IL-33在β-淀粉样蛋白刺激视网膜色素上皮细胞中的表达及作用研究

为探讨年龄相关性黄斑变性的免疫炎症反应机制,采用real-time PCR及ELISA检测β-淀粉样蛋白1-40(Aβ_((1-40)))刺激RPE细胞后IL-33 mRNA及蛋白水平表达,并采用ELISA检测IL-33刺激RPE细胞后炎症因子表达情况及调控产生炎症因子的信号通路。结果发现,Aβ_((1-40))刺激人视网膜色素上皮细胞后能够诱导IL-33因子转录及蛋白水平表达上调,且在人视网膜色素上皮细胞中,IL-33因子分别通过p38 MAPK、ERK1/2信号通路调控促炎症因子IL-6、IL-8的表达。据此说明,IL-33通过调控人视网膜色素上皮细胞产生促炎症因子IL-6、IL-8参与年龄相关性黄斑变性的发生发展。     

幽门螺杆菌黏附素A(HpaA)诱导T细胞高表达IL-21促进胃黏膜损伤

目的研究幽门螺杆菌黏附素A(HpaA)是否能通过诱导T细胞分泌白细胞介素21(IL-21)加剧幽门螺杆菌(H.pylori)感染后的炎症反应及黏膜损伤。方法收集H.pylori感染确诊患者临床内窥镜取得的胃黏膜,以及经根治后再次通过内窥镜取得胃黏膜,通过反转录PCR和Western blot法检测胃黏膜IL-21、基质金属蛋白酶2(MMP2)和MMP9的mRNA水平及蛋白水平;同时克隆构建表达并纯化HpaA,并取健康未感染成年人外周血单个核细胞(PBMC),磁珠分选获得CD3~+T细胞。使用重组HpaA刺激分选获得的细胞, ELISA检测其上清中IL-21的水平;培养胃黏膜AGS细胞系并使用抗IL-21抗体中和培养液中的IL-21 24 h, Western blot法检测AGS细胞中MMP2及MMP9的蛋白水平。结果 H.pylori感染的胃黏膜中IL-21、MMP2和MMP9蛋白水平显著高于根治后的胃黏膜。重组HpaA蛋白能显著诱导CD3~+ T细胞分泌高水平的IL-21, IL-21处理增加AGS细胞MMP2及MMP9的表达水平;使用抗体阻断IL-21后, MMP2及MMP9的蛋白水平显著降低。结论 HpaA通过诱导T细胞产生IL-21促进H.pylori感染导致的胃黏膜损伤。     

结核分支杆菌培养上清诱导呼吸道上皮细胞IL-8表达

目的：观察呼吸道上皮细胞对结核杆菌产物产生的炎症反应，探讨炎症反应信号传递的机制。方法：制备结核分枝杆菌强毒力H37RV株培养上清，用培养上清刺激肺上皮细胞株SPC-A-1和A549，酶联免疫吸附实验检测细胞IL-8表达量。用突变MyD88表达重组质粒转染SPC-A-1细胞，观察MyD88与结核杆菌培养上清诱导上皮细胞炎症反应信号传递的关系。结果：结核分枝杆菌强毒力H37RV株培养上清明显增加SPC-A-1和A549细胞IL-8的表达。用突变MyD88表达重组质粒pcDNA3．1-MyD88转染SPC-A-1。结果显著抑制了结核杆菌培养上清对SPC-A-l细胞的炎症刺激作用。结论：结核杆菌培养上清能够诱导呼吸道上皮细胞IL-8表达，，Toll／IL-1受体信号通路及MyD88分子与细胞炎症反应的信号传递相关，提示在肺结核病发病过程中。结核杆菌的代谢产物可能刺激肺上皮细胞产生炎症细胞因子，参与肺结核炎症病变的形成。     

