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。     

嗜酸粒细胞对支气管上皮细胞表达细胞间黏附分子1的诱导作用

目的探讨嗜酸粒细胞与支气管上皮细胞联合培养对支气管上皮细胞表达细胞间黏附分子(ICAM)-1的影响。方法人嗜酸粒细胞与支气管上皮细胞(BEAS-2B)联合培养4和12h,提取BEAS-2B细胞总RNA;通过逆转录聚合酶链反应(RT-PCR)方法分析与嗜酸粒细胞联合培养对BEAS-2B细胞中ICAM-1基因表达的影响;BEAS-2B细胞表面ICAM-1蛋白质的变化用荧光抗体流式细胞分析。结果经嗜酸粒细胞活化后,BEAS-2B细胞中ICAM-1基因表达上调;BEAS-2B细胞表面ICAM-1蛋白质量显著增加(34.23±4.19和57.60±7.62,P<0.05)。结论嗜酸粒细胞与支气管上皮细胞接触培养可诱导支气管上皮细胞表达ICAM-1。     

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。     

可溶性细胞黏附分子1、白细胞介素8与哮喘关系的临床研究

哮喘是一种由多种细胞特别是肥大细胞、嗜酸粒细胞及淋巴细胞参与的慢性气道炎症性疾病。可溶性细胞间黏附分子(sICAM)主要是介导细胞聚集，特别是嗜酸粒细胞的聚集和跨内皮转移至炎症部位，释放多种炎性介质而致气道高反应性。白细胞介素8(IL-8)作为一种前炎因子通过调节内皮上的黏附因子，介导炎性细胞的趋化、黏附，参与哮喘的慢性     

支气管哮喘患者血清嗜酸粒细胞阳离子蛋白和IL-5测定的意义

支气管哮喘是气道慢性变应性炎症性疾病,其变应性炎症与多种免疫细胞有关,包括嗜酸粒细胞、嗜碱粒细胞、T淋巴细胞和肥大细胞等[1]。其中嗜酸粒细胞及其所释放的嗜酸粒细胞阳离子蛋白(ECP)以及与嗜酸粒细胞密切相关的细胞因子白介素-5(IL-5)在支气管哮喘的发病中起重要的作用[2]。本研究测定支气管哮喘患者和正常对照组血清的ECP和IL-5水平,分析两者的相关性,探讨两者在支气管哮喘发病中的意义。资料与方法一、研究对象收集我院2005年1月至2005年12月在门诊就诊的发作期支气管哮喘患者50例,诊断符合中华医学会呼吸学会的支气管哮喘的诊…     

T细胞淋巴瘤与外周血嗜酸粒细胞增多相关性及其发生机制研究进展

T细胞淋巴瘤与外周血嗜酸粒细胞增多之间的关系已经确立，是由肿瘤性T细胞或具有异常免疫表型的T细胞群分泌大量Th2型细胞因子，包括粒-单核细胞集落刺激因子（GM—CSF）、IL-4、IL-5等导致外周血嗜酸粒细胞增多。具有异常表型的T细胞克隆已被证实具有恶性转化潜能，很容易发展成T细胞淋巴瘤。有些诊断为特发性高嗜酸粒细胞综合征（IHES）或高嗜酸粒细胞综合征（HES）的病例现已证明是由于免疫表型异常、克隆性或非克隆性的T细胞造成的嗜酸粒细胞增多，故此类病例不再分类为HES。不管何种原因引起嗜酸粒细胞增多，都将导致器官、组织受损。     

