系统性红斑狼疮病人外周血中TNF和IL—1的检测

肿瘤坏死因子（ＴＮＦ）和白细胞介素１（ＩＬ－１）是一对介导炎性反应的主要细胞因子，用细胞生物法，ＥＬＩ０Ａ法和３Ｈ－ＴｄＲ渗入法对２０例系统性红斑狼疮（ＳＬＥ）患者的外周血单个核细胞（ＰＢＭＣ）培养上清和血清进行了ＴＮＦ和ＩＬ－１水平的检测。结果表明：ＳＬＥ病人的ＰＢＭＣ在体外对有丝分裂原的刺激不敏感。其上清液中的ＴＮＰ的活性和上清液及血清中ＴＮＦ－α蛋白含量的水平与疾病的活动性及是否合并感染均无相关性。ＳＬＥ病人ＩＬ－１的生物活性和健康人无明显差异。活动期病人ＩＬ－１的分泌水平低于恢复期患者，ＩＬ－１分泌能力的降低和病情活动有关。     

分泌抗rHuIL—6McAb杂交瘤细胞系的建立及其应用研究

应用常规方法建立了４株稳定分泌抗重组人ＩＬ－６（ｒＨｕＩＬ－６）单克隆抗体（ＭｃＡｂ）小鼠杂交瘤细胞系１Ｈ３、２Ａ１０、３Ａ３和４Ｂ１。其中，１Ｈ３为ＩｇＧ２ｂ（Ｋ），２Ａ１０为ＩｇＧ１（Ｋ），３Ａ３和４Ｂ１为ＩｇＧ２ａ（Ｋ）。４株ＭｃＡｂ特异性强，与细胞因子ＩＬ－１β、ＩＬ－３、ＩＬ－８、ＴＮＦ－α、ＧＭ－ＳＣＦ、ＩＣＡＭ－１，以及受体菌菌体蛋白成分均无交叉反应。间接ＥＬＩＳＡ测定小鼠腹水ＭｃＡ     

细胞因子对HUVEC分泌IL—6的影响

采用多种细胞因子作用于人脐静脉内皮细胞（ＨＵＶＥＣ），测其ＩＬ－６的分泌。结果表明，ＩＦＮ－γ既可单独诱导又可促进ＴＮＦ－α诱导ＨＵＶＥＣ分泌ＩＬ－６。协同诱导的动力学表明，４－ｈ后ＩＬ－６分泌明显提高。ＩＬ－１ｒａ对ＩＬ－１诱导的ＩＬ－６分泌具有抑制作用，而对ＬＰＳ诱导的ＩＬ－６分泌无影响。ＩＬ－１０可单独诱导ＨＵＶＥＣ分泌ＩＬ－６，工协同促进ＩＬ－１诱导的ＨＵＶＥＣ分泌ＩＬ－６。     

不同病期支气管哮喘血清及PBMC条件培养液IL-8、TNF-α变化

目的：探讨ＩＬ－８、ＴＮＦ－α在儿童支气管哮喘发病中的作用。方法：采用酶联免疫吸附试验（ＥＬＩＳＡ）检测３５例发作期、１８例稳定期哮喘患儿血清及外周血单个核细胞（ＰＢＭＣ）条件培养液中ＩＬ－８、ＴＮＦ－α水平。结果：哮喘发作期血清ＩＬ－８及ＴＮＦ－α明显增高,随病情缓解逐渐降低,达到或接受对照组水平。与对照组比较,哮喘发作期、稳定期ＰＢＭＣ条件培养液ＩＬ－８、ＴＮＦ－α水平显著增高;无论发作期抑或     

重型乙型肝炎患者TNF和IL—1的检测及意义

检测３０例重型乙型病毒性肝炎（重型肝炎）患者血清和外周血单个核细胞培养上清中肿瘤坏死因子水平及ＰＢＭＣ诱生的白细胞介素１活性，结果：重型肝炎患者ＴＮＦ和ＩＬ－１水平均增高，且二者呈正相关。提示ＴＮＦ和ＩＬ－１在重型肝炎的发病机制中起重要作用。     

