IFN-γ/IL-4对呼吸道合胞病毒感染呼吸道上皮细胞TLR3和TSLP mR-NA表达的影响

目的:探讨呼吸道合胞病毒(RSV)感染呼吸道上皮细胞16HBE对Toll样受体3(TLR3)和胸腺基质淋巴细胞生成素(TSLP)mRNA表达的影响,并探索Th1/Th2细胞因子IFN-γ/IL-4对RSV感染16HBE细胞TLR3和TSLPmRNA表达的作用.方法:利用Hep-2细胞扩增病毒,并进行病毒毒力鉴定;不同浓度人工合成双链RNA(dsRNA)聚肌胞加入细胞培养基中,实时荧光定量RT-PCR检测16HBE细胞TLR3mRNA和TSLP mRNA表达水平变化;RSV感染16 HBE细胞试验分组:对照组、RSV感染组(MOI为2的RSV病毒感染16HBE6h)、RSV/IFN-γ组(RSV病毒感染,同时重组人IFN-γ 100μg/L加入培养基)、RSV/IL-4组(RSV病毒感染,同时重组人IL-4 100μg/L加入培养基),实时荧光定量RT-PCR检测TLR3mRNA和TSLPm RNA表达水平变化.结果:经Hep-2细胞培养扩增获得足量Long株RSV病毒;聚肌胞能有效刺激16HBE细胞TLR3mRNA和TSLPmRNA表达水平升高(P<0.01),并随聚肌胞浓度增加提高;RSV感染16HBE细胞6 h,TLR3和TSLPmRNA表达水平均升高(P<0.01);Th2细胞因子IL-4可以加强RSV感染引起的TSLPmRNA表达水平升高(P<0.01),而Th1细胞因子IFN-γ能抑制RSV感染引起的TSLP mRNA表达水平升高(P<0.01).结论:TLR3刺激物聚肌胞能有效刺激人呼吸道上皮细胞TLR3 mR-NA和TSLPmRNA表达水平升高;RSV感染16HBE细胞引起TLR3mRNA和TSLPmRNA表达水平升高;Th2细胞因子IL-4能协同病毒感染增加TSLPmRNA表达水平;Th1细胞因子IFN-γ能抑制RSV感染引起的TSLPmRNA表达水平升高.     

γδ T细胞参与呼吸道合胞病毒感染对气道变态炎症反应的影响作用

目的 证实γδ T细胞在呼吸道合胞病毒(respiratory syncytial virus,RSV)不同感染时间所致哮喘鼠不同气道炎症反应中的作用.方法 建立致敏前后不同时间感染RSV的哮喘动物模型,HE染色观察哮喘鼠肺炎症反应;real-time RT-PCR法检测γδ T细胞Th1/Th2型细胞因子IFN-γ和IL-4 mRNA表达;流式细胞仪分析脾及肺组织中γδ T细胞数量;γδ T细胞过继回输进一步明确其功能.结果 致敏前感染RSV显著减轻哮喘鼠肺炎症;减少脾及肺组织细胞中γδ T细胞总数及其活化细胞数量;增加γδ T细胞IFN-γ mRNA表达;降低IL-4 mRNA表达,但致敏后感染RSV对哮喘鼠肺炎症反应及ΥδT细胞数量和生物学活性无明显影响.尾静脉回输γδ T细胞导致模型鼠肺炎症反应明显增强,提示γδ T细胞参与哮喘鼠气道炎症的发生发展.结论 致敏前RSV感染可能通过影响γδ T细胞数量及其生物学活性改变变应原诱发的气道炎症反应.     

RNAi技术对改善小鼠呼吸道合胞病毒感染后气道炎症的研究

目的 探讨小鼠呼吸道合胞病毒(respiratory syncytial virus,RSV)感染后气道的炎症反应以及采用小分子RNA技术（smallinterference RNA,siRNA）治疗对气道炎症的改善作用。方法 采用针对RSV -M2基因的特异性siRNA滴鼻治疗RSV感染的RALB/c鼠,通过显微镜观察支气管肺泡灌洗液(BALF)白细胞计数及ELASA方法检测BALF中细胞因子IFN-γ、IL-12和IL-10水平的变化情况。结果 RSV感染后,BALF白细胞总数显著增加,分类以淋巴细胞为主;细胞因子IFN－γ、IL-12和IL-10水平升高;siRNA治疗后,白细胞总数、淋巴细胞、百分数随着siRNA浓度的增加而下降。IFN-Y、IL-12和IL-10水平随着siRNA浓度的增加而下降,与对照组比较差异有统计学意义(P＜0.05)。结论 RSV感染小鼠后,气道发生炎症反应;siRNA技术能够减轻RSV感染后的气道炎症反应。     