长链非编码RNA LINC00260抑制幽门螺杆菌感染胃上皮细胞系引起的炎性反应和细胞迁移

目的探讨长链非编码RNA-LINC00260在幽门螺杆菌(HP)感染引起的胃癌中的作用及可能机制。方法实时荧光定量PCR检测LINC00260在HP感染的胃黏膜正常上皮细胞(GES1)中的表达,在胃癌细胞系SGC-7901中通过瞬时转染siRNA敲低LINC00260后与HP共培养,用q PCR检测炎症相关细胞因子肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、白介素6(IL-6)及白介素8(IL-8)的表达;在敲低LINC00260的表达后,通过划痕愈合实验,观察对胃癌细胞迁移能力的影响。结果 1)HP感染细胞后,GES1和SGC-7901细胞形态学改变;2)LINC00260在HP感染的胃黏膜正常上皮细胞表达显著下降(P0.05);3)敲低LINC00260可抑制HP引起的炎症相关因子TNF-α、IL-1β、IL-6和IL-8的表达;4)敲低LINC00260抑制胃癌细胞的迁移能力。结论 LINC00260可能是一个癌基因,在HP感染引起的胃癌中发挥重要作用。     

细粒棘球蚴早期感染促进小鼠体内IL-22的产生

目的探讨小鼠腹腔细粒棘球蚴感染早期主要产生IL-22的CD4~+T细胞Th1、Th17和Th22细胞以及IL-22R1的表达情况。方法建立小鼠腹腔细粒棘球蚴感染模型,分别在感染后第3、6、9、12天收集外周血、小鼠脾淋巴细胞以及肝脏和肠管组织。ELISA检测外周血IFN-γ和IL-17、IL-22的蛋白表达量;qRT-PCR检测脾淋巴细胞中Th1细胞相关因子(Ifng、Tbx21基因)、Th17细胞相关因子(IL-17、Rorc基因)、Th22细胞相关因子(IL-22、Ahr基因)以及肝脏和肠管组织中IL-22R1的mRNA表达水平;流式细胞术检测脾淋巴细胞中Th1细胞(CD4~+IFN-γ~+)、Th17细胞(CD4~+IFN-γ~-IL-22~+IL-17~+)及Th22细胞(CD4~+IFN-γ~-IL-22~+IL-17~-)的百分比情况。结果与对照组相比,ELISA、qRT-PCR和流式细胞术检测细粒棘球蚴感染后Th1细胞、Th17细胞、Th22细胞增高:qRT-PCR检测细粒棘球蚴感染后肝脏和肠管IL-22R1的表达增高。结论细粒棘球蚴感染小鼠早期,主要产生IL-22的CD4~+T细胞Th1、Th17、Th22细胞比例以及IL-22R1表达增加,可能参与了宿主的免疫防御反应。     

CXCR1/CXCR2拮抗剂G31P抗中性粒细胞介导的炎症作用研究

目的 建立肺炎动物模型,使用人CXCR1/CXCR2受体拈抗剂G31P,治疗与中性粒细胞相关的炎性疾病.方法 检测G31P能否阻断人IL-8对中性粒细胞趋化以及阻断支气管上皮细胞A549释放IL-8;建立pcDNA3.0-CXCR1、2、4转染的肾细胞HEK293,检测G31P阻断IL-8对细胞株的趋化作用;建立肺炎动物模型,计数肺泡灌洗液(BALF)中性粒细胞数,进行肺组织病理学观察.结果 实验证实G31P可以阻断中性粒细胞趋化和IL-8介导的CXCR1、CXCR2转染的HEK293细胞株的趋化作用,抑制A549释放炎性介质;G31P治疗组中性粒细胞比例下降,病理检测有明显差异.结论 G31P可以阻断ELR+CXC趋化因子对中性粒细胞的趋化作用,阻断ELR+CXC趋化因子与中性粒细胞表面受体CXCR2的结合,阻断肺泡上皮细胞和血管内皮细胞表面的CXCR2,从而进一步阻止中性粒细胞介导的炎症反应.     

