SLE患者外周血IFN-γ及IL-4细胞因子mRNA的表达研究

为利用实时监测定量PCR技术检测细胞因子mRNA的表达 ,从mRNA水平探讨IFN γ、IL 4mRNA的变化。对初诊SLE患者外周血IFN γ、IL 4的mRNA表达进行检测 ,并同时使用LPS及PHA作刺激剂研究其对SLE患者细胞因子mRNA表达的影响。结果是PHA刺激后 ,正常组IFN γmRNA的表达明显高于SLE组 (P =0 0 2 4 ) ;LPS对正常对照的IFN γ和IL 4的mRNA表达均能明显上调 ,但SLE患者仅表现为IFN γ升高 ,IL 4升高不明显 ;未刺激组SLE患者与正常对照的比较结果显示IFN γ的表达及IFN γ/IL 4有明显降低 (P =0 0 14 ,P =0 0 33)。故SLE患者与正常对照相比 ,IFN γmRNA的表达下降 ,同时SLE患者的IFN γ、IL 4mRNA表达对LPS及PHA的反应下降。由于不同的患者受累系统不同 ,临床表现不一 ,可能T细胞的异常并不相同 ,其免疫系统紊乱的机制也不一致     

IFN-γ受体Ig融合蛋白对ConA诱导小鼠肝损伤的保护作用

本文研究γ 干扰素受体免疫球蛋白融合蛋白 (IFN γR Ig )对ConA诱导的小鼠细胞免疫性肝损伤的保护作用及机制。在Balb/c小鼠体内一次性静脉注射ConA 2 0mg/kg诱导细胞免疫性肝损伤模型 ,分别于模型建立前后不同时间腹腔注射 10mg/kgIFN γR Ig,观察该融合蛋白对小鼠血清谷丙转氨酶 (GPT )水平 ,细胞因子IFN γ、TNF α和IL 10分泌以及肝组织病理学变化的影响。结果表明IFN γR Ig预防给药明显改善肝脏损伤的组织学和血清学变化 ,降低GPT水平 ,减少肝脏中性粒细胞、单核细胞浸润 ;同时与模型对照小鼠相比血清IFN γ水平下降 ,TNF α分泌合成减少 ,IL 10水平明显增加。而IFN γR Ig通过早期结合并阻断内源性IFN γ ,提高IL 10的抗炎作用 ,减轻炎症细胞对肝脏的侵袭及IFN γ、TNF α的肝细胞破坏作用 ,保护免疫性肝损伤。     

碘化钠对人甲状腺细胞IL-6、IFN-γ和TNF-α基因表达的影响

本研究以 10 4 M碘化钠 (NaI)刺激单层培养的人Graves病 (GD )甲状腺滤泡上皮细胞 (TEC ) ,采用定性及半定量PCR技术检测刺激前后白介素 6 (IL 6 )、γ 干扰素 (IFN γ )和肿瘤坏死因子 α(TNF α )在细胞中的表达水平。结果表明 :(1) 3例GD组织均含有IL 6及IFN γmRNA ,2例检测到TNF α基因表达 ;(2 )基础状态下 ,3例TEC均表达IL 6基因 ,2例表达TNF α ,而所有样本均无IFN γmRNA ;(3)NaI不能诱导TEC产生IFN γmRNA ,对IL 6mRNA的表达亦无明显影响 ;(4 ) 1例TNF α阴性的TEC样本 ,经NaI刺激后 ,可表达mRNA ,2例原含有TNF α的TEC经刺激后 ,其mRNA的表达水平显著增加。提示碘可通过诱导或增强TEC表达TNF α ,导致自身免疫性甲状腺疾病的发生与发展。     

细胞因子对肾癌细胞株786-0Fas表达及其凋亡的调控

为研究细胞因子对肾癌细胞株 786 0Fas表达以及FasAb介导凋亡的影响 ,单独或联合应用IFN γ ,IFN α ,IL 2 ,TNF α刺激 786 0细胞株 ,并以Fas单克隆抗体 (FasAb )诱导其凋亡。结果发现 ,IFN α、IFN γ均能显著上调 786 0细胞的Fas表达 (P <0 0 1,P <0 0 1)并促进FasAb诱导的凋亡 (P <0 0 1,P <0 0 1) ;IL 2、TNF α对 786 0的Fas表达及FasAb诱导的凋亡均无影响 ;IL 2不能增强IFN α、IFN γ诱导的Fas表达 ,亦不能促进凋亡。TNF α能增强IFN α诱导的Fas表达并促进凋亡 ,但不影响IFN γ诱导的Fas表达及其凋亡。结果表明IFN γ、IFN α可增强肾癌细胞株 786 0的Fas表达 ,并促进FasAb介导的凋亡。但其对FasAb介导凋亡的敏感性并不完全取决于Fas表达水平     

