IL-38 及MIP-2 在大鼠肺纤维化中的作用

目的:根据IL-38及MIP-2在博来霉素致大鼠肺纤维化肺组织中的表达水平,探讨IL-38及MIP-2在大鼠肺纤维化中的意义。方法:按照随机、对照原则将45只Wistar大鼠分为生理盐水对照组(N组)、博来霉素组(B组)、地塞米松组(D组)。于第7天、14天、28天,每组处死大鼠5只,肺组织苏木精-伊红(HE)染色观察肺组织的病理动态改变,酶联免疫法(ELISA)测定大鼠肺组织IL-38、羟脯氨酸(HYP)的表达量及RT-PCR法测定大鼠肺组织MIP-2的表达量。结果:(1)HE染色提示B组及D组肺组织从正常到炎性改变再到肺纤维化形成。(2)B、D组IL-38的表达量逐渐下降,第28天时下降最明显,均低于N组,差异具有统计学意义(P0. 05); B组IL-38的表达量较同期D组均下降,差异具有统计学意义(P0. 05)。(3)B、D组MIP-2、HYP的表达量逐渐上升,均高于N组,差异具有统计学意义(P0. 05); B组的MIP-2及HYP表达量较同期D组均上升,差异具有统计学意义(P0. 05)。结论:IL-38及MIP-2在博来霉素致大鼠肺纤维化的发生、发展中有重要作用,应用地塞米松可以改善大鼠肺纤维化程度,其作用可能与上调IL-38及下调MIP-2有关。     

1,25(OH)2VD3 对大鼠肺纤维化中IL-9 及PU-1 表达水平的影响

目的:探讨肺纤维化发生发展中IL-9 及PU.1 的作用以及活性维生素D3 [1,25(OH)2 VD3 ]在纤维化过程中对两种因子表达水平的影响。方法:90 只SPF 级雄性SD 大鼠随机分成三个组:对照组、模型组和治疗组(n =30)。模型组和治疗组经气管注入博来霉素(5 mg/ kg)建立肺纤维化模型,对照组注入等体积生理盐水。治疗组于手术后第2 天腹腔注射活性维生素D3 ,模型组注射等量的活性维生素D3 溶剂(丙二醇),对照组注射等量的生理盐水。各种处理均为两天一次。分别于手术后第14、21 和28 天处死大鼠取材,各小组每个时间点10 只大鼠。HE 染色法观察各组实验大鼠肺部病理变化,Masson染色法观察胶原纤维的差异,碱水解法检测羟脯氨酸含量的变化。应用Real-time PCR 和免疫组化技术分别从mRNA 水平和蛋白质水平检测大鼠肺组织中IL-9 及PU.1 的表达,ELISA 法检测血清中IL-9 的表达水平。结果:博来霉素处理后,第14 天大鼠肺部已经出现纤维化,随着时间推移,纤维化进一步加重。在三个时间点,模型组和治疗组的羟脯氨酸含量明显高于对照组,而治疗组明显低于模型组。在三个时间点,治疗组和模型组中IL-9 及PU.1 的表达量均逐渐增高,且都显著高于对照组;第14、21 天治疗组两种因子的表达量均明显低于相应时间点的模型组,第28 天时治疗组与模型组比较差异无统计学意义(P>0.05)。第21 天模型组和治疗组IL鄄9 和PU郾1 的表达水平明显高于第14 天,第28 天与第21 天比较差异无统计学意义。结论:IL-9 和PU.1 在博来霉素引起的大鼠肺纤维化早期可能发挥促纤维化作用。活性维生素D3 可能通过降低PU.1 的表达水平,进而减少IL-9 的分泌,从而对大鼠肺纤维化的发生发展起一定的抑制作用。     

