重组白细胞介素-17A鼻黏膜免疫抗肺炎链球菌感染的实验研究

目的 观察小鼠重组白细胞介素-17A (rIL-17A)鼻腔黏膜免疫对感染肺炎链球菌(Streptococcus pneumoniae,Sp)小鼠防御素β-2(Defb2)、巨噬细胞炎症蛋白(MIP)等表达的影响,探讨rIL-17A抗肺炎链球菌感染的机制.方法 SPF级BALB/c小鼠随机分为3组:肺炎组、干预组、对照组,以鼻腔接种的方法建立肺炎模型,采用鼻黏膜免疫的方法进行rIL-17A干预.以real-time荧光定量PCR法检测肺组织Defb2、MIP-1α、MIP-2β mRNA的表达,ELISA法检测支气管肺泡灌洗液(BALF)、脾细胞以及纵膈淋巴结细胞培养上清中MIP-1α、MIP-2β、IFN-γ、IL-4的浓度,并对BALF进行白细胞分类计数和Sp菌落计数,对肺组织进行病理分析.结果 rIL-17A干预组BALF中Sp菌落数较肺炎组明显降低(2.18±0.94 vs 4.37+0.57,P＜0.01),中性粒细胞及巨噬细胞数量显著高于肺炎组(P＜0.01);干预组肺组织Defb2、MIP-1α mRNA表达上调,其中Defb2 mRNA表达量为对照组的53.93倍,与肺炎组比较差异有统计学意义(53.93±4.80 vs 14.49±5.84,P＜0.01);rILL-17A干预组与肺炎组比较,淋巴结细胞培养上清中MIP-1α浓度增高明显(431.80±31.57 vs 291.10±5.62,P＜0.01);MIP-2β浓度在脾细胞和淋巴结细胞培养上清中较肺炎组显著增高(246.20±11.50 vs 183.70±10.64,508.50+20.26 vs 290.90+15.20,P＜0.01),而肺泡灌洗液中浓度变化不显著(P＞0.05);IFN-γ浓度在BALF和细胞培养上清中均较肺炎组显著升高;ILM除淋巴结细胞培养上清中外,BALF和脾细胞培养上清中均较肺炎组显著升高(92.42±3.82 vs 80.68±4.83,106.80±8.07 vs 73.57+7.43,P＜0.01);干预组与肺炎组小鼠支气管及血管周围炎症细胞浸润无显著差异,但干预组组织损伤较轻.结论rIL-17A可通过促进Ddb2以及MIP、IFN-y、IL-4等炎症因子的表达,增加炎症部位白细胞募集,提高Sp清除率来增强肺炎小鼠的抗感染能力.     

重组白细胞介素13诱导成纤维细胞分泌巨噬细胞炎症蛋白1α

目的 观察重组白细胞介素13对3T3成纤维细胞巨噬细胞炎症蛋白-1αmRNA表达的影响.方法 分别用DMEM(对照组)和40mg/ml、80ng/ml重组白细胞介素13(干预组)培养液作用于3T3成纤维细胞,24h和48h后MTT法观察细胞生长情况,用细胞增殖率表示;两组细胞24h后原位杂交方法检测巨噬细胞炎症蛋白-1...     