RNAi技术对改善小鼠呼吸道合胞病毒感染后气道炎症的研究北大核心CSCD

目的探讨小鼠呼吸道合胞病毒（respiratory syncytial virus,RSV）感染后气道的炎症反应以及采用小分子RNA技术（smallinterferenceRNA,siRNA）治疗对气道炎症的改善作用。方法采用针对RSV-M2基因的特异性siRNA滴鼻治疗RSV感染的hALB／c鼠,通过显微镜观察支气管肺泡灌洗液（BALF）白细胞计数及ELASA方法检测BALF中细胞因子IFN-γ、IL-12和IL-10水平的变化情况。结果RSV感染后,BALF白细胞总数显著增加,分类以淋巴细胞为主;细胞因子IFN-γ、IL-12和IL-10水平升高;siRNA治疗后,白细胞总数、淋巴细胞、百分数随着siRNA浓度的增加而下降。IFN-γ、IL-12和IL-10水平随着siRNA浓度的增加而下降,与对照组比较差异有统计学意义（P〈0．05）。结论RSV感染小鼠后,气道发生炎症反应;siRNA技术能够减轻RSV感染后的气道炎症反应。     

γδT细胞与呼吸道合胞病毒感染后支气管哮喘的研究进展

根据T细胞受体(TCR)不同T淋巴细胞分为αβT淋巴细胞和γδT淋巴细胞,γδT细胞在支气管哮喘的发病中起着重要作用。呼吸道合胞病毒(RSV)是一种单股负链非节段性的RNA病毒,其感染与哮喘的发病和加重存在明显的相关性。本文综述了γδT细胞与RSV感染后支气管哮喘的关系。     

呼吸道合胞病毒感染的研究进展

呼吸道合胞病毒是在世界范围内引起婴幼儿呼吸道感染的最常见病原微生物,它不仅可以引起呼吸系统的一些常见症状和体征,而且在肺外器官如中枢神经系统、心血管系统、内分泌系统等各器官也可引起一些尚未被人们普遍认识的肺外表现,而这些临床特点的重要性也愈来愈受到人们的重视.     

呼吸道合胞病毒感染后婴幼儿喘息患者血清leptin水平的变化

目的 通过测量呼吸道合胞病毒肺炎儿童患者瘦素(leptin)的水平,探讨瘦素与呼吸道合胞病毒感染后婴幼儿喘息间的相互关系.方法 43例呼吸道合胞病毒感染后婴幼儿分别于入院后24h内、治疗结束及出院后12周用放射免疫法检测血清leptin水平,并随访2年.根据患儿喘息发作的情况,分为婴儿哮喘组和非哮喘组;另选10名健康儿童血清标本作对照.结果 BSV感染后喘息发作≥3次的婴幼儿患儿,占41.9%.治疗前,哮喘组和非哮喘组血清leptin水平均高于对照组,差异有统计学意义(t=3.41、2.64,P<0.05).治疗后,哮喘组血清leptin水平高于非哮喘组和对照组,差异有统计学意义(t=5.74、6.23,P<0.05).出院12周后复查,哮喘组血清leptin水平仍高于非哮喘组和对照组,差异有统计学意义(t=6.32、6.11,P<0.05);而非哮喘组血清leptin水平和对照组比较,差异无统计学意义(t=0.81,P>0.05).结论 呼吸道合胞病毒感染后喘息发作≥3次的婴幼儿血清leptin水平较健康同龄儿童及非哮喘儿童明显升高,持续高leptin水平可能是BSV感染后婴儿哮喘的高危因素之一.     