人脐带血干细胞抑制大鼠肺纤维化及肺巨噬细胞TGF-β1的表达

目的 研究人脐血间充质干细胞对博来霉素所致的大鼠肺纤维化及TGF-β1的影响.方法 取清洁级健康雄性SD大鼠60只随机分为博来霉素组(P组)、干细胞治疗组(M组)、地塞米松治疗组(D组)和阴性对照组(N组),取第2代人脐血间充质干细胞培养至第4代;P组、M组和D组分别经气管注入博来霉素制造肺纤维化模型,M组造模后立即经鼠尾静脉注入5-溴-2-脱氧尿嘧啶核苷(BrdU)标记的干细胞,D组造模后第2天开始连续7d腹腔注射地塞米松,N组经气管注入等量生理盐水,在第7、14、28天处死各组大鼠,行HE、Masson染色,免疫组织化学法观察TGF-β1表达及标记细胞情况.结果 M组第7、14、28天肺组织均可见标记的干细胞;HE及Masson染色后观察,与N组相比,P组7d时肺泡炎最明显,28 d肺纤维化程度最重,M、D组较P组轻且病理切片可见M组较D组肺泡炎及纤维化程度稍轻;肺组织中TGF-β1在P组7d时最高,M、D组明显少于P组,M组减少更明显,组间差异有统计学意义.结论 人脐血间充质干细胞可以定植于肺组织中,在肺纤维化早期通过抑制TGF-β1的表达,可能减轻肺泡炎及肺纤维化.     

博来霉素致大鼠肺纤维化模型肺组织的动态病理变化及其发生机制

目的： 了解博来霉素（BLM）致大鼠肺纤维化模型肺组织的动态病理变化,探讨BLM致肺纤维化的作用机制。方法：60只雄性SD大鼠采用随机数字表法分为正常对照组（N组）和肺纤维化模型组（B3、B7、B14、B28、B56组）,每组10只。除N组外,其余各组采用气管内注入BLM致大鼠肺纤维化模型, 分别于3、7、14、28、56 d处死各组大鼠,右肺行苏木精-依红（HE）、Masson胶原及天狼猩红染色,测定左肺羟脯氨酸（HYP）的含量。 RT-PCR法半定量测定转化生长因子-1（TGF-β1）、基质金属蛋白酶-9（MMP-9）和基质金属蛋白酶组织抑制物-1（TIMP-1）mRNA在肺内的表达。免疫组化法观察TGF-β1、MMP-9及TIMP-1蛋白在大鼠肺组织的表达。结果：(1) 模型组大鼠肺组织HYP含量显著高于N组（P<0.05）,模型组大鼠肺组织肺泡炎症的程度也明显重于N组,B14、B28和B56组大鼠肺纤维化的程度明显重于N组,大鼠在灌注BLM后不同时点其肺组织有着不同的病理变化。 (2) TGF-β1、MMP-9及TIMP-1在正常组大鼠肺脏中即有表达,但表达较弱,灌注BLM后它们的表达均增强,不同时点它们在肺组织内的分布有不同的特点。结论：给予后不同时点大鼠肺组织有着不同的病理变化特点,TGF-β1、MMP-9和TIMP-1在BLM诱导的肺纤维化形成过程中起着重要的调节作用。     

替普瑞酮诱导肺纤维化大鼠肺HSP70表达及干预肺纤维化的研究

目的:研究替普瑞酮(GGA)对博莱霉素(BLM)诱导的肺纤维化大鼠肺组织HSP70表达的影响及对大鼠肺纤维化的干预作用。方法:SD大鼠30只,随机分为假手术组(SO)、模型组(M)和替普瑞酮组(GGA)。M组和GGA组大鼠气管内一次性注射BLM 5 mg/kg,SO组大鼠气管内注射等体积的生理盐水。造模后第1天开始隔天灌胃给予GGA,持续到处死动物的前1天,模型组灌服等体积的生理盐水。记录大鼠每日体重,造模后第28天时处死大鼠,测定肺纤维化大鼠的肺系数、肺组织内HSP70表达及羟脯氨酸(HYP)的含量,观察肺组织病理改变。结果:GGA能提高博莱霉素处理后大鼠肺组织HSP70的表达(P<0.01);与M组相比,大鼠体重下降得到明显恢复(P<0.01),而肺组织的肺系数和羟脯氨酸(HYP)含量则明显降低(P<0.05),病理结果显示肺泡内结构完整,未出现类似M组肺组织实变现象,肺纤维化程度较轻。结论:替普瑞酮能诱导BLM肺纤维化模型大鼠肺组织HSP70表达,减轻肺纤维化程度。     