白细胞介素17 在狼疮性肾炎小鼠中的表达及抗白细胞介素17抗体的干预作用

 目的:探讨白细胞介素17（IL-17）在狼疮性肾炎(LN）小鼠中的表达及抗IL-17抗体的干预作用。方法:11周龄的雌性MRL/lpr小鼠36只随机分为实验组和干预组,另有同龄雌性昆明小鼠18只为对照组。干预组每只小鼠腹腔注射抗小鼠IL-17多克隆抗体20 μg,每2周1次,至实验观察点结束。各组分别于第1次给药后的24 h、14 d及28 d处死6只小鼠。光镜下观察肾脏病理情况,ELISA法检测小鼠血清IL-17的含量,免疫组化法检测肾组织IL-17的表达水平。结果:MRL/lpr小鼠肾小球系膜细胞及基质弥漫增生,肾小管上皮细胞颗粒及空泡变性,灶状萎缩,肾间质淋巴及单核细胞浸润伴纤维化;而干预组肾脏病理改变较实验组为轻。MRL/lpr小鼠的血清IL-17含量在实验组各时点均显著高于对照组（P<0.05）,而在干预组各时点均显著低于实验组（P<0.05）。IL-17在实验组小鼠肾小管上皮细胞中的表达明显增强,在各时点均显著高于对照组（P<0.05）;在干预组,IL-17在各时点的表达均显著低于实验组（P<0.05）。结论:IL-17在MRL/lpr小鼠血清与肾组织中的表达显著增加,而抗IL-17抗体可通过抑制IL-17的表达而抑制LN的炎症免疫反应,减轻肾脏病理损害。     

低剂量白细胞介素17A对糖尿病肾病大鼠肾功能保护作用的研究

目的 观察并探讨小剂量白细胞介素17A对糖尿病大鼠肾脏的保护作用.方法 雄性SD大鼠45只,随机分3组:正常对照组、DN组及IL-17A组.DN组及IL-17A组均以高脂高糖饮食联合腹腔注射链脲佐菌素建立糖尿病肾病大鼠模型.IL-17A组给予小剂量IL-17A重组蛋白,连续给药12周后,下腔静脉取血,检测糖化血红蛋白、肌酐、尿素氮等;将大鼠放入水平代谢笼中收集各组大鼠24h尿液,测定大鼠24h尿蛋白;实时荧光定量PCR检测肾组织炎症因子表达;Western blot测定IL-17A蛋白表达情况.结果 DN组及IL-17A组尿蛋白、HbA1c、肌酐、尿素氮均显著高于正常对照组(P＜0.05);IL-17A组尿蛋白、肌酐、尿素氮均显著低于DN组(P＜0.05).DN组大鼠在注射IL-17A后促炎因子表达显著减少,抑制性炎症因子表达显著降低.糖尿病肾病模型大鼠IL-17A蛋白显著减少.结论 IL-17A具有改善糖尿病肾病大鼠肾功能作用,并有可能逆转糖尿病肾病,是治疗糖尿病肾病的有效靶点.     

IL-17A通过上调ABCA1的表达减少RAW264.7巨噬细胞脂质的积聚

目的:探讨白细胞介素17A(IL-17A)对RAW264. 7巨噬细胞三磷酸腺苷结合盒转运体A1(ABCA1)表达的影响。方法:用不同浓度及的IL-17A处理小鼠RAW264. 7细胞6 h或24 h,或用同一浓度IL-17A处理RAW264. 7细胞不同时间;采用RT-qPCR及Western blot法检测细胞ABCA1的mRNA和蛋白表达情况;采用NBD-胆固醇法检测细胞胆固醇流出情况;采用油红O染色法测定细胞脂质蓄积情况。结果:与对照组相比,IL-17A可以增加RAW264. 7巨噬细胞ABCA1蛋白的表达水平,但不影响ABCA1 mRNA的表达。经IL-17A干预,RAW264. 7巨噬细胞内胆固醇向Apo A-1流出增多,同时细胞脂质蓄积显著减少(P 0. 01)。结论:IL-17A可以增加RAW264. 7巨噬细胞ABCA1的表达水平,其机制并不是通过转录水平实现的,这种影响可能与其抗动脉粥样硬化作用有关。     

Th17细胞及白细胞介素17A在慢性气道炎症性疾病中的作用

长期以来,人们对于T淋巴细胞的研究集中在辅助性T细胞1型、2型(Th1、Th2)、调节性T细胞(Treg)以及细胞毒性T细胞(Tc)等亚群上。传统理论认为,Th1细胞介导细胞免疫,在抗胞内菌感染的过程中发挥作用;而Th2细胞介导体液免疫,与过敏性疾病以及抗寄生虫感染的过程紧密相关。Th1/Th2失衡被认为是许多疾病产生和发展的重要因素。     