用紫外线灭活的呼吸道合胞病毒复制小鼠哮喘动物模型

目的：探讨用紫外线灭活的呼吸道合胞病毒（RSV）复制小鼠哮喘动物模型的方法。方法：随机将小鼠分为3组：正常对照组（A组，6只）、卵清蛋白（OVA）致敏组（B组，6只）、RSV致敏组（C组，24只）。观察C组与其他两组小鼠哮喘发作症状程度及病理切片上浆细胞和嗜酸性粒细胞（EOS）浸润情况的不同。另按末次激发时间与处死小鼠时间之间的时间间隔，以0、24、48、72 h为时间点，随机将C组分为C₁-C₄组，每组6只小鼠。观察用紫外线灭活的RSV致敏的小鼠多次抗原刺激后病理切片上浆细胞和EOS浸润的变化情况。结果：C组小鼠哮喘发作症状及浆细胞和EOS浸润较A、B组明显且支气管粘膜下浆细胞和EOS的浸润随时间增加：0 h浆细胞[ 1.60±0.75（平均每视野计数，下同）]和EOS（2±1）浸润极少；24 h浸润开始增加[ 浆细胞（8±1），EOS（12±1）]；48 h浸润达高峰[ 浆细胞（10±2），EOS（15±3）]；72 h开始下降[ 浆细胞（3±1），EOS（4±2）]。结论：用紫外线灭活的RSV复制小鼠哮喘动物模型是一种较理想的方法。     

呼吸道合胞病毒对致敏小鼠气道炎症和CD8+ T细胞功能的影响

目的观察呼吸道合胞病毒（RSV）对致敏小鼠气道炎症和CD8^＋T细胞功能的影响.方法BALB/c小鼠40只,随机分成4组,分别为磷酸盐缓冲液（PBS）对照组、鸡卵白蛋白（OVA）组、RSV组、OVA/RSV组;应用OVA腹腔注射致敏、OVA气道雾化结合RSV滴鼻激发哮喘;支气管肺泡灌洗液（BALF）作细胞分类计数;酶联免疫吸附试验（ELISA）测定BALF上清中白细胞介素（IL）-4、IL-5、干扰素（IFN）-γ含量;苏木精-依红（HE）染色观察肺病理变化;采用三色光流式细胞分析法测定气管旁淋巴结（PBLN）中CD4^＋、CD8^＋T细胞及细胞内细胞因子IFN-γ、IL-4、IL-5表达与TH2/TH1、Tc2/Tc1比值变化.结果（1）BALF中细胞总数及分类：与OVA组比较,OVA/RSV组细胞总数、嗜酸性粒细胞、淋巴细胞均明显增加（分别P＜0.01）;与RSV组比较,OVA/RSV组细胞总数、嗜酸性粒细胞明显增加（分别P＜0.01）.（2）BALF上清中细胞因子含量：与OVA组比较,OVA/RSV组IFN-γ、IL-4、IL-5含量均明显升高（分别P＜0.01）;与RSV组比较,OVA/RSV组IFN-γ无明显变化,而IL-4、IL-5显著上升（分别P＜0.01）.（3）肺组织病理：OVA/RSV组与其他各组比较气道黏膜增厚,管腔狭窄、收缩,上皮破坏,管壁周围炎症细胞浸润明显加重.（4）PBLN中CD4^＋（WN-γ^＋、IL-4^＋、IL-5^＋）、CD8^＋（IFN-γ^＋、IL-4^＋、IL-5^＋）T细胞各占CD3^＋T细胞百分比及TH2/TH1、Tc2/Tc1比值变化：与OVA组比较,OVA/RSV组CD8^＋（IFN-γ^＋、IL-4^＋、IL-05^＋）T细胞百分比、Tc2/Tc1比值增加（分别P＜0.01）,TH2/TH1比值无明显变化.与RSV组比较,OVA/RSV组CD4^＋（IL-4^＋、IL-5^＋）T细胞、TH2/TH1比值、CD8^＋（IL-4^＋、IL-5^＋）T细胞、Tc2/Tc1比值均明显上升（分别P＜0.01）.结论（1）OVA致敏小鼠RSV感染后可明显加重气道炎症,TH2、TH1型炎症均加强,且以TH2型炎症加重为主.（2）OVA致敏小鼠RSV感染后可引起CD8^＋T细胞数量及功能改变,即由产生IFN-γ^＋为特征的Tc1细胞向产生IL-4^＋、IL-5^＋为特征Tc2细胞转化,并可能与气道内IL-4、IL-5升高及嗜酸性粒细胞的大量募集有关.     

