地塞米松对视网膜色素上皮细胞IL—6表达的抑制作用

目的观察视网膜色素上皮(RPE)细胞中,IL-6的表达及地塞米松对IL-6表达的抑制作用.方法培养的人RPE经IL-1β(10μg/L)刺激及地塞米松作用后,采用ELASA、免疫组化和原位杂交等方法,检测培养的人RPEIL-6mRNA及其蛋白的表达.结果用IL-1β刺激后8h,RPE细胞表达的IL-6约为2000ng/L@106细胞;地塞米松可明显抑制IL-6蛋白的产生(200ng/L@106细胞),与对照组相比较差异显著(P＜0.01).免疫组化染色显示,表达的IL-6在RPE细胞胞浆中呈浓淡不一的棕黄色,地塞米松作用组染色较浅.原位杂交表明,IL-6mRNA在RPE细胞胞浆中的表达呈浓淡不一的紫蓝色,地塞米松作用组染色与对照组相同.图像分析显示,二者的吸光度(A)值无明显差别(P＞0.05).结论在IL-1β刺激下,人RPE细胞可表达IL-6的mRNA及其蛋白,地塞米松能有效地抑制RPE细胞中IL-6蛋白的表达.     

人IL-6受体胞外区真核表达载体的构建及体外表达

目的 构建人IL-6受体(IL-6R)胞外区真核表达载体,检测其在体外培养细胞中的表达.方法 利用PCR扩增IL-6R胞外区,克隆到pcDNA3.1(+)中,用双酶切、测序鉴定.重组质粒通过脂质体转染HL-60细胞,用G418进行筛选,利用Western印迹检测IL-6R蛋白表达.结果 PCR扩增出1 218 bp的目的 片段,双酶切和测序结果显示重组质粒正确.Western印迹结果显示转染细胞能够表达目的 蛋白.结论 成功构建了人IL-6R胞外区真核表达载体,并且能够在真核细胞中表达.     

IL-6/IL-6受体与类风湿关节炎关联性研究新进展

IL-6是一种多效性前炎症细胞因子,有多种生物活性,包括介导炎症反应,免疫反应等。IL-6在许多炎症性疾病中高表达,如RA,SLE,Crohn’s病等。IL-6的产生受多种因素的调节如NF-κB,Lin28,let-7 microRNA,IL6阳性反馈环,JunB等。IL-6受体(IL-6R)包括特异性IL-6Rα及IL-6Rβ即IL-6家族成员共有的信号转导蛋白gp130。IL-6可通过两种途径向细胞内传导信号:传统途径及反式信号途径,LMO4可参与反式信号途径的调节。IL-6/IL-6R在T、B细胞的发生中起重要作用。IL-6可抑制Th1、Treg的分化,促进Th2的分化;而且IL-6是CD4+T细胞分化为Th17细胞所必需;在B细胞中IL-6可诱导RAG基因的表达,促进抗体的产生。近年来越来越多的证据表明IL-6/IL-6R与RA存在密切关联性。RA患者血清及滑液中IL-6及sIL-6R的浓度升高,并且IL-6水平与疾病活性及临床表现密切相关,这可以解释RA患者的许多症状。因此可将IL-6信号转导途径相关的分子作为RA的治疗靶点,目前也研制出几种药物,其中之一为抗sIL-6R的人源化抗体Tocilizumab。临床试验中,该药可减轻RA患者的症状,降低疾病活性,可是也带来一定的副作用,这就限制了该药在临床中的应用。正在研制的另一种抗IL-6R的抗体可能为干预RA带来新的希望。     

白细胞介素6促人脾NK细胞活性及其机制初探

本文采用~(51)Cr释放试验测定人重组白细胞介素6(HrIL-6)处理正常成人睥单个核细胞(MNC)24小时后,NK细胞抗K562靶细胞的杀伤活性。结果证实,IL-6能明显增强脾NK细胞的抗瘤活性(P<0.05,与对照组比较),且可诱导MNC产生IL-2,NK活性的增强效应和MNCIL-2的合成水平均与IL-6的处理剂量呈依赖关系,提示IL-6促人脾NK活性的机制可能是通过诱导脾细胞产生IL-2而实现的。     

