Toll-like Receptor9在大鼠胰腺表达及与大鼠急性胰腺炎相关性的研究

目的 (1)建立急性胰腺炎大鼠模型,定性检测Toll-like Receptor 9(TLR 9)在大鼠胰腺的表达、分布情况;(2)定量测定TLR 9在大鼠急性胰腺炎不同时间点的表达变化情况;(3)结合TLR 9在大鼠胰腺的组织分布、表达情况及在雨蛙素诱导性胰腺炎(cerulein-induced pancreatitis,CIP)早期24 h的表达改变,探讨TLR9与CIP发生发展的相关性.方法(1)采用Wistar大鼠,并随机分配进入实验组或对照组;通过皮下注射雨蛙素建立急性胰腺炎模型;(2)采用免疫组化方法检测TLR 9在正常大鼠胰腺及CIP时大鼠胰腺的表达TLR 9在大鼠胰腺的组织分布情况;(3)提取总RNA,采用实时荧光定量逆转录-多聚酶链反应(Quantitative-Real-Time;QRT-PCR)法测定TLR9基因的表达.(4)分析TLR9的分布特征及可能的意义(5)统计分析TLR 9 mRNA的表达情况与CIP发生、发展的关系.结果 (1)TLR 9主要分布于胰管上皮、血管内皮和胰岛;(2)外分泌腺泡细胞没有明显的表达;(3)QRT-PCR结果显示TLR9 mRNA在正常大鼠胰腺组织呈现低水平表达;(4)CIP早期TLR9 mRNA表达出现快速上调并在1 h时达到最高值;TLR 9 mRNA表达在CIP前4 h内维持于高水平;其后下降缓慢,至到CIP的第24小时也未降至正常,保持相对较高的表达水平.结论 (1)TLR 9在大鼠胰腺有表达,且表达具有一定的组织特异性;(2)TLR9在CIP胰腺组织中的表达明显升高,提示TLR 9在胰腺炎早期炎症反应的发生、发展中具有重要作用,与之存在相关性.

Toll样受体7,9与人细小病毒B19在桥本甲状腺炎发病机理中的作用

目的:探讨桥本甲状腺炎(Hashimoto's thyroiditis,HT)中人细小病毒B19(parvovirus B19,B19)抗原与TLR7,9(Toll-like receptor7,9)表达的相关性。方法:采用连续切片免疫组织化学方法,检测41例单纯桥本甲状腺炎及20例甲状腺腺瘤旁正常甲状腺组织中B19抗原及TLR7和TLR9的表达,并对其进行相关性分析。收集3例新鲜HT组织做免疫荧光双标记染色,用激光共聚焦显微镜分别检测B19抗原和TLR7,B19抗原和TLR9的共定位关系。另外,用免疫共沉淀检测3例新鲜标本中B19抗原和TLR9的相互作用。结果:B19抗原在桥本甲状腺炎组的阳性表达率为80.49%(33/41);TLR9在桥本甲状腺炎组的阳性表达率为82.93%(34/41);正常甲状腺组两者均阴性,两者差异非常显著(P<0.01)。B19抗原的阳性表达与TLR9的阳性表达存在非常显著的正相关关系(r=0.594,P=0.001)。免疫荧光双标记激光共聚焦显微镜观察发现,B19抗原和TLR9蛋白明确共定位于桥本甲状腺炎嗜酸性变的滤泡上皮细胞的胞质中。免疫共沉淀结果进一步显示B19抗原与TLR9存在相互作用。TLR7在桥本甲状腺炎组的嗜酸性变的甲状腺滤泡上皮细胞和正常甲状腺组织表达均阴性。结论:TLR9通过识别B19抗原在HT的发病中起作用,TLR9很可能是B19病毒感染的识别受体。

TLR9在成牙本质细胞中的表达

目的:研究成牙本质细胞中TLR9、DSPP基因表达特征及信号转导途径。方法:采用RT-PCR检测TLR9在小鼠牙髓组织中及TLR9、DSPP在成牙本质细胞系中的表达。用CpGODN-A和CpGODN-B刺激细胞,在时间梯度0、3、6、9、12、24h,检测TLR9、DSPP基因的表达特征。结果:TLR9mRNA在小鼠牙髓组织中有表达。TLR9、DSPPmRNA在成牙本质细胞中都有显著的表达,在CpGODN刺激下显著上调,在6h处于峰值。结论:TLR9在成牙本质细胞中有表达;TLR9的特异性表达对DSPP基因表达量有影响。