白介素-33在支气管哮喘发病机制中的作用

白介素（IL）-33属于IL-1细胞因子家族，它通过ST2膜受体诱导辅助型T细胞（Th）2免疫反应，在调节免疫系统中起重要作用。IL-33主要由上皮细胞分泌，也可由其他细胞如骨髓细胞、树突状细胞、巨噬细胞和肥大细胞产生。IL-33受体ST2在多种细胞广泛表达。Th2细胞、肥大细胞、嗜碱性粒细胞和嗜酸性粒细胞等多种Th2阳性细胞均参与支气管哮喘的发展过程。IL-33被认为是Th2型免疫反应始发因素，通过激活固有免疫及适应性免疫，诱导哮喘等过敏性疾病的发生发展。进一步了解IL-33的生物学特性有助于阐明其在哮喘治疗中干预作用。     

哮喘患者诱导痰嗜酸细胞趋化因子的改变及其意义

目的探讨嗜酸细胞趋化因子（嗜酸细胞趋化因子）在嗜酸细胞性气道炎症和气流阻塞发生中的作用。方法收集支气管哮喘（A组）急性发作期患者30例和健康对照者（B组）20例，分别给予肺功能检测和痰诱导检查。用酶联免疫吸附法（ELISA）测定诱导痰上清液中嗜酸细胞趋化因子浓度。结果A组诱导痰嗜酸细胞占白细胞百分比（EOS／Leu％）与B组比较，差异有显著性（P〈0．05）；A组嗜酸细胞趋化因子浓度与B组比较，差异有显著性（P〈0．05）。A组EOS／Leu％、嗜酸细胞趋化因子浓度与一秒钟用力呼气容积占预计值百分比（FEV1％）均呈负相关；A组嗜酸细胞趋化因子浓度与EOS／Leu％呈正相关。结论嗜酸细胞趋化因子可能通过对嗜酸细胞（EOS）的选择性趋化作用使其释放一系列炎性介质参与了嗜酸细胞性气道炎症和气流阻塞的发生机制。     

人参五味子汤预防小鼠哮喘的作用机制研究

目的 探讨中药人参五味子汤预防小鼠哮喘的作用及其机制。方法 40只小鼠被随机分为正常对照组（A）、模型组（B）、布地奈德治疗组（C）、人参五味子汤治疗组（D）、人参五味子汤和布地奈德联合治疗组（E）5组，每组8只。用卵白蛋白致敏建立哮喘小鼠模型；分别给予布地奈德、人参五味子汤对哮喘小鼠进行早期治疗4周。观察支气管肺泡灌洗液（BALF）中细胞学变化；采用酶联免疫吸附法（ELISA）测定BALF中自细胞介素-4（IL-4）与γ-干扰素（IFN-γ）；免疫组化测定肺组织中基质金属蛋白酶-9（MMP-9）和金属蛋白酶组织抑制剂-1（TIMP-1）；原位杂交结合免疫组化测定骨髓细胞表达IL-5Rα mRNA^＋的CD34^＋细胞。结果 与B组比较，C、D、E组BALF中细胞总数、中性粒细胞、淋巴细胞、嗜酸粒细胞比例及IL-4、IL-4／IFN-γ比值均显著降低，IFN-γ升高；肺组织中MMP-9、TIMP-1、MMP-9／TIMP-1比值亦显著降低（P均〈0．01）；C组和E组IFN-γ较D组升高，其余指标均较D组明显下降（P〈0．05或P〈0．01），而C组与E组间差异无统计学意义。与A组比较，B、C、D、E组小鼠骨髓CD34^＋细胞数和IL-5Rα mRNA^＋的CD34^＋细胞比例均明显升高（P〈0．05或P〈0．01）；C组与B组差异无统计学意义，D组和E组较B组显著降低（P均〈0．01）。结论 人参五味子汤可通过影响小鼠骨髓CD34^＋造血细胞和IL-5Rα mRNA^＋的CD34^＋造血细胞向嗜酸粒细胞的转化和对气道炎症过程等途径，影响小鼠哮喘的病理过程。     