用7TD_1细胞检测IL-6生物学活性方法的改良

本文对IL-6生物学活性检测试验的条件进行了研究。着重比较了4组不同状态的IL-6依赖细胞系7TD_1对IL-6的反应。~3H-TdR掺入结果显示,重组人IL-6(10u/ml)对长期培养组、饥饿组、复苏组和PH4.0枸橼酸缓冲液处理组7TD_1细胞的刺激指数分别为5.0、3.0、20.0和13.0。检测IL-6的敏感性以复苏组和pH4.0处理组7TD_1细胞为高。7TD_1细胞的密度以2×10~2/孔最佳。     

卵巢癌细胞IL-6、IL-8及其受体表达的研究

目的分析比较五种常见的上皮性卵巢癌细胞系IL-6、IL-8及其受体表达的差异。方法IL-6、IL-8的表达分别采用RT-PCR和ELISA法进行检测,IL-6受体（IL-6Rα和gp130）及IL-8受体（IL-8RA和IL-8RB）的表达采用免疫印迹技术进行测定。结果①五种上皮性卵巢癌细胞均组成性表达IL-6和IL-8。IL-6和IL-8在CAOV-3细胞中的表达水平均最高,而在HO-8910PM细胞中的表达水平均最低,IL-6在SKOV-3、HO-8910、OVCAR-3细胞中的表达水平依次降低,IL-8在OVCAR-3、SKOV-3、HO-8910细胞中的表达水平依次降低。②五种上皮性卵巢癌细胞均表达IL-6Rα、gp130及IL-8RA;除CAOV-3细胞外,其它细胞均表达IL-8RB。结论本研究旨在筛选表达IL-6和IL-8及其相应受体的细胞株,为研究IL-6、IL-8与卵巢癌发生、发展关系奠定基础,同时也为今后卵巢癌的免疫治疗提供一个新的思路。     

小鼠IL-6和B细胞活化因子融合蛋白真核表达质粒对干燥综合征动物模型的体内治疗作用

目的探讨IL-6和B细胞活化因子(B cell activating factor, BAFF)的融合蛋白真核表达质粒对干燥综合征动物模型非肥胖糖尿病小鼠(non-obese diabetic mouse, NOD)唾液腺、泪腺组织病理的影响, 阐明IL-6/BAFF融合蛋白真核表达质粒对NOD小鼠可能的治疗机制。方法构建IL-6/BAFF融合蛋白真核表达质粒, 转染CHO细胞, Western blot检测融合蛋白的表达水平。IL-6/BAFF融合蛋白真核表达质粒免疫BALB/c小鼠, 每2周1次, 共3次, 免疫结束后处死小鼠, ELISA法检测血清中抗IL-6和抗BAFF抗体效价。将21只NOD小鼠随机数字表法分为空白组、阴性对照组、治疗组。治疗组小鼠肌肉注射IL-6/BAFF融合蛋白真核表达质粒, 阴性对照组小鼠肌肉注射对照质粒, 空白组不做处理, 每周1次, 6次免疫后处死NOD小鼠, 心脏采血取血清, ELISA法检测血清中抗IL-6和抗BAFF抗体及细胞因子IL-6、BAFF、IFN-γ、IL-10、IL-17A的表达水平;取脾组织流式细胞术检测小鼠脾细胞中Th17、Tr...     

CHO细胞无血清培养研究

目的:用无血清培养基培养中华仓鼠卵巢(Chinese hamster ovary,CHO)细胞。方法:应用无血清培养基采用小方瓶培养CHO细胞,观察细胞维持时间、培养过程的形态变化。结果:应用无血清培养基培养CHO细胞可维持细胞正常生长。结论:无血清培养基可以完全取代含血清培养基用于培养CHO细胞。     

人IL—6和CHO细胞表达及其实验室生产工艺的建立

本文以ＰＫＣＲ６质粒为载体，构建了ＩＬ－６的次级克隆ＰＫＣＲ６－ＩＬ－６，并成功地转染了ＣＨＯ细胞，获得高表达，稳定分泌人ＩＬ－６的转基因细胞，由此建立了实验室生产的ＩＬ－６的基本工艺，即从方瓶过渡到转瓶，经转瓶高密度培养，ＣＨＯ上清中ＩＬ－６含量可以提高近６倍，这为扩大规模生产奠定了一定的基础。     