胃癌和肺癌病人PBMC产生抗肿瘤细胞因子能力变化的研究

应用＾３Ｈ－ＴｄＲ参入及病变抑制等测定１０名正常人和７２例肿瘤病人ＰＢＭＣ受ＰＨＡ刺激后产生ＩＬ－２、ＩＦＮ－γ、ＴＮＦ的能力扩手术切除肿瘤后产生三种细胞因子的水平。结果发现：１）胃癌、肺癌和其它肿瘤患者ＰＢＭＣ产生ＩＬ－２、ＩＦＮ－γ能力极度降低，只有正常人的１／２９￣１／３５和１／１６￣１／１９；２）胃癌、肺癌和其它肿瘤患者ＰＢＭＣ产生ＴＮＦ能力明显增加，是正常人的８￣１２倍；３）手术切除肿瘤     

内皮细胞源性IL—8对人树突状细胞作用的研究

目的：探讨内皮源性白细胞介素－８（ＩＬ－８）对人树突状细胞（ＤＣ）的作用。方法：用ＴＮＦ－α分别诱导内皮细胞和单核细胞产生ＩＬ－８,并将这２种不同来源的ＩＬ－８分别加入ＤＣ常规诱导培养的早期（第３天）及后期（第７天）,培养至第９天收获细胞。采用流式细胞术（ＦＣＭ）分析ＤＣ的表型;体外混合淋巴细胞反应（ＭＬＲ）测定ＤＣ刺激Ｔ细胞增殖能力;ＥＬＩＳＡ检测培养上清中ＩＬ－１２的含量;ＰＩ染色观察ＤＣ凋亡。结果：在ＤＣ培养的早期（第３天）加入内皮源性ＩＬ－８可以抑制ＤＣ的成熟,ＦＣＭ分析表明：ＣＤ１ａ,ＣＤ４０,ＣＤ８０,ＣＤ８３,ＨＬＡ－ＤＲ等ＤＣ表面标志显著下降,而ＣＤ１４表达率明显高于常规培养组。同时,激发同种Ｔ细胞增殖的能力及分泌的ＩＬ－１２量均显著低于常规组（Ｐ〈０．０１）,而加入单核细胞来源的ＩＬ－８则无此     

TNF—α诱导分泌型IL—1和膜型IL—1生成的实验研究

本实验应用丙酸杆菌和内毒素引起的肝坏死模型研究了肿瘤坏死因子α（ＴＮＦα），分泌型白细胞介素１（ｓＩＬ－１）和膜型白细胞介素１（ｍＩＬ－１）在肝坏死形成中的相互作用。其结果：①枯否细胞培养上清液中的ＴＮＦα、ｓＩＬ－１和枯否细胞的ｍＩＬ－１活性较对照组明显增高。ＴＮＦα首先增高，随之为ｍＩＬ－１，最后为ｓＩＬ－１。②ＴＮＦα可以诱导ｓＩＬ－１和ｍＩＬ－１生成。③未发现ＩＬ－１诱导ＴＮＦα生成的作用     

人胎胸腺素对T细胞增殖及IL—2分泌的影响

胸腺素具有广泛的免疫调节作用。本文观察了人胎胸腺素（ＨＦＴ）在体外对人周围血Ｔ细胞的增殖及对周围血单个核细胞（ＰＢＭＣ）分泌ＩＬ－２的影响，实验结果表明：ＨＦＴ能刺激Ｔ细胞增殖，１％ＨＦＴ的作用最强，但未见ＨＦＴ与ＰＨＡ对Ｔ细胞增殖的协同促进作用；而ＨＦＴ对ＰＢＭＣ分泌ＩＬ－２的影响不明显，ＨＦＴ与ＰＨＡ共同作用的效应约有提高，亦仅相当于ＩＬ－２在１．９￣７．９ｕ／ｍｌ时的效应。     

慢性肝病患者血清细胞因子变化与临床意义

目的：为探讨慢性乙型肝炎病毒性肝病患者血清细胞因子ＴＮＦ－α、ＩＬ－１、ＩＬ－６、ＩＬ－８活性变化及其在慢性肝病发生发展中的作用及临床意义。方法：采用ＥＬＩＳＡ法对慢性乙型肝炎（ＣＨ）、慢性乙型重型肝炎（ＣＳＨ）、乙型肝炎性肝硬化（ＨＣ）患者血清中细胞因子ＴＮＦ－α、ＩＬ－１、ＩＬ－６、ＩＬ－８活性进行了测定。结果：慢肝患者血清ＴＮＦ－α、ＩＬ－１、ＩＬ－６、ＩＬ－８水平明显高于健康对照组（Ｐ〈０     