Toll-like receptor 9 polymorphisms and Helicobacter pylori influence gene expression and risk of gastric carcinogenesis in the Brazilian population

BACKGROUND Toll-like receptors(TLRs)are the first line of host defense,and are involved in Helicobacter pylori(H.pylori)recognition and activation of both inflammatory and carcinogenic processes.The presence of single nucleotide polymorphisms(SNPs)in genes that activate the immune response may modulate the risk of precancerous lesions and gastric cancer(GC).Among them,Toll-like receptor 9(TLR9)polymorphisms have emerged with a risk factor of infectious diseases and cancer,however the studies are still inconclusive.AIM To evaluate whether TLR9 rs5743836 and rs187084 SNPs contribute to the risk of gastric carcinogenesis,and its influence on mRNA expression.METHODS A case-control study was conducted to evaluate two TLR9 SNPs(TLR9-1237 TCrs5743836 and TLR9-1486 CT-rs187084)in chronic gastritis(CG)and GC patients.A total of 609 DNA samples of peripheral blood[248 CG,161 GC,and 200 samples from healthy individuals(C)]were genotyped by polymerase chain reaction-restriction fragment length polymorphism.All samples were tested for the H.pylori infection using Hpx1 and Hpx2 primers.Quantitative polymerase chain reaction by TaqMan?assay was used to quantify TLR9 mRNA from fresh gastric tissues(48 GC,26 CG,and 14 C).RESULTS For TLR9-1237,the TC+CC or CC genotypes were associated with a higher risk of GC than C[recessive model odds ratio(OR)=5.01,95%confidence interval(CI):2.52-9.94,P<0.0001],and the CG(recessive model OR=4.63;95%CI:2.44-8.79,P<0.0001)groups.For TLR9-1486,an association between the CT+TT genotypes and increased risk of both GC(dominant model OR=2.72,95%CI:1.57-4.72,P<0.0001)and CG(dominant model OR=1.79,95%CI:1.15-2.79,P=0.0094)was observed when compared to the C group.Moreover,the presence of TLR9-1237 TC/CC+TLR9-1486 CC genotypes potentiate the risk for this neoplasm(OR=18.57;95%CI:5.06-68.15,P<0.0001).The TLR9 mRNA level was significantly higher in the GC group(RQ=9.24,P<0.0001)in relation to the CG group(RQ=1.55,P=0.0010)and normal mucosa(RQ=1.0).When the samples were grouped according to the polymorphic genotypes and the presence of H.pylori infection,an influence of TLR9-1237 TC+CC polymorphic genotypes(P=0.0083)and H.pylori infection(P<0.0001)was observed on the upregulation of mRNA expression.CONCLUSION Our findings show that TLR9 rs5743836 and rs187084 polymorphisms are associated with a higher risk of carcinogenesis gastric,and that TLR9 mRNA levels can be modulated by TLR9-1237 TC+CC variant genotypes and H.pylori infection.

Tumor necrosis family receptor superfamily member 9/tumor necrosis factor receptor-associated f

Hepatitis C virus(HCV)infection is an excellent immunological model for understanding the mechanisms developed by non-cytopathic viruses and tumors to evade the adaptative immune response.The antigen-specific cytotoxic T cell response is essential for keeping HCV under control,but during persistent infection,these cells become exhausted or even deleted.The exhaustion process is progressive and depends on the infection duration and level of antigenemia.During high antigenic load and long duration of infection,T cells become extremely exhausted and ultimately disappear due to apoptosis.The development of exhaustion involves the impairment of positive co-stimulation induced by regulatory cytokines,such as transforming growth factor beta 1.This cytokine downregulates tumor necrosis factor receptor(TNFR)-associated factor 1(TRAF1),the signal transducer of the T cell co-stimulatory molecule TNFR superfamily member 9(known as 4-1BB).This impairment correlates with the low reactivity of T cells and an exhaustion phenotype.Treatment with interleukin-7 in vitro restores TRAF1 expression and rescues T cell effector function.The process of TRAF1 loss and its in vitro recovery is hierarchical,and more affected by severe disease progression.In conclusion,TRAF1 dynamics on T cells define a new pathogenic model that describes some aspects of the natural history of HCV,and sheds light on novel immunotherapy strategies for chronic viral infections and cancer.