Th17细胞在肺纤维化小鼠中的动态变化及作用机制

目的:研究Th17细胞在博来霉素致肺纤维化小鼠中的动态变化及作用机制。方法:小鼠随机分为对照组、博来霉素组和全反式维甲酸组。博来霉素组和维甲酸组利用博来霉素建立小鼠肺纤维化模型,维甲酸组在造模第0天每只腹腔注射全反式维甲酸。评价小鼠肺组织病理形态变化;检测肺组织中羟脯氨酸含量和维甲酸相关孤儿受体(RORγt)mRNA的表达;应用流式细胞术分析脾中Th1、Th2、Th17细胞的动态变化。结果:博来霉素组和维甲酸组羟脯氨酸(HYP)含量逐渐升高,博来霉素组第7、14、28天高于对照组和维甲酸组,第28天时达最高;博来霉素组、维甲酸组在4个时间点与对照组比较,Th17、Th2细胞均明显增高,Th1细胞低于对照组,RORγt mRNA表达水平上调。其中维甲酸组Th17、Th2细胞及RORγt mRNA表达水平低于博来霉素组,Th1细胞高于博来霉素组。结论:Th17细胞可能参与特发性肺纤维化的发病机制,并通过调节Th1、Th2细胞分化来发挥作用。     

IL-17A促进博来霉素诱导的肺纤维化大鼠肺组织的炎症形成

目的研究IL-17A在肺纤维化发病机制中的作用。方法 20只雌性Wistar大鼠,随机分为生理盐水(NS)组和博来霉素(BLM)组,NS组大鼠气管内灌注NS,BLM组大鼠气管内灌注BLM,两组分别于气管内灌注药物后第7天和第28天各处死一半动物。HE和Masson染色观察肺组织病理变化;免疫组织化检测肺组织IL-17A表达;收集支气管肺泡灌洗液(BALF),一部分行细胞数测定并进行分类分析,ELISA检测BALF中IL-17A的含量,另外一部分行分离、纯化得到肺泡巨噬细胞(AM),对其进行培养得到AM培养上清液(AMS),ELISA检测上清液IL-17A的含量,反转录PCR(RT-PCR)检测AM IL-17A mRNA表达。结果与NS组相比,BLM第7天组肺泡炎较明显,BALF中细胞总数增加,AM减少、中性粒细胞增加;第28天组肺泡炎减轻,而肺纤维化程度较重。与NS组比较,BLM第7天组和第28天组肺组织IL-17A表达明显增加(P0.05),第28天较第7天的表达有所降低。与NS组比较,BLM组BALF中细胞总数第7天明显增高(P0.05),第28天恢复至正常水平;BLM组BALF中IL-17A含量第7天和第28天明显增高,但第28天时较第7天降低;与NS组比较,BLM第7天组和第28天组AM培养前12 h、前24 h,前48 h上清液中IL-17A的含量均明显增加(P0.05);RT-PCR结果显示,与NS组比较,BLM第7天组和第28天组各时间点AM IL-17A mRNA表达均明显增加(P0.05)。结论 IL-17A促进了肺纤维化大鼠肺组织的炎症形成,进而参与了肺纤维化。     

甘草甜素抑制肺纤维化大鼠肺组织单核细胞趋化蛋白-1的表达

目的: 观察甘草甜素对博来霉素(BLM)诱导的大鼠肺纤维化的干预作用及可能机制。方法: 随机将大鼠分为对照组、肺纤维化模型组、甘草甜素干预组。气管内注入博莱霉素造成动物模型后,于当天开始每天给药,分别于7 、28 d处死,取肺组织,行嗜伊红染色、Masson染色；检测肺组织匀浆中羟脯氨酸(HYP) 的变化; RT-PCR法测肺组织单核细胞趋化因子-1 mRNA的表达,免疫组化测肺组织单核细胞趋化因子-1蛋白的表达。结果: 干预组肺纤维化程度轻于模型组; 肺组织匀浆羟脯氨酸(HYP)含量显著低于模型组( P<0.01) ;在模型组 MCP-1第7 d表达就明显升高，第28 d下降，但仍高于对照组，干预组与模型组有同一规律，但均减弱，第7、28 d与模型组(M组)比较，P<0.05。结论: 甘草甜素能减轻博莱霉素诱导的大鼠肺纤维化，这种作用可能部分是通过抑制MCP-1的表达而实现。     

