45.5±5.1)%,(48.1±6.1)%。HepG2组P-gp在细胞膜上的表达较照射前后差异有显著性意义(P<0.01)。结论辐射可诱导肝癌细胞HepG2 mdr1基因表达增强,细胞膜表面Pgp表达增加。对HepG2/ADM耐药性影响较小。     

嗜酸性粒细胞凋亡与鼻息肉发病的研究

目的考察嗜酸性粒细胞在鼻腔正常下鼻甲黏膜及鼻息肉标本中的分布情况及凋亡情况,研究嗜酸性粒细胞凋亡与鼻息肉发病间的关系。方法通过流式细胞仪观察鼻息肉组(A组,21例)和对照组(B组,7例)嗜酸性粒细胞分布情况,AnnexinV-PE染色后,在流式细胞仪下检测嗜酸性粒细胞凋亡情况。结果A组嗜酸性粒细胞个数(12.83±5.20)明显高于B组(4.38±1.99)(P<0.05);A组凋亡的嗜酸性粒细胞比例(0.81±0.15)%明显低于B组(24.73±1.22)%(P<0.05)。结论嗜酸性粒细胞的浸润及凋亡受抑制可能在鼻息肉的发生、发展中具有重要作用。     

Bystander transfer of functional human CD40 ligand from gene-modified fibroblasts to B-chronic lymphocytic leukemia cells

CD40 ligand (CD40L) is a good candidate molecule for the immunotherapy of B cell malignancies including B-chronic lymphocytic leukemia (B-CLL), because it may increase the capacity of the malignant cells to present tumor antigens. However, efforts to manipulate expression of the human CD40L (hCD40L) molecule have foundered on problems associated with lack of consistent gene transfer into the malignant target cells. We now describe a new, highly reproducible method for inducing hCD40L surface expression on malignant B cells, which is dependent on intercellular transfer of the hCD40L protein from donor gene-modified fibroblasts to patient tumor cells. Ten B-CLL samples were cocultured with MRC-5 fibroblasts (a human embryonic lung cell line) previously transduced with an adenoviral vector encoding the hCD40L gene. The malignant cells expressed high levels of surface hCD40L, B7-1, B7-2, and ICAM-1 after coculture. Upregulation of B7-1 and B7-2 was cycloheximide inhibitable and was a consequence of CD40 activation. However, inhibition of protein synthesis had no effect on the ability of B-CLL cells to acquire surface expression of hCD40L. hCD40L surface expression required cell-to-cell contact, but was independent of CD40 engagement. hCD40L transfer was not mediated by membrane fusion. The transferred hCD40L was functionally intact and B-CLL cells expressing this molecule induced increased interferon-gamma production from autologous peripheral blood T lymphocytes. This approach does not use any direct gene transfer to primary leukemia cells and can readily be scaled up for production of clinical B-CLL vaccines.     

长链非编码RNA-PVT1介导JAK/STAT3信号通路对肺纤维化的研究

目的 探讨长链非编码RNA-浆细胞瘤变异易位基因1/蛋白质酪氨酸激酶/信号转导和转录激活因子3（lnc-PVT1/JAK/STAT3）信号通路参与肺纤维化的作用机制。方法 采用不同浓度脂多糖（LPS）作用于人正常肺上皮细胞BEAS-2B,用CCK-8法检测细胞活性、蛋白免疫印迹法检测α-SMA的蛋白相对表达量,对LPS...     

Antimicrobial and anti-inflammatory effects of clarithromycin on Mycobacterium avium complex replication in cultured human bronchial epithelial cells

The Mycobacterium avium complex (MAC) invades cultured human bronchial cells, can replicate intracellularly, and facilitates the release of inflammatory cytokines and chemokines from cells. The purpose of this study was to examine the effects of clarithromycin (CAM) on MAC invasion, replication, and the release of cytokines and chemokines. A human bronchial epithelial cell line (BEAS-2B) monolayer grown on a tissue culture plate was infected with MAC. After 24 h, the cells were washed with Hanks’ buffered salt solution, and extracellular bacteria were killed. The monolayer was further cultured for 5 days in medium containing CAM and subjected to a replication assay. The supernatants were assessed using a microchemotaxis assay and enzyme-linked immunosorbent assay (ELISA). mRNA expression was evaluated using a DNA array. The amount of intracellular MAC on day 5 of culture was significantly lower in the presence of CAM at the levels of 1× and 4× MIC. CAM inhibited the release of chemotactic activity and the production of interleukin (IL)-8 and macrophage chemotactic protein (MCP)-1. DNA array analysis of mRNA expression in BEAS-2B cells showed that CAM inhibited the expression of inflammatory cytokines and chemokines, involving IL-6, MCP-1, and IL-8 mRNA. MAC invaded and replicated in BEAS-2B cells and induced the production of chemotactic factors. In contrast, CAM may have bactericidal and bacteriostatic effects leading to the inhibition of inflammatory events.     