IL-13对肾小球系膜细胞NF-κB的抑制作用

为了观察IL 13对肾小球系膜细胞核因子 κB (NF κB )活性的影响 ,探讨IL 13抑制免疫及炎症反应的分子机制。体外培养的大鼠系膜细胞经LPS激活及不同浓度IL 13处理后 ,利用凝胶电泳迁移率试验 (EMSA )检测系膜细胞NF κB的活性。在含 5 %FCS的RPMI 1640培养状态下的肾小球系膜细胞存在一定的NF κB活性 ;LPS激活后系膜细胞NF κB活性显著增高 ;IL 13可抑制基础状态及LPS激活的系膜细胞NF κB活性。结果表明 ,IL 13可抑制体外培养系膜细胞的NF κB活性。从而抑制NF κB参与调控的细胞因子的表达而最终对系膜细胞炎症效应产生调节作用。     

低密度脂蛋白对培养的人肾小球系膜细胞的增殖作用及冬虫夏草对其的影响

目的：探讨肾小球硬化的发病机理;并发现治疗肾小球硬化的治疗方法。方法：（１）通过测定［３Ｈ］胸腺嘧啶的掺入率,检测体外培养的人肾小球系膜细胞的增殖;（２）对人肾小球系膜细胞进行纯化和培养并对低密度、高密度脂蛋白进行［１２５Ｉ］标记。结果：（１）肾小球系膜细胞选择性结合和摄取［１２５Ｉ］ＬＤＬ,（２）ＬＤＬ刺激系膜细胞增殖且增殖作用具有双向性,（３）冬虫夏草对ＬＤＬ引起的细胞增殖具有抑制作用。结论：ＬＤＬ刺激系膜细胞增殖可能直接参与肾小球硬化的发病,高脂血症对肾脏的损害可能由肾小球系膜细胞介导;抑制系膜细胞增殖可能对肾小球硬化的防治具有一定的可行性。     

NF-κB介导脂氧素A4拮抗TNF-α所致的系膜细胞白介素的合成

为研究脂氧素A4(LXA4)拮抗肿瘤坏死因子α(TNF-α)对肾小球系膜细胞的白介素(IL)-1β(IL-1β)、IL-6合成的作用。对体外培养大鼠肾小球系膜细胞,用不同浓度的LXA4预刺激,再加入TNF-α共同孵育;或单用TNF-α刺激肾小球系膜细胞。在孵育后用ELISA法检测培养上清中的IL-1β、IL-6蛋白表达量;用RT-PCR法检测IL-1β、IL-6的mRNA表达量。应用凝胶电泳迁移率试验(EMSA)测定核因子-κB(NF-κB)的DNA结合活性。结果发现,LXA4呈剂量依赖性地抑制TNF-α诱导的肾小球系膜细胞IL-1β和IL-6蛋白的合成与mRNA表达,抑制NF-κB的DNA结合活性。说明LXA4通过下调NF-κB的DNA结合活性,拮抗TNF-α对肾小球系膜细胞的IL-1β、IL-6合成的促进作用。     

Binding capacity and pathophysiological effects of IgA1 from patients with IgA nephropathy on human glomerular mesangial cells