呼吸道合胞病毒感染与细胞凋亡的研究进展

呼吸道合胞病毒感染和细胞凋亡的关系是错综复杂的,既可以表现为抗凋亡作用,以利于病毒在感染细胞内复制;也可以表现为促凋亡作用,此可能是机体对病毒感染的防御反应,亦可能是病毒感染导致宿主组织细胞严重损伤的重要机制.研究呼吸道合胞病毒感染与细胞凋亡关系及凋亡的可能机制,将有助于进一步认识呼吸道合胞病毒感染发生、发展及转归机制,为RSV感染的防治提供一些新的思路.     

胸腺基质淋巴生成素受体及其抗体在哮喘小鼠气道炎症反应中的作用

目的 探讨胸腺基质淋巴生成素受体(TSLPR)及其抗体在实验性哮喘小鼠气道炎症反应中的作用以及对气道树突状细胞(DCs)成熟和活化的影响.方法 BALB/c小鼠随机分成A、B、C三组.B和C组小鼠腹腔注射卵清白蛋白(OVA)致敏,A组腹腔注射PBS作为正常对照.B和C组小鼠在OVA激发哮喘发作前分别吸入非特异性IgG和TSLPR IgG.采集各组小鼠支气管肺泡灌洗液(BALF)进行细胞分类计数,采用ELISA定量检测BALF中IL-4、IL-5、IFN-γ和IL-10浓度.采集各组小鼠肺组织标本进行病理学检查,采用流式细胞术分别检测各组小鼠淋巴结和肺组织中DCs数量和表型.结果 与A组小鼠比较,8和C组小鼠BALF中各种细胞因子水平均明显升高(P<0.01).C组小鼠BALF中IL-4和IL-5水平低于8组(P<0.05,P<0.01),IFN-γ和IL-10水平高于8组(P<0.05,P<0.01).C组小鼠BALF中细胞总数、嗜酸性粒细胞和淋巴细胞数也明显低于B组(P<0.01).B组小鼠支气管周围有大量炎性细胞浸润以及杯状细胞增生,黏液分泌增强,C组小鼠仅见微弱的炎性细胞浸润和杯状细胞增生.B组小鼠膈淋巴结中DCs数量以及肺组织中DCs的I-Ad、CD40、CD80和CD86表达水平均高于C组小鼠(P<0.05).结论 TSLP/TSLPR具有促哮喘效应并与其调节气道DCs活性的作用密切相关,TSLPR抗体干预可明显减弱TSLP/TSLPR上述作用,故有作为抗哮喘药物的前景.     

Respiratory syncytial virus-induced CCL5/RANTES contributes to exacerbation of allergic airway inflammation

Severe respiratory syncytial virus (RSV) infection has a significant impact on airway function and may induce or exacerbate the response to a subsequent allergic challenge. In a murine model combining early RSV infection with later cockroach allergen (CRA) challenge, we examined the role of RSV-induced CCL5/RANTES production on allergic airway responses. RSV infection increased CCL5 mRNA and protein levels, peaking at days 8 and 12, respectively. Administration of CCL5 antiserum during days 0-14 of the RSV infection did not significantly alter viral protein expression when compared to mice treated with control serum. In mice receiving the combined RSV-allergen challenge, lungs collected on day 22 exhibited significantly increased numbers of CD4- and CD8-positive T cells. This increase in T cell numbers was not observed in mice receiving alpha-CCL5. On day 43, peribronchial eosinophilia and leukotriene levels were increased in RSV-allergen mice. Pretreatment with CCL5 antiserum resulted in decreased recruitment of inflammatory cells to bronchoalveolar and peribronchial regions of the lungs and these reductions were associated with a reduction in both T cell recruitment into the bronchoalveolar space, leukotriene release and chemokine generation. Thus, CCL5 released during RSV infection has a significant effect on the inflammatory response to subsequent allergic airway challenges.     