小鼠白细胞介素17A的克隆、表达及活性鉴定

目的:克隆小鼠白细胞介素17A(mIL-17A)基因,构建mIL-17A原核表达质粒在E.coil Rosetta(DE3)PlysS表达,得到有活性的mIL-17A蛋白。方法:提取人IL-1β免疫的小鼠胸腺总RNA,反转录成cDNA,以此为模板,根据GenBank报道的mIL-17A序列设计引物,进行巢式PCR,得到成熟mIL-17A的编码序列并构建到pMD18-T载体中,再亚克隆至原核表达载体pHisSUMO Express中,经DNA测序鉴定后转化Rosetta感受态细胞,IPTG诱导表达,Western blot鉴定。纯化后的蛋白处理3T3-L1前体脂肪细胞,real time PCR鉴定白细胞介素6(IL-6)的表达变化。结果:DNA测序证明所克隆基因序列与GenBank报道的完全一致。成功构建了pHisSUMO Express-mIL-17A原核表达质粒以包涵体形式表达了重组mIL-17A蛋白,且Western blot证实为目的蛋白。所得蛋白上调3T3-L1细胞表达IL-6。结论:获得具有生物活性的mIL-17A蛋白,为进一步研究其蛋白特性及其生物活性奠定基础。     

IL-17A在急性呼吸窘迫综合征肺泡炎症中的表达及意义

为探讨急性呼吸窘迫综合征肺泡炎症中IL-17A的表达及意义,选择急性呼吸窘迫综合征肺泡炎症患者98例,根据病情严重程度分为A组(轻度,36例)、B组(中度,32例)及C组(重度,30例),均进行IL-17A水平及中性粒细胞计数检测,比较3组患者血清IL-17A水平、中性粒细胞计数、急性生理学与慢性健康状况评分系统Ⅱ(acute physiology and chronic health evaluation scoring systemⅡ,APACHEⅡ)评分、序贯器官功能衰竭(sequential organ failure assessment,SOFA)评分及治疗前后血清IL-17A水平,分析不同预后患者血清IL-17A水平及其价值。结果显示,C组患者血清IL-17A水平、中性粒细胞计数、APACHEⅡ评分及SOFA评分均高于A组和B组,且B组高于A组(P0.05);3组患者治疗后血清IL-17A水平均低于治疗前,但C组血清IL-17A水平仍高于A组和B组,且B组高于A组(P0.05);经Pearson分析,IL-17A水平与APACHEⅡ评分、SOFA评分呈正相关(P0.05);经28 d随访,98例患者中存活60例,死亡38例,存活者血清IL-17A水平明显低于死亡者(P0.05);经受试者工作特征(receiver operating characteristic,ROC)曲线分析,AUC为0.859。研究提示,急性呼吸窘迫综合征肺泡炎症患者IL-17A的表达水平升高,动态监测IL-17A水平可判断病情进展及预后。     