IgA deposition in glomerular mesangium and the interaction with mesangial cells may well be the final common pathway to IgA nephropathy (IgAN). Altered hinge-region O-glycosylation of IgA1 from patients with IgAN may predispose to mesangial deposition and activation of the mesangial cell (MC) by IgA1, via a novel IgA1 receptor, and may be a key event in the pathogensis of IgAN. The aim of this study was to investigate the binding capacity and biological effects of IgA1, from both patients with IgAN and healthy controls, on human mesangial cells (HMC). Serum IgA1 was isolated with jacalin affinity chromatography, heated to aggregated form (aIgA1) and labelled with (125)I. Binding capacity of aIgA1 in vitro to cultured primary HMC was evaluated by a radioligand binding assay and the specificity of binding was determined by a competitive inhibition assay. Intracellular calcium release was studied by confocal analysis and phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis. Change of cell cycles was demonstrated by flow cytometry and HMC proliferation was evaluated by direct cell count. Expression of TGF-beta mRNA and production of supernatant fibronectin were tested by RT-PCR and indirect competitive ELISA, respectively. aIgA1 from both the patients with IgAN and normal controls bound to HMC in a dose-dependent, saturable manner, and was saturated at approximately 500 pmoles per 0.5 ml of aIgA1. aIgA1 from patients with IgAN, however, bound to HMC at a higher speed and Scatchard analysis revealed a Kd of (8.89 +/- 2.1) x 10(-8)m versus (4.3 +/- 1.2) x 10(-7)m for aIgA1 from healthy controls (P = 0.026).The binding was specific because it was only inhibited by unlabelled Mono-IgA1 (mIgA1) and not by serum albumin or IgG. aIgA1 from patients with IgAN could induce release of intracellular calcium, phosphorylation of ERK, DNA synthesis, proliferation of HMC, expression of TGF-betamRNA and secretion of fibronectin in HMC in a similar time-dependent manner as aIgA1 from healthy controls, but the effects were much stronger and the durations were much longer (P < 0.05, respectively). We conclude that aIgA1 from patients with IgAN has a higher binding capacity to HMC and stronger biological effects than aIgA1 from healthy controls. This suggests that direct interaction between IgA1 and HMC and subsequential pathophysiological responses may play an important role in the pathogenesis for IgAN.     

Differential binding characteristics of native monomeric and polymeric immunoglobulin A1 (IgA1) on human mesangial cells and the influence of in vitro deglycosylation of IgA1 molecules

Recent studies had demonstrated that serum and mesangial immunoglobulin A1 (IgA1) in patients with IgA nephropathy (IgAN) were polymeric and deglycosylated. The current study was to investigate the binding characteristics of monomeric and polymeric normal human IgA1 on mesangial cells and the influence of in vitro deglycosylation of IgA1 molecules. The normal human IgA1 was desialylated and degalactosylated with specific enzymes, respectively. The monomeric IgA1 (mIgA1) and polymeric IgA1 (pIgA1) were separated by Sephacryl S-300 chromatography. The binding capacities of the mIgA1 and pIgA1 to primary human mesangial cells (HMC) were evaluated by classical radioligand assay. Both the native mIgA1 and pIgA1 could bind to HMC in a dose-dependent and saturable manner. The maximal binding capacity of the native pIgA1 were significantly higher than that of the native mIgA1 (P < 0.05). However, the affinity of the native mIgA1 was almost 100 times higher than that of the native pIgA1. After deglycosylation, binding of the two deglycosylated mIgA1 to HMC could not be detected. However, the maximal binding capacities of the two deglycosylated pIgA1 to HMC were increased significantly compared with that of native pIgA1. The affinity of the two deglycosylated pIgA1 was similar to that of native pIgA1 (P > 0.05). The current study suggests differential binding characteristics of native monomeric and polymeric IgA1 on mesangial cells. Glycosylation of IgA1 molecules could significantly affect the binding of IgA1 on HMC.     

稳定高表达A型清道夫受体的人肾小球系膜细胞系的建立及其受体功能

目的探讨A型清道夫受体(SR-A)在人肾小球系膜细胞中的表达功能和受体特异性。方法采用脂质体转染法,将人SR-A cDNA转染入人肾小球系膜细胞,建立稳定高水平表达SR-A的人肾小球系膜细胞系(HMC-SCR)。RT-PCR法检测SR-A mRNA的表达,使用免疫荧光法、油红“O”细胞化学染色及激光共聚焦显微镜检测HMC-SCR对配体氧化低密度脂蛋白(Ox-LDL)和乙酰化低密度脂蛋白(Ac-LDL)的摄取及配体间的竞争作用。结果与未转染细胞相比,转染SR-A的HMCL对修饰低密度脂蛋白的摄取功能明显增强;40倍过量未标记的Ox-LDL、Ac-LDL竞争荧光D il标记的Ac-LDL的摄取,但未修饰的LDL对该受体无明显竞争抑制作用。结论Ox-LDL和Ac-LDL通过同一SR-A进入系膜细胞内,SR-A对修饰的低密度脂蛋白具有较高的亲和力和特异性。     