宫颈癌细胞通过分泌TSLP促进自身细胞活力

目的:探讨胸腺基质淋巴细胞生成素(TSLP)对宫颈癌细胞活力的自分泌调节作用。方法:体外培养宫颈癌细胞株HeLa和CasKi,分别收集培养24、48和72小时的培养上清,ELISA法检测培养上清中TSLP的分泌水平;流式细胞术分析HeLa和CasKi细胞表面TSLP受体(TSLPR)的表达水平;再用MTT法分别检测HeLa和CasKi细胞经人重组细胞因子TSLP(1、10和100 ng/ml)处理24、48和72小时后细胞活力的水平变化。结果:HeLa和CasKi细胞均呈时间依赖性分泌TSLP(P<0.01),且HeLa细胞在24和48小时分泌TSLP的水平高于CasKi细胞(P<0.01或P<0.05);HeLa和CasKi细胞TSLPR阳性表达率分别为23.61±1.30和27.07±2.13,前者低于后者(P<0.05);此外,10或100 ng/ml TSLP作用HeLa和CasKi细胞48小时或72小时均能上调其细胞活力(P<0.05)。结论:宫颈癌细胞可能通过TSLP自分泌作用促进自身的活力,进而利于宫颈癌细胞的生长,因而参与宫颈癌的发病和进展。     

Skin thymic stromal lymphopoietin promotes airway sensitization to inhalant house dust mites leading to allergic asthma in mice

Asthma is often preceded by atopic dermatitis (AD), a phenomenon known as 'atopic march'. It has been suggested that sensitization to common inhalant allergens, which is developed in a majority of patients with AD and often during the course of AD, may play a critical role in triggering the atopic march. Yet, what signal(s) delivered by AD skin could promote sensitization to inhalant allergens remains elusive. Here, by employing an experimental mouse asthma protocol, which is induced by airway sensitization and challenge to inhalant house dust mite (HDM), we demonstrate that the overproduction of cytokine thymic stromal lymphopoietin (TSLP) by AD skin promotes airway sensitization to HDM, thereby triggering subsequently an allergic asthma. Together, this study provides, for the first time, the experimental proof that TSLP represents an AD skin-delivered signal to promote sensitization to inhalant aeroallergen, which may account for one mechanism underlying the 'atopic march'.     

The level of thymic stromal lymphopoietin and its gene polymorphism are associated with rheumatoid arthritis

ObjectiveTo study the correlation between TSLP gene SNPs and RA in a Han Chinese population.MethodsThe genotypes of TSLP genes rs11466749, rs11466750 and rs10073816 among 197 RA patients and 197 controls were analysed by direct sequencing. ELISA was used to detect the plasma TSLP level. Logistic regression analysis was also conducted to identify risk factors for RA.ResultsThe rs11466749 locus GG genotype (OR = 5.30, 95% CI: 1.76–15.95, P < 0.01), dominant model (OR = 1.68, 95% CI: 1.03–2.73, P = 0.04), recessive model (OR = 5.15, 95% CI: 1.72–15.43, P < 0.01), and G allele (OR = 2.02, 95% CI: 1.33–3.09, P < 0.01) were associated with an increased risk of RA. The rs1073816 locus AA genotype (OR = 4.58, 95% CI: 1.49–14.01, P < 0.01), dominant model (OR = 1.75, 95% CI: 1.09–2.79, P = 0.03), recessive model (OR = 4.27, 95% CI: 1.40–13.00, P = 0.03) and A allele (OR = 1.94, 95% CI: 1.29–2.91, P < 0.01) were associated with an increased risk of RA. The rs1073816 locus GA genotype (OR = 0.29, 95% CI: 0.18–0.45, P < 0.01), dominant model (OR = 0.32, 95% CI: 0.21–0.49, P < 0.01) and A allele (OR = 0.45, 95% CI: 0.32–0.63, P < 0.01) were related to a decreased risk of RA susceptibility. The rs1466749 locus GG genotype, rs11466750 AA genotype, and rs10073816 GG genotype were independent risk factors for RA (P < 0.05). The AUC of plasma TSLP level in the diagnosis of RA was 0.8661 (95% CI: 0.8301–0.9002, P < 0.001). There were statistically significant differences in plasma TSLP levels among subjects with different genotypes at rs11466749, rs11466750, and rs10073816 in the TSLP gene (P < 0.05).ConclusionPlasma TSLP levels are a potential molecular marker of RA. SNPs at rs11466749, rs11466750 and rs10073816 of the TSLP gene are related to the susceptibility of the Han Chinese population to RA.     