IL-1β和IL-6对椎间盘髓核细胞NGF表达的影响

目的:研究白细胞介素1β(IL-1β)和IL-6对体外培养的椎间盘髓核细胞神经生长因子(NGF)表达的影响。方法:分离培养人腰椎间盘髓核细胞,经过7d的预培养后,实验组分别加IL-1β(10μg/L,50μg/L)和IL-6(10μg/L,50μg/L)进行刺激,对照组加0.3%FBS,48h后应用免疫组织化学法、RT-PCR和Western blotting检测NGF的表达。结果:培养的椎间盘髓核细胞经鉴定为类软骨细胞。对照组髓核细胞很少表现NGF免疫活性,IL-1β和IL-6刺激组明显上调了髓核细胞NGF mRNA及蛋白质的表达,在相同的浓度下IL-6的上调作用更明显。结论:正常情况下NGF在椎间盘髓核细胞呈低表达状态,IL-1β和IL-6能促进髓核细胞NGF的表达,相同浓度下IL-6刺激髓核细胞分泌NGF的作用更强。     

Tumour necrosis factor, IL-1 and IL-6 in bronchoalveolar washings and their in vitro production during Nippostrongylus brasiliensis infection.

Bronchoalveolar washings (BAW) were obtained from rats primarily infected with N. brasiliensis during the early infection stage that coincides with the lung passage of the parasite and the recruitment of inflammatory cells. BAW were tested for IL-1, IL-6 and tumour necrosis factor (TNF) activities. We found that IL-1 production occurred only on day 1 post infection and ceased thereafter. IL-6 activity was present as from day 1 with a maximum on day 3 post infection and then returned to its normal levels on day 5 post infection. TNF activity was not recovered in BAW at any time of the early infection. Results obtained from the in vitro culture of BAW-adherent cells demonstrated that on day 1 post infection IL-1, but also large amounts of TNF were produced spontaneously, whereas IL-6 was continuously released. Bacterial lipopolysaccharide (LPS) stimulation of the cell culture resulted in an amplification of the cytokine production. Our results suggest that pulmonary cytokines detected in BAW were at least in part produced by alveolar macrophages. Furthermore, the kinetics of IL-1, TNF and IL-6 production show that these monokines are induced at different times during the course of infection, suggesting that cytokine production may follow different regulation patterns during the early phase of N. brasiliensis infection.     

Interferon-gamma stimulates the secretion of IL-1, but not of IL-6, by glomerular mesangial cells.

IL-1 activity in culture supernatant and cell lysate from rat mesangial cells stimulated with interferon-gamma (IFN-gamma) was measured by a thymocyte proliferation assay. While IFN-gamma alone had no effect on the secretion or the intracellular pool of IL-1, the enhancement by IFN-gamma of IL-1 secretion in response to lipopolysaccharide (LPS) was observed. The stimulatory effect of culture supernatant on thymocyte proliferation was abrogated by preincubation with the anti-IL-1 antibody. At least 4-h incubation with IFN-gamma and LPS was required to detect enhancing effect of IFN-gamma. The addition of as little as 1 U/ml IFN-gamma significantly increased IL-1 secretion in the presence of 10 micrograms/ml LPS. The IL-6 activity in culture supernatants was determined by measurement of thymidine uptake in mouse IL-6-dependent cell line (MH60.BSF2). Mesangial cells secreted IL-6 in culture supernatant without additional stimuli and LPS distinctly increased it as described previously. However, in contrast to IL-1 production, no effect of IFN-gamma on IL-6 secretion was observed in the presence or absence of LPS. Moreover, we determined whether enhanced IL-1 release is associated with Ia expression on mesangial cells. IFN-gamma alone and the combination with LPS induced marked expression of Ia antigen, whereas LPS alone did not. We conclude that IFN-gamma stimulates the production of IL-1, but not IL-6, by mesangial cells and suggest an important role of IFN-gamma in the pathogenesis of glomerulonephritis by regulating the mesangial production of IL-1 and the accessory cell function of mesangial cells.     

不同杂交瘤饲养细胞上清和条件培养液中IL-2和IL-6水平的比较

本文应用细胞因子依赖细胞株CTLL和7TD1及。~3H-TDR掺入方法,分别对6种常用培养杂交瘤的饲养细胞培养上清和条件培养液中IL-2和IL-6的生物学活性水平进行了定量检测和比较。结果发现,除大鼠混合胸腺细胞培养上清中含有约1u/ml IL-2外,其它均未测出IL-2的生物学活性。但6种上清中都含有IL-6,其中人成纤维细胞CRL_(1506)和人内皮细胞中含有很高水平的IL-6,分别为10000u/ml和2512u/ml;大鼠混合胸腺细胞和小鼠腹腔巨噬细胞培养上清分别为158u/ml和126u/ml;小鼠脾脏细胞和胸腺细胞培养上清中IL-6相对较低,分别为79u/ml和16u/ml。此外,本文还对常用饲养细胞及条件培养液在杂交瘤细胞生长中可能起的主要作用进行了讨论。     

Serum levels of IL-1, IL-6 and tumour necrosis factors in patients undergoing coronary artery bypass grafts or cholecystectomy.