β淀粉样蛋白诱导的阿尔茨海默病模型大鼠脑内骨桥蛋白和白细胞介素17表达的变化及相关性分析

目的 探讨骨桥蛋白(OPN)和白细胞介素17(IL-17)在β淀粉样蛋白(Aβ)神经毒性及阿尔茨海默病(AD)病理机制中的作用.方法 36只成年雄性SD大鼠随机分为3组:假手术组(Sham,S),AD模型7天组(AD7d),AD模型14d组(AD14d).Morris水迷宫测试各组大鼠认知功能;酶联免疫吸附试验(ELISA)检测各组大鼠脑脊液和血清IL-17和OPN含量变化.结果 Morris水迷宫显示与S组(12.27±3.28)比较,AD7d组(33.12±7.76)和AD14d组(45.03±6.85)大鼠潜伏期均显著延长(P =0.002、P=0.000),AD 14 d组比AD7 d组潜伏期显著延长(P=0.000).与S组(37.72±6.46)比较,AD7d组(31.48±6.18)和AD14d组(28.55±9.22)大鼠目标象限停留时间百分比显著减少(P=0.005、P=0.031).与S组(9.47±1.15)比较,AD7d组(13.35±1.74)和AD14d组(15.86±2.13)大鼠脑脊液IL-17含量显著增加(P=0.000、P=0.000).与AD7d组比较,AD14d组大鼠脑脊液IL-17含量显著增加(P=0.002).与S组(8.44 ±0.92)比较,AD7d组(10.38±2.15)和AD14d组(11.64±1.60)大鼠血清IL-17含量显著增加(P =0.033、P=0.000).与S组(5.90±0.81)比较,AD7d组(18.51±7.57)和AD14d组(41.54±6.68)大鼠脑脊液OPN含量显著增加(P=0.000、P=0.000).与AD7d组比较,AD14d组脑脊液OPN含量显著增加(P=0.000).与S组(176.54±23.69)比较,AD7d组(227.15±20.56)和AD14d组(302.19±12.19)大鼠血清OPN含量显著增加(P=0.000、P=0.000).与AD7d组比较,AD14d组血清OPN含量显著增加(P=0.000).AD7 d组和AD14 d组脑脊液和血清中IL-17和OPN含量变化均呈显著正相关(P=0.000).结论 Aβ1-42脑室内注射能够引起大鼠脑脊液、外周血IL-17和OPN含量增加,且脑脊液和血清中IL-17和OPN含量在AD大鼠模型中呈明显的正相关.     

白细胞介素-17参与固有免疫应答的研究进展

白细胞介素(IL)-17是一类重要的细胞因子,通常认为IL-17主要由Th17细胞分泌,参与机体的适应性免疫应答.近年来大量研究表明,在肺、胃肠黏膜及皮肤等屏障组织中存在多种固有免疫细胞可以分泌IL-17,作为的机体免疫系统第一道防线的重要成员,一方面通过模式识别受体迅速感知外来抗原的刺激,趋化募集到损伤部位,不经克隆扩增即可发挥清除病原,参与炎症、应激或者超敏反应的作用.另一方面,分泌IL-17的固有免疫细胞能够与慢性炎症的记忆细胞相互作用,启动适应性免疫应答,发挥调节机体免疫稳态的功能.     

Interleukin-17A is required to suppress invasion of Salmonella enterica serovar Typhimurium to enteric mucosa

Salmonella enterica serovar Typhimurium (S. typhimurium) causes a localized enteric infection and its elimination is dependent on a T helper type 1 immune response. However, the mechanism of the protective immune response against the pathogen in gut‐associated lymphoid tissue (GALT) at an early stage of the infection is not yet clarified. Here, we show that interleukin‐17A (IL‐17A) was constitutively expressed in GALT; it was also detected on crypt and epithelial cells of the small intestine. Neutralization of the IL‐17A in the intestinal lumen exacerbated epithelial damage induced by intestinal S. typhimurium infection at an early stage of the infection. The result suggests that IL‐17A has a pivotal role in the immediate early stage of protection against bacterial infection at the intestinal mucosa. As IL‐17A neutralization also suppressed the constitutive localization of β‐defensin 3 (BD3), an IL‐17A‐induced antimicrobial peptide, at the apical site of the intestinal mucosa, it is estimated that IL‐17A constitutively induces the expression of the antimicrobial peptide to kill invading pathogens at the epithelial surface immediately after the infection. In contrast, interferon‐γ is induced around 3 days after S. typhimurium infection, and its expression level increases thereafter. Taken together, the findings lead to the hypothesis that IL‐17A participates in the immediate early stage of protection against S. typhimurium intestinal infection whereas interferon‐γ is important at a later stage of the infection.     