对白细胞介素-1β激活核转录因子-κB介导A549细胞分泌细胞间黏附分子-1作用的研究

目的 研究白细胞介素-1β(IL-1β)对A549细胞表达细胞间黏附分子-1(ICAM-1)的诱导作用及相关细胞内信号通路的激活和转导机制.方法 以1 μg/L终浓度的IL-1β刺激经SC-514[核转录因子-κB(NF-κB)的IKK-2复合物抑制物]预处理的A549细胞,用蛋白质免疫印迹法(Western blotting)检测IL-1β刺激5、10、30、60 min时细胞内磷酸化NF-κB抑制蛋白(pIκBα)和IκBα蛋白表达水平;激光扫描共焦显微镜(LSCM)显像检测NF-κB p65的核转移过程;按试剂盒说明测定NF-κB DNA结合活性;p65抗体染色质免疫沉淀结合聚合酶链反应(ChIP-PCR)技术检测乙酰化组蛋白H4和p65与ICAM-1基因启动子的结合;逆转录-聚合酶链反应(RT-PCR)检测4 h后的ICAM-1 mRNA表达;酶联免疫吸附法(ELISA)检测24 h后细胞表面的ICAM-1蛋白表达.结果 IL-1β刺激后细胞内pIκBα蛋白表达水平明显升高,IκBα蛋白表达水平明显下降;LSCM检测显示激活的p65蛋白从胞质向胞核转移,p65与DNA结合活性明显升高(P<0.01).ChIP-PCR扩增发现,IL-1β促使乙酰化组蛋白H4和p65与ICAM-1基因启动子区域的DNA片断结合.IL-1β刺激4 h后ICAM-1 mRNA表达明显升高,刺激24 h后A549细胞表面ICAM-1蛋白表达亦明显升高(P均<0.01).SC-514阻断了pIκBα蛋白的升高和IκBα蛋白的下降;减少了p65蛋白的核转移和DNA结合活性;降低了ICAM-1的mRNA及蛋白表达水平(P均<0.01).结论 IL-1β通过激活NF-κB 介导了A549细胞表达ICAM-1.     

Type IV pilus protein PilA of Pseudomonas aeruginosa modulates calcium signaling through binding the calcium-modulating cyclophilin ligand

The lungs are a major site of Pseudomonas aeruginosa infection in patients with compromised immune systems. We have shown that a large number of cells of the P. aeruginosa wild-type PAO1 strain invade cultured human bronchial epithelial cells (BEAS-2B). In the present study, we investigated whether P. aeruginosa pilus protein PilA might modulate cellular functions by binding to unknown factor(s) in human host cells. Using a yeast two-hybrid screen, we showed that the calcium-modulating cyclophilin ligand (CAMLG), which is involved in Ca²⁺ signaling, was the major host cell PilA binding protein. Overexpression of the pilA gene in BEAS-2B cells resulted in a significant increase in cytoplasmic Ca²⁺ and consequent upregulation of the activity of the nuclear factor of activated T cells, followed by induction of cyclooxygenase 2 gene expression. Infection of BEAS-2B cells with the P. aeruginosa wild-type strain, but not with the pilA gene knockout strain (ΔpilA), caused a significant increase in intracellular Ca²⁺ concentration in infected cells. Therefore, we propose a novel bacterial strategy for PilA modulation of intracellular Ca²⁺ signaling through intracellular PilA-CAMLG interaction.     

Dermatan sulfate activates nuclear factor-kappab and induces endothelial and circulating intercellular adhesion molecule-1

Proteoglycans (PGs) can influence cell behaviors through binding events mediated by their glycosaminoglycan (GAG) chains. This report demonstrates that chondroitin sulfate B, also known as dermatan sulfate (DS), a major GAG released during the inflammatory phase of wound repair, directly activates cells at the physiologic concentrations of DS found in wounds. Cultured human dermal microvascular endothelial cells exposed to DS responded with rapid nuclear translocation of nuclear factor-kappaB (NF-kappaB), increased expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, and increased ICAM-1 cell surface protein. Heparan sulfate and chondroitin sulfates A and C had no effect on activation of NF-kappaB or induction of ICAM-1. Inhibition of NF-kappaB activation blocked the effect of DS. The increase in cell surface ICAM-1 did not involve TNF-alpha or IL-1 release by endothelial cells, but it was facilitated by autocrine factors whose release was initiated by DS. The ICAM-1-inductive activity of DS was confirmed in vivo. Injection of DS, but not heparin or other chondroitin sulfates, into mice greatly increased circulating levels of soluble ICAM. These data provide evidence that DS, but not other GAGs, initiates a previously unrecognized cell signaling event that can act during the response to injury.