博来霉素致大鼠肺纤维化后神经营养因子4和酷氨酸激酶受体B表达及其意义

目的:探讨博来霉素致大鼠肺纤维化后神经营养因子4(NT4)和酷氨酸激酶受体B(Trk B)的表达及其意义。方法:用36只健康雄性SD大鼠,分为对照组和模型组,每组18只。对照组大鼠为气管内注射生理盐水,模型组大鼠按要求用博来霉素进行气管内灌注。两组大鼠分别于造模后第3、7和14 d各处死6只,取出肺组织,分别用Masson和HE染色检测肺纤维化程度。对照组和模型组大鼠中NT4及Trk B的含量用PCR和免疫组化检测,结果进行统计学分析。结果:与对照组比较,模型组造模后第3、7和14 d NT4和Trk B含量都有不同程度升高,并且随肺纤维化的严重程度而增高(P0.05),同时NT4和Trk B二者含量的升高呈正相关(r=0.568,P0.05)。结论:实验大鼠肺纤维化其肺组织中NT4和Trk B表达明显增加,表明NT4和Trk B可能参与肺纤维化的发生发展。这种改变可能与肺上皮细胞增生和Trk B/NT4轴信号传递受影响有关。     

绿色荧光蛋白标志的同种异体骨髓间充质干细胞对博来霉素致大鼠肺组织纤维化损伤的影响

目的制备并用绿色荧光标志骨髓间充质干细胞(BM⁃MSC),观察其对博来霉素所致大鼠肺纤维化损伤的影响。方法60只雄性SD大鼠随机分为5组,A组于气管内注入生理盐水,B、C、D、E组于气管内注入5 mg/kg博来霉素0.1 ml。建模后1 d,A、B组经尾静脉注射生理盐水1.0 ml,C组经尾静脉注射1.0 ml BM⁃MSC(1.0×106细胞),D组尾静脉注射1.0 ml BM⁃MSC(5.0×10^(5)细胞),建模后8 d,再给1.0 ml BM⁃MSC(5.0×10^(5)细胞)。E组尾静脉注射1.0 ml BM⁃MSC(1.0×107细胞)。建模后28、42 d处死各组一半大鼠,留取肺组织。行病理学检查,对HE染色切片评定肺组织肺泡炎程度,对Masson染色切片评定肺组织纤维化程度;测定肺组织的转化生长因子⁃β1(TGF⁃β1)、羟脯氨酸(HYP)、基质金属蛋白酶⁃2(MMP⁃2)和组织金属蛋白酶抑制剂⁃1(TIMP⁃1)的表达水平,分析TGF⁃β1、HYP、MMP⁃2/TIMP⁃1与肺泡炎程度及肺纤维化程度评分的相关性。结果A组肺泡结构正常,无明显的炎症或纤维化改变,B组博来霉素可致肺泡结构严重紊乱,大量胶原纤维沉积,与B组比较,C、D、E组注射BM⁃MSC后肺泡炎性和肺纤维化程度较轻。与A组比较,B组肺泡炎程度评分、肺纤维化程度评分、TGF⁃β1、HYP、MMP⁃2/TIMP⁃1水平增高(均P<0.05),MSC治疗后的C、D、E 3组可使上述指标下降(均P<0.05)。肺组织TGF⁃β1与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.82、0.89,HYP与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.86、0.92,MMP⁃2/TIMP⁃1与肺泡炎程度评分、肺纤维化程度评分相关系数r=0.83、0.92,均存在正相关(均P<0.05)。结论BM⁃MSC可减低博来霉素致肺纤维化损伤,较高剂量和分次移植效果较好,其机制与移植细胞长期存活无关,可能与下调TGF⁃β1水平、改善MMP/TIMP失衡有关。     

The extended IL-10 superfamily: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29

Cytokines are involved in virtually every aspect of immunity and inflammation. A cascade of responses evolves after cytokine activation, although optimal function might ultimately involve several complementary cytokines. Understanding the function of individual cytokines is complicated because their role can vary depending on the cellular source, target, and phase of the immune response. In fact, numerous cytokines have both proinflammatory and anti-inflammatory potential, with the contrasting outcome observed being determined by the immune cells present and their state of responsiveness to the cytokine. These issues make the study of cytokine biology daunting, particularly so for IL-10 and IL-10-related genes. The IL-10 superfamily is highly pleiotropic. These genes are linked together through genetic similarity and intron-exon gene structure. Significant commonality exists not only through shared receptors but also through conserved signaling cascades. However, its members mediate diverse activities, including immune suppression, enhanced antibacterial and antiviral immunity, antitumor activity, and promotion of self-tolerance in autoimmune diseases.     