The polymorphisms of interleukin 17A (IL17A) gene and its association with pediatric asthma in Taiwanese population

Background:  The interleukin 17A ( IL17A ) gene, located on chromosome 6p and linked to asthma phenotype, is a highly potential candidate gene conferring asthma susceptibility. The purpose of this study was to investigate the genetic association between single nucleotide polymorphisms (SNPs) of IL17A and asthma in Taiwanese children.
Methods:  We selected and performed genotyping on nine SNPs that encompass the genomic region of IL17A in Taiwanese children with or without asthma. A total of 1939 subjects containing 1027 subjects in testing group and 931 subjects in validation group were recruited in this study.
Results:  After Bonferroni correction, SNP rs8193036 was found to have a weak association ( P  = 0.0074 × 9 = 0.066) in genotype frequency test. This association was confirmed by validation group. Logistic regression adjusted allergy comorbidity and gender showed a slightly weaker association.
Conclusions:  The results indicated an independent role of IL17A promoter polymorphism rs8193036 in the association with pediatric asthma in Taiwanese population.     

人白介素17F真核蛋白表达及促炎症作用的研究

利用真核表达载体pCEP4,在人肾上皮293T细胞中表达hIL-17F/mFc融合蛋白并初步研究IL-17F生物学特性。实验应用RT-PCR法克隆hIL-17F CDS段基因序列。将测序正确的hIL-17F序列插入pCEP4质粒构建pCEP4/hIL-17F真核表达载体。转染人肾上皮293T细胞后,筛选阳性表达细胞株。RT-PCR、ELISA和Western blot等法鉴定hIL-17F分子的mRNA和蛋白表达。体外实验验证其促炎症作用。结果表明,构建的pCEP4/hIL-17F重组表达载体,能在293T细胞中稳定表达。获得的hIL-17F重组蛋白能稳定结合Raji细胞上的IL-17RC受体。体外刺激ECV304细胞,能显著促进IL-2等炎症因子的分泌。稳定表达hIL-17F重组蛋白的293T细胞系的建立,为进一步研究hIL-17F的生物学功能奠定了良好的基础。     

The nature of innate and adaptive interleukin-17A responses in sham or bacterial inoculation

Streptococcus pyogenes is the causative agent of numerous diseases ranging from benign infections (pharyngitis and impetigo) to severe infections associated with high mortality (necrotizing fasciitis and bacterial sepsis). As with other bacterial infections, there is considerable interest in characterizing the contribution of interleukin‐17A (IL‐17A) responses to protective immunity. We here show significant il17a up‐regulation by quantitative real‐time PCR in secondary lymphoid organs, correlating with increased protein levels in the serum within a short time of S. pyogenes infection. However, our data offer an important caveat to studies of IL‐17A responsiveness following antigen inoculation, because enhanced levels of IL‐17A were also detected in the serum of sham‐infected mice, indicating that inoculation trauma alone can stimulate the production of this cytokine. This highlights the potency and speed of innate IL‐17A immune responses after inoculation and the importance of proper and appropriate controls in comparative analysis of immune responses observed during microbial infection.     

Endogenous interleukin (IL)‐17A promotes pristane‐induced systemic autoimmunity and lupus nephritis induced by pristane

Interleukin (IL)-17A is increased both in serum and in kidney biopsies from patients with lupus nephritis, but direct evidence of pathogenicity is less well established. Administration of pristane to genetically intact mice results in the production of autoantibodies and proliferative glomerulonephritis, resembling human lupus nephritis. These studies sought to define the role of IL-17A in experimental lupus induced by pristane administration. Pristane was administered to wild-type (WT) and IL-17A^−/− mice. Local and systemic immune responses were assessed after 6 days and 8 weeks, and autoimmunity, glomerular inflammation and renal injury were measured at 7 months. IL-17A production increased significantly 6 days after pristane injection, with innate immune cells, neutrophils (Ly6G⁺) and macrophages (F4/80⁺) being the predominant source of IL-17A. After 8 weeks, while systemic IL-17A was still readily detected in WT mice, the levels of proinflammatory cytokines, interferon (IFN)-γ and tumour necrosis factor (TNF) were diminished in the absence of endogenous IL-17A. Seven months after pristane treatment humoral autoimmunity was diminished in the absence of IL-17A, with decreased levels of immunoglobulin (Ig)G and anti-dsDNA antibodies. Renal inflammation and injury was less in the absence of IL-17A. Compared to WT mice, glomerular IgG, complement deposition, glomerular CD4⁺ T cells and intrarenal expression of T helper type 1 (Th1)-associated proinflammatory mediators were decreased in IL-17A^−/− mice. WT mice developed progressive proteinuria, but functional and histological renal injury was attenuated in the absence of IL-17A. Therefore, IL-17A is required for the full development of autoimmunity and lupus nephritis in experimental SLE, and early in the development of autoimmunity, innate immune cells produce IL-17A.     