中医药治疗儿童支气管哮喘进展

<正>支气管哮喘是儿科常见病,是由多种细胞(如嗜酸性粒细胞、肥大细胞、T淋巴细胞、中性粒细胞及气道上皮细胞等)和细胞组分共同参与的气道慢性     

骨髓瘤细胞和骨髓基质细胞均参与多发性骨髓瘤白细胞介素6过？…

目的：探讨白细胞介素６（ＩＬ－６）在多发性骨髓瘤（ＭＭ）中过度表达的机制。方法：采用多聚甲醛分别固定骨髓瘤细胞ＫＭ３和骨髓基质细胞，再将其置于同一体系中共同培养，用Ｂ９细胞增殖试验测量培养上清中ＩＬ－６活性。结果：骨髓基质细胞及骨髓瘤细胞均能自发分泌ＩＬ－６，两种细胞经固定后几乎均不能分泌ＩＬ－６。固定的骨髓瘤细胞与未经固定的骨髓基质细胞共培养后，可使共培养上清中的ＩＬ－６活性明显增加，同时固定后     

Production of the novel C-C chemokine MCP-4 by airway cells and comparison of its biological activity to other C-C chemokines.

Monocyte chemotactic protein-4 (MCP-4) is a newly identified C-C chemokine with potent eosinophil chemoattractant properties. We describe studies of its biological activity in vitro to induce chemotaxis of peripheral blood eosinophils and to induce histamine release from IL-3-primed peripheral blood basophils. MCP-4 and eotaxin caused a similar rise in eosinophil intracytoplasmic Ca2+ and complete cross-desensitization. MCP-4 also abolished the eosinophil Ca2+ response to MCP-3 and partially desensitized the response to macrophage inflammatory protein-1alpha. MCP-4 activated cell migration via either CCR2b or CCR3 in mouse lymphoma cells transfected with these chemokine receptors. MCP-4 inhibited binding of 125I-eotaxin to eosinophils and CCR3-transfected cells and inhibited 125I-MCP-1 binding to CCR2b-transfectants. MCP-4 mRNA was found in cells collected in bronchoalveolar lavage of asthmatic and nonasthmatic subjects and was prominently expressed in human lung and heart. MCP-4 mRNA was expressed in several human bronchial epithelial cell lines after cytokine stimulation. Pretreatment of BEAS-2B epithelial cells with the glucocorticoid budesonide inhibited MCP-4 mRNA expression. These features make MCP-4 a candidate for playing a role in eosinophil recruitment during allergic respiratory diseases.     

Inhibition of human rhinovirus-induced cytokine production by AG7088, a human rhinovirus 3C protease inhibitor

Symptom severity in patients with human rhinovirus (HRV)-induced respiratory illness is associated with elevated levels of the inflammatory cytokines interleukin-6 (IL-6) and IL-8. AG7088 is a novel, irreversible inhibitor of the HRV 3C protease. In this study, AG7088 was tested for its antiviral activity and ability to inhibit the production of IL-6 and IL-8 in a human bronchial epithelial cell line, BEAS-2B. Infection of BEAS-2B cells with HRV 14 resulted in the production of both infectious virus and the cytokines IL-6 and IL-8. Treatment of HRV 14-infected cells with AG7088 resulted in a statistically significant (P, <0.05) dose-dependent reduction in the levels of infectious virus as well as IL-6 and IL-8 released into the cell supernatant compared to the results obtained for compound-free infected cells. AG7088 was also able to inhibit the replication of HRV 2 and 16 in BEAS-2B cells. In time-of-addition studies, AG7088 could be added as late as 14 to 26 h after HRV 14 infection of BEAS-2B cells and still result in a statistically significant (P, <0.05) reduction in the levels of infectious virus, IL-6, and IL-8 compared to the results obtained for compound-free infected cells. These findings have implications for the development of an antirhinovirus agent that may not only block virus replication but also diminish symptoms.     

Expression of CD28 and CD86 by human eosinophils and role in the secretion of type 1 cytokines (interleukin 2 and interferon gamma): inhibition by immunoglobulin a complexes.