IL-10 subfamily members: IL-19, IL-20, IL-22, IL-24 and IL-26

It has been reported that the CD4+ T cell is a very important source of interleukin 10 (IL-10), while CD8+ cells produce low amounts. IL-10 exerts several immune stimulating, as well as inhibitory effects. There are at least five novel human IL-10 family-related molecules: IL-19, IL-20, IL-22, IL-24, and IL-26. Activated T cells produce IL-19, IL-22 and IL-26, while IL-24 is produced by activated monocytes and T-cells. IL-20 induces cheratin proliferation and Stat-3 signal transduction pathway, while IL-22 induces acute-phase production by hepatocytes and neonatal lethality with skin abnormalities reminiscent of psoriasic lesions in humans. In addition, IL-22 mediates inflammation and binds class II cytokine receptor heterodimers IL-22 RA1/CRF2-4. This cytokine is also involved in immuno-regulatory responses. IL-26 (AK155) is a novel cytokine generated by memory cells and is involved in the transformed phenotype of human T cells after infection by herpes virus. All these new IL-10 subfamily member cytokines are strongly involved in immune regulation and inflammatory responses.     

IL-21 and IL-21 receptor

Interleukin (IL)-21 is a new member of the type I cytokine superfamily. Although it is most homologous to IL-15, it has a unique receptor chain, IL-21R, that pairs with the γ-common cytokine receptor chain. The first experiments examining the biology of the IL-21 pathway reveal that it is a cytokine with effects on natural killer (NK) cells, T cells, and B cells. Mice deficient in the IL-21 R have also been made, and are being examined for the effects of the IL-21/IL-21R pathway in vivo. Here we summarize our current knowledge of this new cytokine pathway, and its role in innate and adaptive immunity.     

Ovarian follicular concentration of IL-12, IL-15, IL-18 and p40 subunit of IL-12 and IL-23

BACKGROUND: The aim of the study was to determine the presence of interleukin (IL)-12, IL-15, IL-18 and p40 subunit of IL-12/IL-23 in follicular fluid from spontaneous cycles and the relation between the concentration of selected cytokines and IVF-embryo transfer outcome. METHODS: IVF-embryo transfer and enzyme immunoassay (EIA) (R&D Systems, Minneapolis, MN, USA and MBL, Nagoya, Japan) were used. RESULTS: Follicular fluid of women included in the IVF-embryo transfer procedure contained common p40 subunit of IL-12/IL-23 (median 70.1 pg/ml), IL-15 (median 1.3 pg/ml) and IL-18 (median 38.2 pg/ml). There was a significant negative correlation between follicular fluid concentrations of IL-15 and IL-18 (R=-0.392, P=0.003). Significantly higher concentrations of common p40 subunit of IL-12/IL-23 (median 79.8 pg/ml) were found in the follicular fluid taken from follicles containing oocytes, when compared with those without an oocyte (median 44.5 pg/ml, P=0.006). Patients who achieved clinical pregnancy had significantly decreased concentration of IL-15 (median 0.8 pg/ml) compared with patients without successful IVF-embryo transfer outcome (median 1.4 pg/ml, P=0.047). CONCLUSION: Follicular fluid collected from spontaneous cycles contains detectable levels of p40 subunit of IL-12/IL-23, IL-15 and IL-18. Increased concentrations of p40 subunit of IL-12/IL-23 in follicles containing oocytes suggest an important role of this cytokine in reproduction. Possible negative value of IL-15 as a predictor of IVF-embryo transfer success remains to be determined.     