新生小鼠巨细胞病毒感染早期脾淋巴细胞Toll样受体2和9与髓样分化因子88及Th1／Th2类细胞因子的表达变化

目的探讨新生小鼠巨细胞病毒（cytomegalovirus，CMV）感染后脾淋巴细胞Toll样受体（Toll—like receptor，TLR）2、9，髓样分化因子88（myeloid cell differentiation 88，MyD88），白细胞介素（interleukin，IL）-2，干扰素-γ（interferon-γ，IFN-γ）和IL-10的表达变化及其在小鼠CMV感染中的作用。方法64只新生小鼠随机分为对照组（32只）和模型组（32只）。生后4～6h，模型组小鼠腹腔接种病毒致半数细胞感染量为104．31U／0．1 ml的CMV悬液20μl，建立新生小鼠CMV全身播散型感染模型。2组小鼠于3、5、7、10日龄时各随机处死8只。采用聚合酶链反应技术检测7日龄小鼠肝脏、脾脏、心脏、肺脏、肾脏和唾液腺中的CMV—DNA；采用Western印迹技术检测3、5、7、10日龄小鼠脾淋巴细胞TLR2、TLR9及MyD88蛋白的表达水平；采用酶联免疫吸附试验检测脾淋巴细胞培养上清液中IL-2、IFN-γ和IL-10水平。统计分析采用两独立样本t检验及Pearson相关分析。结果模型组脾淋巴细胞TLR2、TLR9和MyD88蛋白表达量在7日龄时达高峰（分别为1．06±0．09、1．19±0．03和0．89±0．07），均高于对照组（分别为0．47±0．04，0．71±0．02和0．43±0．03），差异均有统计学意义（t分别为21．69、50．40和42．06，P均〈0．01）。模型组脾淋巴细胞培养上清液IL-2水平7日龄时降至最低[（16．9±7．3）pg／ml]，IFN-γ5日龄达高峰[（242．9±8．4）pg／ml]，而IL-10在5日龄时才开始升高[（62．4±11．1）pg／ml]，与正常组相应日龄时[分别为（51．9±10．8）pg／ml、（135．3±10．5）pg／ml和（36．3±5．1）pg／ml]比较，差异均有统计学意义（t分别为-15．74、19．00和9．60，P均〈0．05）。7日龄时，TILR2、TLR9和MyD88蛋白水平与IL-2水平呈负相关（r分别为-0．978、-0．960和0．979，P均〈0．01）；与IL-10水平呈正相关（r分别为0．954、0．914和0．892，P均〈0．01）。结论在小鼠CMV感染早期（7d内），机体可通过TLR／MyD88信号通路，激活免疫应答，在感染早期起到一定抗病毒作用。但7d后则表现为Th1／Th2细胞免疫平衡失调。使病毒易于进一步复制并导致炎症扩散。