Eosinophils are the source of various immunoregulatory cytokines, but the membrane molecules involved in their secretion have not been clearly identified. Here we show that peripheral blood eosinophils from hypereosinophilic patients could express membrane CD86 but not CD80. The T cell costimulatory molecule CD28 is also detected on the eosinophil surface. CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected. In contrast to T cells requiring two signals for effective stimulation, CD28 ligation alone was sufficient for optimal eosinophil activation. Eosinophil-derived IL-2 and IFN-gamma were biologically active, as supernatants from anti-CD28-treated cells were able to induce CTLL-2 proliferation and major histocompatibility complex class II expression on the colon carcinoma cell line Colo 205, respectively. Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release. These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals. They confirm that through activation of costimulatory molecules, eosinophils could function as an immunoregulatory cell involved in the release of both type 1 and type 2 cytokines.     

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...     

Dendritic cells induce T lymphocytes to release B cell-stimulating factors by an interleukin 2-dependent mechanism

Dendritic cells (DC) are essential accessory cells for T-dependent antibody responses in culture (1). We have outlined a three-stage mechanism to explain the capacity of DC to stimulate primary antibody responses to heterologous erythrocytes. First, DC induced T cells to produce and to become responsive to interleukin 2 (IL-2). This stage corresponded to the syngeneic mixed leukocyte reaction (2) and involved the clustering of DC and T cells into discrete aggregates. Isolated clusters, representing 5-10% of the culture, were critical for IL-2 release and the production of IL-2-responsive T blasts. In the second stage, IL-2 directly triggered the responsive T cells to release B cell helper factors. This role for IL-2 was documented with a rabbit anti-IL- 2 reagent, purified IL-2, and T cells that had been rendered IL-2 responsive by an initial co-culture with DC. T cell growth was not required for IL-2-mediated helper factor release, since irradiated and untreated responders produced similar levels of factor and did so within 3 h of the addition of IL-2. In the final stage, helper factors stimulated the development of antibody-secreting cells from purified B lymphocytes. The helper factors were not H-2 restricted, but for both sheep and horse erythrocytes, the response to factors was antigen dependent and specific. The IL-2 that was present in the DC/T cell- conditioned medium did not act on B cells, since helper activity was neither neutralized nor absorbed by our anti-IL-2 reagent. We conclude that the ability of the DC to induce IL-2 release and responsiveness underlies its capacity to trigger both T and B lymphocyte reactions.     

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.     

Expression of interleukin 2 receptors on activated human B cells

Using anti-Tac, a monoclonal anti-interleukin 2 (IL-2) receptor antibody, we have explored the possibility that certain activated B cells display receptors for IL-2. Resting normal B cells and unselected B cell lines established from normal individuals were Tac antigen negative. In contrast, the cell surface Tac antigen expression was demonstrable on 6 of 10 B cell lines from patients with Burkitt's lymphoma, 5 of 6 B cell lines derived from patients with HTLV-I- associated adult T cell leukemia (including all four that had integrated HTLV-I into their genome), and on certain normal B cells activated with pokeweed mitogen. Furthermore, cloned Epstein-Barr virus- transformed B cell lines derived from Tac-positive normal B cells continued to express the Tac antigen in long-term cultures and manifested high affinity IL-2 receptors identified in binding studies with purified radiolabeled IL-2. The line 5B4 developed in the present study could be induced with purified JURKAT-derived or recombinant IL-2 to express a larger number of IL-2 receptors. Furthermore, the addition of IL-2 to the 5B4 B cell line augmented IgM synthesis, which could be blocked by the addition of anti-Tac. The size of the IL-2 receptors expressed on the cloned normal B cell lines was similar (53,000-57,000 daltons) to that of receptors on phytohemagglutinin-stimulated T cell lymphoblasts. Thus, certain malignant and activated normal B cells display the Tac antigen and manifest high affinity receptors for IL-2. These data suggest that IL-2 may play a role in the differentiation of activated B cells into immunoglobulin-synthesizing and -secreting cells.