A tissue specific IL-1 receptor antagonist homolog from the IL-1 cluster lacks IL-1, IL-1ra, IL-18 and IL-18 antagonist activities

Interleukin (IL)-1-like protein 1 (IL-1L1) is a 155-amino acid protein that shares 27% identity with IL-1beta and 47% with IL-1 receptor antagonist (IL-1ra). A 2.7-kb IL-1L1 mRNA was cloned from human placenta and is detectable in the trophoblastic cell line JEG-3, in macrophages and in endotoxin-stimulated monocytes. Expression of IL-1L1 is much less abundant and less widespread than IL-1ra. We have determined the human and mouse IL-1L1 cDNA sequences and the complete sequence of the human gene, IL1L1. IL1L1 consists of four coding exons, has two alternative non-coding first exons, lies between IL1B and IL1RN, is orientated in the same direction as IL1RN and is separated from it by approximately 53 kb. The predicted IL-1L1 protein lacks both signal sequence and glycosylation signals. A 17-kDa protein was recovered by immunoprecipitation with IL-1L1-specific antibodies from JEG-3. IL-1L1 did not stimulate IL-6 production from primary human fibroblasts or human umbilical vein endothelial cells nor did it block the IL-1alpha or IL-1beta-dependent activation of IL-6 expression. We conclude, contrary to a recent suggestion made by others, that IL-1L1 is not a functional IL-1ra. IL-1L1 also had no detectable agonistic or antagonistic effect on IFN-gamma production in response to IL-18 in KG-1 cells.     

IL-1, IL-18, and IL-33 families of cytokines

Summary: The interleukin-1 (IL-1), IL-18, and IL-33 families of cytokines are related by mechanism of origin, receptor structure, and signal transduction pathways utilized. All three cytokines are synthesized as precursor molecules and cleaved by the enzyme caspase-1 before or during release from the cell. The NALP-3 inflammasome is of crucial importance in generating active caspase-1. The IL-1 family contains two agonists, IL-1α and IL-1β, a specific inhibitor, IL-1 receptor antagonist (IL-1Ra), and two receptors, the biologically active type IL-1R and inactive type II IL-1R. Both IL-1RI and IL-33R utilize the same interacting accessory protein (IL-1RAcP). The balance between IL-1 and IL-1Ra is important in preventing disease in various organs, and excess production of IL-1 has been implicated in many human diseases. The IL-18 family also contains a specific inhibitor, the IL-18-binding protein (IL-18BP), which binds IL-18 in the fluid phase. The IL-18 receptor is similar to the IL-1 receptor complex, including a single ligand-binding chain and a different interacting accessory protein. IL-18 provides an important link between the innate and adaptive immune responses. Newly described IL-33 binds to the orphan IL-1 family receptor T1/ST2 and stimulates T-helper 2 responses as well as mast cells.     

支气管哮喘病人胸导管淋巴液与血清IL-6、IL-8、 IL-10、IL-12、TNF-α水平的对比研究

目的：探讨哮喘病人胸导管淋巴液和血清IL-6、IL-8、IL-10、IL-12及TNF-α水平变化。方法：采用酶联免疫吸附试验（ELISA）检测31例行胸导管引流治疗的中、重度哮喘病人术后0 d、3 d、5 d淋巴液及0 d、5 d血清IL-6、IL-8、IL-10、IL-12以及TNF-α水平。结果：哮喘病人胸导管引流淋巴液中IL-6、IL-8、IL-10、TNF-α水平均高于正常对照组血清水平，而IL-12则明显低于正常血清水平；引流5 d淋巴液中IL-6、IL-8、IL-10及TNF-α明显低于，IL-12则显著高于引流0 d时水平；同时哮喘病人血清IL-10、TNF-α含量低于，血清IL-12则高达正常对照组水平。结论：哮喘病人淋巴液中存在以IL-12产生受抑和炎性细胞因子产生亢进为特征的细胞因子失调，且淋巴液中CK变化与血清变化大致平行。     

IL-4、IL-13蛋白表达及抗IL-4、IL-13人源单链抗体的筛选

目的:表达IL-4和IL-13蛋白,从人源单链抗体文库中分别筛选抗IL-4和抗IL-13单链抗体.方法:采用RT-PCR从健康志愿者外周血单核细胞(PBMC) mRNA中扩增IL-4和IL-13 cDNA;构建硫氧还蛋白融合表达载体,转化大肠杆菌BL21,IPTG诱导表达并对表达产物进行纯化鉴定.以生物素化的IL-4和IL-13为抗原从前期构建的人源抗体文库中采用噬菌体展示技术分别筛选抗IL-4和抗IL-13人源单链抗体(scFv).结果:扩增的IL-4 cDNA大小为280 bp,表达的融合蛋白大小为27 kD左右.扩增的IL-13 cDNA大小为252 bp,表达的融合蛋白大小为25 kD左右.分别以生物素化的IL-4和IL-13蛋白为抗原,采用噬菌体展示技术对人源抗体文库进行3轮富集后,分别有大约37％的scFvs与IL-4有结合特性,有约27％的scFvs与IL-13有结合特性.筛选了4株分别与IL-4和IL-13结合能力强的单链抗体进行了Westem blot鉴定和测序.结论:成功筛选到抗IL-4和抗IL-13人源性单链抗体.     

Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma

Allergen-reactive T helper type-2 (Th2) cells and proinflammatory cytokines have been suggested to play an important role in the induction and maintenance of the inflammatory cascade in allergic asthma. We compared the plasma concentrations of novel proinflammatory cytokines IL-17 and IL-18, other proinflammatory cytokines IL-6 and IL-12, Th2 cytokines IL-10 and IL-13, and intracellular interferon-gamma (IFN-gamma) and IL-4 in Th cells of 41 allergic asthmatics and 30 sex- and age-matched health control subjects. Plasma cytokines were measured by enzyme-linked immunosorbent assay. Intracellular cytokines were quantified by flow cytometry. Plasma IL-18, IL-12, IL-10, IL-13 concentrations were significantly higher in allergic asthmatic patients than normal control subjects (IL-18: median 228.35 versus 138.72 pg/ml, P < 0.001; IL-12: 0.00 versus 0.00 pg/ml, P = 0.001; IL-10: 2.51 versus 0.05 pg/ml, P < 0.034; IL-13: 119.38 versus 17.89 pg/ml, P < 0.001). Allergic asthmatic patients showed higher plasma IL-17 and IL-6 concentrations than normal controls (22.40 versus 11.86 pg/ml and 3.42 versus 0.61 pg/ml, respectively), although the differences were not statistically significant (P = 0.077 and 0.053, respectively). The percentage of IFN-gamma-producing Th cells was significantly higher in normal control subjects than asthmatic patients (23.46 versus 5.72%, P < 0.001) but the percentage of IL-4 producing Th cells did not differ (0.72 versus 0.79%, P > 0.05). Consequently, the Th1/Th2 cell ratio was significantly higher in normal subjects than asthmatic patients (29.6 versus 8.38%, P < 0.001). We propose that allergic asthma is characterized by an elevation of both proinflammatory and Th2 cytokines. The significantly lower ratio of Th1/Th2 cells confirms a predominance of Th2 cells response in allergic asthma.     

Polymorphisms of IL-1B, IL-1RN, IL-2, IL-4, IL-6, IL-10, and IFN-gamma genes in the Korean population

Cytokines play a crucial role in regulating the immune and inflammatory responses. The collective influence of several cytokines can regulate immune responses as complex as those underlying allograft rejections or autoimmune diseases. Polymorphisms in the regulatory regions of the cytokine genes may influence their expression. Therefore, the polymorphisms of cytokine genes are potentially important as genetic predictors of the disease susceptibility or clinical outcome. In 311 unrelated healthy Korean individuals, we investigated the polymorphisms of cytokine genes (interleukin-1 [IL-1], IL-2, IL-4, IL-6, IL-10, and interferon-gamma [IFN-gamma]), which had been previously reported to be associated with a number of immune diseases, transplant complications, and direct or indirect influences on the level of expression and production. And we also compared the results to those published for other populations. The genotype distributions were consistent with the assumption of the Hardy-Weinberg equilibrium, with the exceptions of IL-1B +3954 and IL-6-174 polymorphisms. The polymorphisms examined in this study were almost similar to that observed in Asian populations. There were significant differences of the polymorphisms, except for IL-4 receptor alpha +1902, between Korean and other populations. Comparing the alleles associated with higher level of expression and production, IL-1B +3954*T, IL-2-330*G, and IL-4-590*T alleles were significantly higher, and IL-1RN*A2, IL-10-1082*G, and IFN-gamma*2 alleles were lower in Koreans than other populations. Especially in IL-6 promoter -174 polymorphism, we found only the G allele associated with higher plasma IL-6 levels. In haplotype analysis of IL-10 promoter polymorphisms, the GCC haplotype, associated with higher expression of IL-10, was significantly lower in Koreans. These results may be helpful for understanding transplant-related complications, immune or autoimmune diseases, and malignant diseases in the Korean population.