Binding capacity of in vitro deglycosylated IgA1 to human mesangial cells

IgA nephropathy (IgAN) is the most common glomerular disease and it is characterized by deposition of IgA1 molecules in mesangium. Recent studies had demonstrated that serum and mesangial IgA1 in IgAN were deglycosylated and IgA1 could bind to human mesangial cells (HMC) through a novel receptor. The aim of the current study is to investigate and compare the binding capacities of different in vitro deglycosylated IgA1 on human mesangial cells. Serum IgA1 was purified by jacalin affinity chromatography and then was desialylated (DesIgA1) and/or degalactosylated (Des/DeGalIgA1) with neuraminidase and/or beta-galactosidase. The efficacy of deglycosylations was assessed by Peanut agglutinin (PNA) and Vicia villosa (VV) lectin. The sizes of normal IgA1 and deglycosylated IgA1 were determined by Sephacryl S-300 chromatography and binding capacities to primary HMC were evaluated by radioligand binding assays. Normal IgA1 and deglycosylated IgA1 could bind to HMC in a dose-dependent, saturable manner. The maximal binding capacities and binding sites/cell of DesIgA1 and Des/DeGalIgA were significantly higher than that of normal IgA1. However, more aggregated IgA1 was found in DesIgA1 and Des/DeGalIgA1. Scatchard analysis revealed a similar Kd of normal IgA1 and deglycosylated IgA1. The current study suggested that the binding capacities of DesIgA1 and Des/DeGalIgA1 to HMC were significantly higher than that of normal IgA1, which at least in part was due to more macromolecular IgA1 in deglycoslated IgA1. However, there were no significant differences in the affinities of normal IgA1, DesIgA1 and Des/DeGalIgA1 with HMC. Deglycosylated IgA1 might play an important role in pathogenesis of IgAN.     

Low-density lipoproteins enhance transforming growth factor-beta 1 (TGF-beta 1) and monocyte chemotactic protein-1 (MCP-1) expression induced by cyclosporin in human mesangial cells.

Cyclosporin (CsA) is widely used in the treatment of renal disease and transplantation, which are often complicated by alterations of lipid metabolism. Both chronic administration of CsA and hyperlipidaemia have been shown to evoke an early macrophage influx and have progressively led to glomerular and interstitial sclerosis. MCP-1 is the major monocyte chemoattractant secreted by stimulated mesangial cells and TGF-beta 1 is a key mediator of fibrogenesis in chronic progressive renal fibrosis. Thus, the combined effect of CsA and low-density lipoprotein (LDL) on the gene and protein expression of MCP-1 and TGF-beta 1 in cultured human mesangial cells (HMC) was explored. Both agents induced an early and persistent increase of MCP-1 and TGF-beta 1 mRNA levels and protein release. The simultaneous addition of CsA and LDL did not display any additive effect on target gene expression, but it caused a synergistic effect on MCP-1 and TGF-beta 1 protein secretion into culture medium. On the other hand, CsA and LDL had different effects on cell proliferation: the latter increased DNA synthesis, whereas CsA inhibited both spontaneous and mitogen-stimulated mesangial cell growth. The study concludes that CsA and LDL display an additive effect on TGF-beta 1 and MCP-1 synthesis and release by HMC, thus possibly co-operating to induce an early macrophage influx and the subsequent mesangial expansion and increased extracellular matrix deposition. However, in contrast they seem to modulate HMC proliferation differently, which is a further critical event intimately involved in the development of glomerulosclerosis.     

IL-13抑制人肾小球系膜细胞IL-12的表达

为了探讨白细胞介素 13(IL 13)对体外培养的人肾小球系膜细胞产生白细胞介素 12 (IL 12 )的影响。我们用脂多糖(LPS 10 μg/ml) ,不同浓度的IL 13对系膜细胞培养 ,分别采用ELISA法和半定量RT PCR法检测细胞上清液的IL 12和系膜细胞IL 12p4 0mRNA表达。结果提示 5 %NCSRPMI 16 4 0基础培养条件下的系膜细胞未检测到IL 12蛋白分泌及其mRNA表达。在LPS刺激下系膜细胞的IL 12p4 0mRNA表达加强 ,并分泌出大量的IL 12。IL 13在 1～ 10 0ng/ml浓度范围内对LPS诱导的系膜细胞IL 12分泌及IL 12p4 0mRNA表达的抑制作用呈剂量依赖趋势。本研究认为IL 13可能通过抑制IL 12的产生 ,而调整了体内Th1/Th2细胞因子平衡 ,作为抗炎性细胞因子在肾小球肾炎发病机制中发挥一定作用