白介素17A受体在系统性红斑狼疮患者外周血B淋巴细胞的表达及意义

目的:研究白介素17A受体(IL-17AR)在系统性红斑狼疮(SLE)患者外周血B淋巴细胞内的表达情况及其临床意义。方法:流式细胞术检测60例SLE患者及33例健康人外周血B细胞上IL-17AR的表达水平。并将其与有关临床及实验室指标进行相关性分析。结果:SLE患者组的IL-17AR+细胞表达比例为(47.58±17.20)%,高于正常对照组(40.71±11.82)%(P<0.05)。在SLE患者中:口腔溃疡组、有浆膜炎组、有肾脏病变组、有免疫异常组分别高于相应的无症状组(P<0.05)。在抗Sm-D1抗体阳性组及抗核小体抗体阳性组分别高于相应的抗体阴性组(P<0.05)。IL-17AR+细胞比例与SLE疾病活动指数(SLEDAI)、C反应蛋白(CRP)、甘油三酯呈正相关;与间接胆红素、血清白蛋白的表达负相关(P<0.05)。B淋巴细胞比例与血清IgG、ALT、直接胆红素的表达呈正相关;与胆固醇、甘油三酯、低密度脂蛋白的表达呈负相关(P<0.05)。结论:IL-17AR在SLE患者B细胞上表达上调,且与病情有一定的相关性。提示IL-17/IL-17AR可能在SLE发病中起着重要作用。     

人白细胞介素2基因在酵母菌中的表达及发酵研究

将一带有人白细胞的介素2cDNA 的大肠杆菌——酵母菌穿酸质粒转化啤酒酵母Saccharomyces cerevisiae 8534-8c,再将转化子与另一啤酒酵母 Saccharomyces cervisiae CSH83—L 进行杂交,得到二倍体菌株.同时,还研究了单倍体转化子和二倍体菌株在表达重组白细胞介素2上的差异以及不同培养条件下二倍体菌株的表达情况,确定了合适的发酵条件,并进行了五立升自动发酵罐的发酵研究.     

Roles of macrophage stimulating protein and tyrosine kinase receptor RON in smoke-induced airway inflammation of rats

Objective: To investigate the roles of macrophage stimulating protein (MSP) and its tyrosine kinase receptor RON in smoke-induced airway inflammation of rats. Methods: Inhalation of combustion smoke was administered in rats to induce airway inflammation. Alveolar macrophages (AM) of healthy and smoking rats were isolated at different time points, cultured and then treated with different concentrations of MSP for 24 h. Results: When compared with healthy rats, MSP increased in the serum and bronchoalveolar lavage fluid (BALF) of smoking rats in a time dependent manner. In smoking rats, the RON expression in the lung and AM was higher than in healthy rats, and these increases were time dependent. MSP stimulated the production of malondialdehyde (MDA) and reduced superoxide dismutase (SOD) activity in rat AM cells in a dose dependent manner. MSP also stimulated the release of inflammatory factors TNF-α, IL-8, IL-1β and IL-10 in rat AM in a dose-dependent manner. Moreover, at the same MSP concentration, the contents of MDA, TNF-α, IL-8 and IL-1β in the AM of smoking rates were higher than in healthy rats, while the IL-10 content and SOD activity were lower than in healthy rats. Conclusion: MSP and its receptor RON are involved in the smoke-induced airway inflammation in rats via promoting AM to release inflammatory cytokines and inducing the increase of oxygen free radical.