阿托伐他汀抑制ox-LDL诱导的人脐静脉内皮细胞表达胸腺基质淋巴细胞生成素TSLP的表达

目的:观察阿托伐他汀对ox-LDL诱导人脐静脉内皮细胞(HUVECs)表达胸腺基质淋巴细胞生成素(TSLP)的影响。方法:将HUVECs分为3组,对照组加50 mg/L的ox-LDL培养12小时;实验组分为2组,分别加50 mg/L的ox-LDL和不同浓度的阿托伐他汀培养12小时,于6小时和12小时进行检测,用ELISA法检测细胞培养上清液中TSLP的浓度,用RT-PCR检测TSLP mRNA的表达,采用免疫荧光检测细胞胞浆中TSLP表达。结果:ELISA和IF结果显示,实验组上清液和细胞浆内的TSLP的表达明显低于对照组,并随浓度的增大及时间的延长,TSLP降低得更明显。结论:阿托伐他汀可抑制ox-LDL诱导的HUVECs表达TSLP,这可能是阿托伐他汀抗动脉粥样硬化炎症反应的机制之一。     

IgE anti-respiratory syncytial virus antibodies detected in serum of pediatric patients with asthma

Respiratory syncytial virus (RSV) causes lower respiratory tract disease in infants and young children, and is a public health concern, as is the increase in pediatric asthma. Respiratory viral infections may trigger asthma exacerbations. However, it remains unknown whether RSV infection may have a specific association with asthma. Total serum IgE, and IgE- and IgG-anti-RSV Ab responses were studied in older asthmatic compared with non-asthmatic children (M/F, mean age: 14) (N = 30, N = 43, respectively). We found: (1) total serum IgE was higher in asthmatic compared with non-asthmatics (P < 0.001); (2) total serum IgE did correlate with IgE anti-RSV Abs (P < 0.001), and with IgG anti-RSV Abs (P = 0.008) in all subjects; (3) total serum IgE levels did correlate with IgE anti-RSV in asthmatics (P = 0.047), but not in non-asthmatics (P = 0.13); (4) IgE anti-RSV Abs did correlate with IgG anti-RSV Abs in all subjects (P = 0.001); (5) IgE- and IgG-anti RSV Abs were higher in asthma compared with no asthma (P = 0.003; <0.001, respectively); (6) there was a significant association between age and IgE anti-RSV in non-asthma (P = 0.008), but not in asthma (P = 0.64). Our findings indicate that IgE-anti-RSV Ab responses may play important roles in RSV infection and asthma.     

Increased pathogenesis and inflammation of airways from respiratory syncytial virus infection in T cell deficient nude mice

Respiratory syncytial virus (RSV) infection is ubiquitous and leads to various outcomes between immunocompetent and immunocompromised individuals. This study aimed to compare RSV infection and inflammatory responses between immunocompetent BALB/c mice and immunodeficient nude mice. RSV titers in both infected BALB/c mice and nude mice peaked on the third day post-inoculation, but the nude mice had longer lasting and higher levels of viral replication. RSV infection induced a more severe grade of pulmonary histopathology and larger numbers of leukocytes in airways of nude mice than that of BALB/c mice. RSV infection increased pulmonary macrophages and natural killer (NK) cells in both strains of mice. Furthermore, infected nude mice had larger numbers of pulmonary macrophages and NK cells than infected BALB/c mice. Whereas the RSV infected BALB/c mice secreted more tumor necrosis factor -alpha (TNF-alpha), interleukin-12 (IL-12), interferon-gamma (IFN-gamma) and IL-10 than control BALB/c mice, the infected nude mice had higher levels of TNF-alpha, IL-12 and IL-10 than the infected BALB/c mice. The inflammation induced by RSV infection did not correspond with the immune response of T cells. Macrophages and NK cells were potent immunocytes and inflammatory cells in RSV infection especially when T lymphocytes were deficient. Therefore, nude mice may be a good model for severe and persistent RSV infection in immunocompromised hosts.