α黑素细胞刺激素保护内毒素休克小鼠的免疫学机制研究

为研究α黑素细胞刺激素 (α MSH)对内毒素休克小鼠发挥保护作用的免疫学机制 ,将BALB/c小鼠经腹腔注射 (ip)LPS5 0 μg/kg和D 半乳糖胺 (D Gal) 90 0mg/kg建立内毒素休克模型后 ,在不同时间给予α MSHip后观察小鼠的存活率 ;用MTT法测定小鼠血清中IL 1、TNF α、IL 6的含量 ;用Griess试剂测定血清中NO的含量。腹腔巨噬细胞 (MΦ)膜表面免疫相关分子以流式细胞仪检测。结果是给予LPS前 1h、同时和后 1hα MSH 2 5mg/kgip可以明显提高内毒素休克小鼠的存活率 ,其中以给予LPS 1h后注射α MSH疗效最佳 ,能显著降低血清中IL 1、TNF α含量 (P <0 0 0 1) ,增加IL 6的产生 (P <0 0 5 ) ,抑制LPS引起的腹腔MΦ高表达CD14、CD40、CD5 4及CD86分子。因此 ,α MSH降低TNF α、IL 1及NO的含量 ,抑制MΦ膜表面CD14、CD5 4、CD40及CD86的表达 ,在其抗内毒素休克效应中起重要作用。     

人生长激素基因在小鼠体内的表达及其对血清中细胞因子浓度的影响

目的 :探讨体内高水平的人生长激素 (GH)对机体免疫功能的影响。方法 :构建人GH基因的真核表达质粒pcDNA3 hGH ,直接注射于小鼠的股四头肌中。注射后分别在第 5、15、2 5天分离血清 ,通过ELISA方法检测血清中GH的水平 ,同时检测血清中IL 2、IFN γ和TNF α水平的变化。结果 :所构建的质粒目的基因插入正确 ,转染细胞能分泌高水平的GH。接受质粒的小鼠血清中人GH的浓度与对照组相比较明显提高 ,但IL 2、IFN γ和TNF α的浓度没有显著变化。结论 :体内较高水平的GH对IL 2、IFN γ和TNF α的分泌没有显著影响     

大鼠胰岛β细胞损伤中IL-18的作用研究

新生Wistar大鼠离体胰岛与细胞因子孵育后 ,观测胰岛素释放和一氧化氮 (NO )生成的变化 ,并用逆转录 聚合酶链反应 (RT PCR )观察IL 18受体信号链 (IL 18Rβ )mRNA的表达水平。结果表明 :(1) 0 6 2 5～ 10nmol/L基因重组小鼠 (rm )IL 18孵育胰岛 2 4h后 ,对累积的和葡萄糖刺激的胰岛素释放以及NO生成均无显著效应 ;(2 ) 2 0 0U/ml基因重组大鼠 (rr)γ干扰素 (IFN γ )或 2 5 0U/ml基因重组人 (rh )肿瘤坏死因子 α (TNF α)单独存在对胰岛素释放和NO生成均无明显效应 ,也不能促使 10nmol/LrmIL 18对胰岛素释放和NO生成产生影响 ;(3) 2 0 0U/mlrrIFN γ +2 5 0U/mlrhTNF α或 15pg/mlrhIL 1β均明显促进NO生成和抑制胰岛素释放 ,而 10nmol/LrmIL 18则不影响IFN γ +TNF α或IL 1β的上述效应 ;(4 )即使经IL 1β和 (或 )IFN γ +TNF α或IL 12孵育后 ,大鼠胰岛素瘤 (RIN )细胞或离体大鼠胰岛仍未见IL 18RβmRNA的表达。提示IL 18在细胞因子所致胰岛β细胞损伤中不发挥直接作用 ,原因是IL 18受体在胰岛 β细胞中不表达。     