番石榴叶总黄酮对慢性胰腺炎小鼠纤维化的影响

目的：探讨番石榴叶总黄酮（total flavonoids from Psidium guajava leaves,TFPGL）对慢性胰腺炎（chronic pancreatitis,CP）小鼠纤维化的影响及其机制。方法：将50只C57BL/6小鼠随机均分为正常组,CP模型组,oxidized ATP（oxATP）组,TFPGL低、高剂量组。后4组小鼠腹腔注射雨蛙素50μg·kg^-1·h^-1（溶于200μL生理盐水）,每天6次,每周3 d,连续6周,建立慢性胰腺炎小鼠模型。6周后,oxATP组腹腔注射oxATP（15μg·kg^-1·d^-1,溶于200μL生理盐水）2周,TFPGL低、高剂量组分别予以TFPGL 0.186,0.372 g·kg^-1·d^-1灌胃2周。同时,正常组小鼠腹腔注射与模型组等体积、等频率的生理盐水。苏木素-伊红（HE）染色评估胰腺损伤程度;天狼星红染色检测胰腺组织胶原含量;免疫组化检测α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）,核苷酸结合寡聚化结构域样受体3（nucleotide binding oligomerization domain-like receptor 3,NLRP3）,半胱氨酸天冬氨酸蛋白酶-1（cysteinyl aspartate specific proteinase-1,Caspase-1）的蛋白表达水平;酶联免疫吸附法测定胰腺组织中IL-1β（interleukin-1β）,IL-18水平。结果：与正常组比较,雨蛙素注射后胰腺损伤加重（P〈0.01）,Caspase-1,NLRP3,α-SMA,IL-1β,IL-18水平升高（P〈0.01）。天狼星红染色显示胰腺组织细胞周围胶原Ⅰ（ColⅠ）,胶原Ⅲ（ColⅢ）含量较正常组升高（P〈0.01）。与模型组比较,oxATP组及TFPGL低、高剂量组炎症损伤及纤维化程度均减轻,表现为天狼星红染色程度减轻（P〈0.05,P〈0.01）,Caspase-1,NLRP3,α-SMA,IL-1β,IL-18水平均降低（P〈0.05,P〈0.01）。结论：TFPGL可通过抑制P2X7R介导NLRP3炎性体信号途径活化显著减轻慢性胰腺炎模型小鼠的慢性炎症及纤维化程度。

Neutrophil extracellular traps contribute to myofibroblast differentiation and scar hyperplasia through the Toll-like receptor 9/nuclear factor Kappa-B/interleukin-6 pathway

Background:Inflammation is an important factor in pathological scarring.The role of neutrophils,one of the most important inflammatory cells,in scar hyperplasia remains unclear.The purpose of this article is to study the correlation between neutrophil extracellular traps(NETs)and scar hyperplasia and identify a new target for inhibiting scar hyperplasia.Methods:Neutrophils were isolated from human peripheral blood by magnetic-bead sorting.NETs in plasma and scars were detected by enzyme-linked immunosorbent assays(ELISAs),immunofluorescence and flow cytometry.Immunohistochemistry was used to assess neutrophil(CD66B)infiltration in hypertrophic scars.To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry.Results:We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs(p<0.05).Hypertrophic scars showed greater infiltration with neutrophils and NETs(p<0.05).NETs activate fibroblasts in vitro to promote their differentiation and migration.Inhibition of NETs with cytochalasin in wounds reduced the hyperplasia of scars in mice.We induced neutrophils to generate NETs with different stimuli in vitro and detected the proteins carried by NETs.We did not find an increase in the expression of common scarring factors[interleukin(IL)-17 and transforming growth factor-β(TGF-β),p>0.05].However,inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts intomyofibroblasts.In vitro,NETs were found to be mediated by Toll-like receptor 9(TLR-9)in fibroblasts and further phosphorylated nuclear factor Kappa-B(NF-κB).We found that IL-6,which is downstream of NF-κB,was increased in fibroblasts.Additionally,IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion.Conclusions:Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway,thereby providing a new target for regulating hypertrophic scars.

Hydroxyapatite nanoparticles drive the potency of Toll-like receptor 9 agonist for amplified innate and adaptive immune response

The potency of Toll-like receptor 9(TLR9)agonist to drive innate immune response was limited due to immune suppression or tolerance during TLR9 signaling activation in immune cells.Herein we addressed this problem by introducing hydroxyapatite nanoparticles(HANPs)to CpG ODN(CpG),a TLR9 agonist.The study revealed that HANPs concentration and durationdependently reprogramed the immune response by enhancing the secretion of immunostimulatory cytokines(tumor necrosis factorα(TNFα)or IL-6)while reducing the production of immunosuppressive cytokine(IL-10)in macrophages in response to CpG.Next,the enhanced immune response benefited from increased intracellular Ca2+in macrophage by the addition of HANPs.Further,we found exposure to HANPs impacted the mitochondrial function of macrophages in support of the synthesis of adenosine triphosphate(ATP),the production of nicotinamide adenine dinucleotide(NAD),and reactive oxygen species(ROS)in the presence or absence of CpG.In vaccinated mice model,only one vaccination with a mixture of CpG,HANPs,and OVA,a model antigen,allowed the development of a long-lasting balanced humoral immunity in mice without any histopathological change in the local injection site.Therefore,this study revealed that HANPs could modulate the intracellular calcium level,mitochondrial function,and immune response in immune cells,and suggested a potential combination adjuvant of HANPs and TLR9 agonist for vaccine development.