Plasma levels of biologically active IL-1, tumour necrosis factor (TNF) and IL-6 were measured before, during and after coronary artery bypass graftings (CABG) (n = 9) and cholecystectomy (CHO, n = 9), and in normal controls (nine healthy volunteers). Mean pre-operative IL-1 concentration in four of the nine CABG patients was 0.452 + 0.03 ng/ml, significantly (P less than 0.001) higher than that of the other five (0.045 +/- 0.009 ng/ml), CHO patients (0.035 +/- 0.005 ng/ml) and controls (0.029 +/- 0.008 ng/ml). Three of the four patients with high pre-operative IL-1 had functional capacity IV, while the other five had functional capacity IIa or IIb. Slight IL-1 elevation after anaesthesia, followed by reduction after initiation of bypass, elevation on completion of surgery and reduction to basal levels after 7 days was found in patients undergoing CABG. Mean basal TNF levels of CABG and CHO patients did not differ, but were higher than those of controls (2.85 +/- 0.5 ng/ml for CABG, 2.05 +/- 0.06 ng/ml for CHO, 0.72 +/- 0.07 ng/ml for normals, P less than 0.001). A unique kinetics of release during CABG was observed also for TNF. Mean pre-operative IL-6 levels were normal (50 +/- 3 ng/ml for CABG, 50 +/- 0.5 ng/ml for CHO and 65 +/- 10 ng/ml for controls). Gradual elevation to a mean peak of 725 +/- 100 ng/ml on completion of CABG was observed as compared with 275 +/- 50 ng/ml in CHO (P less than 0.01). On the seventh post-operative day mean IL-6 levels returned to normal. Two patients with post-operative low-grade fever (38 degrees C) had high, late cytokine levels. One of these two patients had leucocytosis, sterile discharge from the operative wound and was diagnosed as suffering from the Dressler syndrome. In this study elevated cytokine values and unique kinetics of release into the serum were found in patients undergoing CABG.     

IL-6-soluble IL-6 receptor complex inhibits the proliferation of dermal fibroblasts

A number of investigators has reported that there is increased production of interleukin-6 (IL-6) by fibroblasts and monocytes from the patients with systemic sclerosis (SS). However, the precise role of IL-6 in the pathogenesis of SS remains unclear. On the basis of our previous study showing that the complex of IL-6 and soluble IL-6 receptor (sIL-6R) could induce synovial fibroblast proliferation, we examined whether the IL-6-s1L-6R complex could induce the proliferation of normal dermal fibroblastic cells (DF). IL-6 suppressed DF proliferation, and, in the presence of sIL-6R, dose-dependently showed much stronger suppressive effects on DF proliferation. This suppression was completely blocked by either anti-IL-6 or anti-sIL-6R antibody. Furthermore, the IL-6-sIL-6R complex significantly suppressed IL-1β-, TNFα- and PDGF-AA-induced DF proliferation. These lines of evidence suggest that the IL-6-sIL-6R complex may have potential as a useful agent for the treatment of SS.     

GM-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine production in human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1 alpha and TNF-alpha.

The role of mucosal fibroblasts in intestinal inflammatory reactions is not known. In this study, we demonstrate that fibroblasts grown from histologically normal human duodenal biopsy tissues expressed mRNA genes for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) when stimulated with lipopolysaccharide (LPS) or IL-1 alpha. The increased mRNA expression of GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and IL-8 in response to IL-1 alpha and LPS stimulation was time- and dose-dependent. In contrast, IL-10 was weakly expressed when fibroblasts were stimulated with LPS, IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha), but the expression was enhanced in the presence of cycloheximide combined with optimal concentrations of LPS, IL-1 alpha or TNF-alpha, IL-1 alpha was a more potent stimulator than LPS for GM-CSF, IL-6, IL-8 and IL-10 expression, but not for IL-1 alpha and IL-1 beta. Increased GM-CSF, IL-6 and IL-8 gene expression was associated with the production of cytokine proteins in culture supernatant, but IL-1 alpha and IL-1 beta remained undetectable. Dexamethasone suppressed both gene expression and protein production of GM-CSF, IL-6 and IL-8 when fibroblasts were exposed to IL-1 alpha. TNF-alpha stimulated the release of GM-CSF, IL-6 and IL-8 and, combined with IL-1 alpha, cytokine production was enhanced synergistically. Finally, both LPS and IL-1 alpha up-regulated ICAM-1 and VCAM-1 gene expression. These findings implicate duodenal fibroblasts in the initiation and/or regulation of intestinal inflammation.     