小檗碱对小鼠DTH及其体内几种细胞因子的影响

目的 :以二硝基氟苯 (DNFB)所致迟发型超敏反应 (DTH)小鼠模型观察小檗碱对小鼠DTH及其体内几种重要细胞因子的影响。方法 :采用 1%DNFB腹部致敏、耳廓发敏的方法建立DTH小鼠模型 ,以巨噬细胞NO2 -释放法测定血清IFN γ水平 ,胸腺细胞法检测IL 1水平 ,丝裂原激活的淋巴母细胞法检测IL 2水平 ,L92 9细胞结晶紫染色法测定TNF α水平。结果 :发现小檗碱可抑制DNFB诱导的小鼠DTH ,降低其血清IFN γ水平 ,抑制其腹腔MΦ产生IL 1及TNF α ,抑制其脾细胞产生IL 2。结论 :表明小檗碱有抑制小鼠DTH的作用 ,其机制可能是抑制了IFN γ、IL 1、TNF α、IL 2等细胞因子的产生和分泌 ,从而抑制免疫反应 ,减轻炎症损伤。     

烫伤对大鼠白细胞介素-18 mRNA和IFN-γ的影响

探讨烫伤对白细胞介素－18（IL－18）mRNA的表达和IFN－γ的影响。采用大鼠30％体表面积Ⅲ度烫伤模型，IL－18、IFN－γmRNA表达采用转录聚合酶链式反应检测，IFN－γ蛋白含量采用ELISA法测定，烫伤后组织IL－18mRNA表达较伤前值有显著升高，8h达高峰且一直持续至伤后24h。组织IFN－γmRNA表达增加以肠、肺组织明显，伤后24h仍维持较高水平。肠、肺组织IFN－γ蛋白水平与其mRNA表达趋势基本一致。SDD预处理可不同程度降低IL－18、IFN－γmRNA表达和肠、肺组织IFN－γ水平。相关分析表明，IL－18mRNA表达与IFN－γmRNA表达、IFN－γ水平呈显著正相关。组织IL－18mRNA表达在烫伤后显著增多，并对IFN－γ的生成具有重要影响。     

TNF-α、IL-8在脂多糖致大鼠脑水肿中的表达

目的　探讨脂多糖 (lipopolysaccharide ,LPS)致大鼠脑水肿发生过程中 ,地塞米松作用下TNF α、IL 8表达水平的变化及相互关系。方法　利用LPS诱导大鼠脑水肿发生 ,测定脑组织含水量及伊文私蓝 (Evansblue,EB)含量 ,制备脑组织匀浆 ,ELISA法测IL 8含量 ,放射免疫法测TNF α含量。结果　LPS注入后LPS组和DXM组脑组织含水量和伊文思蓝含量均较NS组增高 (P <0 .0 5) ;LPS组TNF α和IL 8含量较NS组显著增高 (P <0 .0 1 ) ,但DXM组较LPS组显著降低 (P <0 .0 5) ;DXM组TNF α和IL 8含量在早期 (≤ 2 4h)较NS组增高 (P <0 .0 5) ,48h时则无显著差异 (P >0 .0 5)。LPS组脑组织含水量与EB含量 ,TNF α与脑组织含水量 ,IL 8与脑组织含水量 ,IL 8与EB含量均呈正相关 (r值分别为 0 .537,0 .42 3 ,0 .473 ,0 .831 ,P <0 .0 5)。结论　TNF α、IL 8参与了脂多糖诱导的脑水肿的发生、发展过程 ,早期应用糖皮质激素可以抑制炎性细胞因子的生成 ,减轻炎症反应与组织损伤。     

Interleukin-12 is required for interferon-γ production and lethality in lipopolysaccharide-induced shock in mice

Several cytokines, in particular tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 γg/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-γ production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-γ production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1–3 ng/ml at 3–6 h after LPS injection, whereas IFN-γ production was observed only in BCG-primed mice. The priming effect of BCG on IFN-γ production appears to be mostly due to its ability to increase TNF-α production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-γ production, as shown by the ability of injection of TNF-α and LPS (1 γg/mouse), but not LPS alone, to induce IFN-γ production. However, in addition to TNF-α, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-γ production, because co-injection of TNF-α and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-γ production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-γ production, also completely protect BCG-primed mice injected with up to 10 μg of LPS from shock-induced death. Thus, IL-12 is required for IFN-γ production and lethality in an endotoxic shock model in mice.     