Coexpression of TLR9 and VEGF-C is associated with lymphatic metastasis in prostate cancer

Prostate cancer(PCa)is one of the most frequent cancers in men,and its biomolecular targets have been extensively studied.This study aimed to analyze the expression of toll-like receptor 9(TLR9)and vascular endothelial growth factor C(VEGF-C)and the clinical value of the coexpression of TLR9 and VEGF-C in PCa.We retrospectively evaluated 55 patients with clinically localized,intermediate-risk,or high-risk PCa who underwent laparoscopic radical prostatectomy(LRP)and extended pelvic lymph node dissection(ePLND)without neoadjuvant hormonal therapy at a single institution from June 2013 to December 2016.In all 55 patients,the median number of lymph nodes(LNs)resected was 23(range:18-31),and a total of 1269 LNs were removed,of which 78 LNs were positive.Seventeen patients had positive LNs,with a positive rate of 30.9%.In addition,the immunohistochemical results in the above patients revealed that high TLR9 expression was correlated with higher Gleason score(GS)(P=0.049),increased LN metastasis(P=0.004),and more perineural invasion(PNI)(P=0.033).Moreover,VEGF-C expression was associated with GS(P=0.040),pathological stage(pT stage)(P=0.022),LN metastasis(P=0.003),and PNI(P=0.001).Furthermore,a significant positive correlation between TLR9 and VEGF-C was found(P<0.001),and the TLR9/VEGF-C phenotype was associated with LN metastasis(P=0.047).Collectively,we propose that TLR9 stimulation may promote LN metastasis in PCa cells through the upregulation of VEGF-C expression,thereby affecting the prognosis of PCa patients.Therefore,these markers may serve as valuable targets for the treatment of PCa.

Tumor necrosis factor receptor superfamily member 9 is upregulated in the endothelium and tumor cells in melanoma brain metastasis

Aim:The cytokine receptor tumor necrosis factor receptor superfamily member 9(TNFRSF9)is mainly considered to be a co-stimulatory activation marker in hematopoietic cells.Several preclinical models have shown a dramatic beneficial effect of treatment approaches targeting TNFRSF9 with agonistic antibodies.However,preliminary clinical phase I/II studies were stopped after the occurrence of several severe deleterious side effects.In a previous study,it was demonstrated that TNFRSF9 was strongly expressed by reactive astrocytes in primary central nervous system(CNS)tumors,but was largely absent from tumor or inflammatory cells.The aim of the present study was to address the cellular source of TNFRSF9 expression in the setting of human melanoma brain metastasis,a highly immunogenic tumor with a prominent tropism to the CNS.Methods:Melanoma brain metastasis was analyzed in a cohort of 78 patients by immunohistochemistry for TNFRSF9 and its expression was correlated with clinicopathological parameters including sex,age,survival,tumor size,number of tumor spots,and BRAF V600E expression status.Results:Tumor necrosis factor receptor superfamily member 9 was frequently expressed independently on both melanoma and endothelial cells.In addition,TNFRSF9 was also present on smooth muscle cells of larger vessels and on a subset of lymphomonocytic tumor infiltrates.No association between TNFRSF9 expression and patient survival or other clinicopathological parameters was seen.Of note,several cases showed a gradual increase in TNFRSF9 expression on tumor cells with increasing distance from blood vessels,an observation that might be linked to hypoxia-driven TNFRSF9 expression in tumor cells.Conclusion:The findings indicate that the cellular source of TNFRSF9 in melanoma brain metastasis largely exceeds the lymphomonocytic pool,and therefore further careful(re-)assessment of potential TNFRSF9 functions in cell types other than hematopoietic cells is needed.Furthermore,the hypothesis of hypoxia-driven TNFRSF9 expression in brain metastasis melanoma cells requires further functional testing.