Dose-dependent induction of IL-6 by plant-derived proteases in vitro

Oral administration of proteases such as bromelain and papain is commonly used in patients with a wide range of inflammatory conditions, but their molecular and cellular mechanisms of action are still poorly understood. The aim of our study was to investigate the impact of these proteases on the release of interleukin-6 (IL-6) and other cytokines in the recently described modified mixed lymphocyte culture (MMLC) test system which is based on the mutual interaction of cells of the innate and adaptive immunity. Bromelain and papain enhanced IL-6 production dose-dependently up to 400-fold in MMLC before and up to 30-fold after neutralization of LPS content of proteases using polymyxin B, indicating that IL-6 induction by protease treatment was attributable to both protease action and LPS content of enzyme preparations. The production of IFNgamma and IL-10 was not altered by bromelain or papain, indicating a selective and differential immune activation. Both proteases impaired cytokine stability, cell proliferation and expression of cell surface molecules like CD14 only marginally, suggesting no impact of these mechanisms on protease-mediated cytokine release. These findings might provide the mechanistic rationale for the current use of proteases in wound healing and tissue regeneration since these processes depend on IL-6 induction.     

人胚胎胰岛分泌白细胞介素6的动力学研究

目的：阐述ＩＬ－６与胰岛功能或胰岛病变的可能关系。方法：采用常规消化方法分离人胚胎胰岛并进行原代培养,培养上清进行ＩＬ－６、胰岛素、胰高血糖素活性测定和ＩＬ－６ＭｃＡｂ中和实验,同时对胚胎胰岛进行体外葡萄糖刺激实现。结果：原代培养的胚胎胰岛在４０ｈ内ＩＬ－６分泌呈上升趋势（３７～１５３ｍＵ／胰岛）,每２４ｈ重新换取新培养液作为一个分泌周期,ＩＬ－６分泌以第１周期最高（１０６ｍＵ／胰岛）,体外连续培养ＩＬ－６的分泌以１ｄ最高（１４５ｍＵ／胰岛）,用ＩＬ－６ＭｃＡｂ可以阻断胰岛培养上清中ＩＬ－６的活性;胰岛素、胰高血糖素的分泌周期与ＩＬ－６不同;葡萄糖刺激明显促进ＩＬ－６的分泌,其分泌趋势与胰岛素分泌一致。结论：原代培养的人胚胎胰岛在无任何刺激情况下可自发分泌ＩＬ－６,而且ＩＬ－６的分泌有自己的分泌环路。     

Interaction of IL-1 beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) in human T cells activated by murine antigens.

We used a mixed leucocyte culture between human T cells and irradiated murine splenocytes which allowed us to distinguish between cytokine production from the responder and stimulator cells by the use of species-specific assays for mRNA up-regulation. Using this model of T cell activation by antigen, we studied the effects of human antigen-presenting cell-derived cytokines IL-1 beta, IL-6 and TNF-alpha on the activation of human T cell subsets. We show in this system that exogenously added IL-1 beta, IL-6 and TNF-alpha induces IL-2 receptor (R) up-regulation and IL-2 production, and proliferation by both CD4+ and CD8+ cells. The addition of IL-1 beta induces IL-6 mRNA, and anti-IL-1 antibodies or an IL-1R antagonist protein completely suppresses IL-6 and TNF-alpha supported proliferation. Similarly, addition of IL-6 or TNF-alpha induces up-regulation of IL-1 beta mRNA. However, anti-IL-6 and anti-IL-6R antibodies only partially block proliferation supported by IL-1 beta. These findings suggest that IL-6 and TNF-alpha will induce IL-2R up-regulation/IL-2 secretion via the induction of IL-1 beta production.