IFN-γ Inhibits the Suppressive Effects of PGE2 on the Production of Tumor Necrosis Factor-α by Mouse Macrophages

Tumor necrosis factor-α (TNF-α) has become known as a central mediator of responses to endotoxin, rheumatoid diseases, and other forms of inflammation. Current investigations indicate that the production of TNF-α is controlled by other mediators, including interferon-γ (IFN-γ) and prostaglandin E₂ (PGE₂). In the present study, we investigated the regulatory effects of IFN-γ and/or PGE₂ on LPS-induced TNF-α production and mRNA expression in mouse peritoneal macrophages using the enzyme immunoassay and Northern blot analysis, respectively. In response to 10 ng/ml of LPS, TNF-α production reached a maximum at approximately 4 hrs, followed by rapid decline. At the molecular level, TNF-α mRNA accumulated rapidly after LPS exposure, reaching a peak by 3 hr, and declined more rapidly than did the production of TNF-α. Exposure of macrophages to 100 U/ml of IFN-γ caused an increase in both the TNF-α production and mRNA expression induced by LPS. Exogenous PGE₂ caused a dose dependent reduction in LPS-induced TNF-α mRNA accumulation as well as TNF-α production. Macrophages primed with IFN-γ showed the reduced responsiveness to the suppressive effect of PGE₂ on the production of TNF-α and the accumulation of TNF-α mRNA. These findings indicate that the suppressive effects induced by PGE₂ on the accumulation of TNF-α mRNA as well as the production of TNF-α can be reduced by the pretreatment of macrophages with IFN-γ. These studies demonstrate the role of IFN-γ as an immunomodulating compound that may effectively regulate TNF-α production by modulation of macrophage responsiveness to PGE₂.     

激活的脐血树突状细胞对肿瘤细胞的直接杀伤作用

探索脐血树突状细胞 (DC)在γ干扰素 (IFN γ )及细菌脂多糖 (LPS )激活前后对肿瘤细胞杀伤活性的差异。分离健康脐血单核细胞 ,用重组粒单细胞集落刺激因子 (GM CSF )、白介素 4 (IL 4 )和α肿瘤坏死因子α (TNF α )诱导为DC。于诱导第11天在合成培养基中加入LPS或IFN γ继续培养 12h ,将其激活。用流式细胞仪检测DC表面共刺激分子的改变 ,以明确LPS或IFN γ对DC的不同刺激作用 ;同时 ,以恶性血液病细胞株Jurkat及Daudi为靶细胞 ,以不同效靶比 (E∶T )与DC共同培养 18h ,采用51Cr释放试验检测DC激活前后抗肿瘤活性的差异。结果 (1)LPS及IFN γ可不同程度地上调DC表面CD86、CD80、CD83及CD1a的表达 ,尤以LPS刺激组明显 ;(2 )DC在未加刺激因子前能有效杀伤Jurkat细胞 ,在效靶比为 2 0∶1时 ,LPS或IFN γ可使其杀伤活性进一步提高至 5 0 0 %、 36 9% ,均与未加刺激因子前有显著差异 (P <0 0 0 1,P <0 0 2 5 ) ;(3)在效靶比为 2 0∶1时 ,LPS可使DC对Daudi的杀伤率提高至 19 8% (P <0 0 2 5 ) ,而未加刺激因子组及IFN γ刺激组DC对Daudi在任何效靶比均未显示明显的杀伤作用。LPS或IFN γ激活的脐血DC对Jurkat及Daudi细胞具有选择性杀伤作用     

Proinflammatory Cytokines Regulate the Gene Expression of Hyaluronic Acid Synthetase in Cultured Rabbit Synovial Membrane Cells

To elucidate the mechanism of accumulation and fragmentation of hyaluronic acid (HA) under inflammatory conditions, we investigated the effect of proinflammatory cytokines on hyaluronic acid synthetase (HAS) mRNA expression using cultured rabbit synovial membrane cells. HASs mRNA levels were determined by real-time PCR. HAS2 mRNA expression was maximally enhanced 3.3- and 2.8-fold after 3-hour stimulation with IL-1β (1 ng/ml) and after 1-hour stimulation with TNF-α (10 ng/ml). HAS3 mRNA expression was increased by a maximum of 4.3 times after 3-hour stimulation with IL-1 β (10 ng/ml), whereas 1-hour stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) induced around a 2.5-fold increase in HAS3 mRNA. Although IFN-γ (1–100 ng/ml) alone showed little effect on HAS2 mRNA expression, the effect was synergized by combined with both IL-lβ and TNF-α, substantially increasing HAS2 mRNA expression.

These results suggest that proinflammatory cytokines regulate the HAS expression, and consequently may contribute to the accumulation and fragmentation of HA.