MIP-1α和MCP-1在沙眼衣原体肺感染中的产生及与机体防御的关系

目的：探讨趋化性细胞因子巨噬细胞炎症蛋白-1(MIP-1α)和单核细胞趋化蛋白-1(MCP-1)在沙眼衣原体肺感染中的产生及与机体防御的关系。方法：用沙眼衣原体小鼠肺炎株(MoPn)通过鼻腔感染小鼠，酶消化法制备小鼠肺组织炎症细胞，Wright-Giemsa染色计数巨噬细胞占肺炎症细胞的百分率；用RT-PCR检测小鼠肺组织趋化性细胞因子及细胞因子mRNA表达；ELISA法检测肺组织匀浆中细胞因子分泌。结果：MoPn感染后7及14天，MIP-1α和MCP-1及其相应的受体CCR1、CCR2在小鼠肺组织中的表达明显增高。与之趋化作用有关的单核．巨噬细胞在肺组织中的浸润也随之增高；而且MoPn感染上调Th1细胞因子IFN-γ及IL-12的表达及分泌，未见Th2细胞因子IL-4在肺组织中的基因表达及分泌；但具有免疫抑制作用的Th2细胞因子IL-10在感染后7天明显表达。结论：衣原体呼吸道感染诱导CC趋化性细胞因子MIP-1α和MCP-1高表达，可能与单核细胞免疫防御及Th1／Th2免疫应答调节有关。     

党参多糖调控miR-361-5p/TLR4/NF-κB通路对胃癌细胞AGS增殖、凋亡和炎症因子表达的影响

目的 探讨党参多糖对胃癌细胞AGS增殖、凋亡和炎症因子表达的影响和机制。方法 采用CCK-8实验、流式细胞术评估党参多糖（10、20、40μmol/L）对胃癌细胞AGS活力和凋亡的影响。RT-qPCR检测miR-361-5p表达;ELISA法检测肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6、IL-1β分泌。蛋白质免疫印记法检测TLR4和磷酸化核因子（NF）-κBp65(p-p65)和磷酸化NF-κB抑制蛋白-α(p-IκBα)蛋白的表达。将miR-361-5p模拟物、miR-361-5p抑制物分别转染AGS细胞,经40μmol/L党参多糖处理后,用上述方法检测AGS细胞活力、凋亡率、Toll样受体4(TLR4)、p-p65和p-IκBα蛋白表达以及TNF-α、IL-6、IL-1β分泌。结果 10、20、40μmol/L党参多糖显著降低AGS细胞活力（P<0.05）,增加细胞凋亡率（P<0.05）,抑制TNF-α、IL-6、IL-1β分泌（P<0.05）,并上调miR-361-5p表达水平（P<0.05）。40μmol/L党参多糖明显降低TLR4、p-p65...     

基质金属蛋白酶14通过促进CD100脱落增强急性B淋巴细胞白血病患者单核细胞功能

目的 检测不同活性形式的CD100在急性B淋巴细胞白血病（B-ALL）患者中的表达，观察基质金属蛋白酶14(MMP14)对CD100的表达调控及对B-ALL患者单核细胞功能的影响。方法 入组44例B-ALL患者和22例对照者，采集外周血并分离血浆和外周血单个核细胞（PBMCs），酶联免疫吸附试验（ELISA）检测血浆MMP14和可溶型CD100(sCD100)水平，流式细胞术检测CD14+单核细胞中膜结合型CD100(m CD100)水平。使用磁珠分离法分选CD14+单核细胞，使用脂多糖刺激培养；反转录实时定量PCR法检测肿瘤坏死因子相关凋亡诱导配体（TRAIL）和Fas配体（FasL）mRNA相对表达量；ELISA法检测颗粒酶A、颗粒酶B、干扰素-γ(IFN-γ)、白细胞介素-1β(IL-1β)、IL-6水平；比较B-ALL患者和对照者单核细胞功能差异。使用重组人MMP14刺激B-ALL患者纯化的单核细胞，加入抗CD72抗体，通过检测TRAIL和FasL mRNA相对表达量以及颗粒酶A、颗粒酶B、IFN-γ、IL-1β、IL-6水平变化评估MMP14对单核细胞功能影响。结果 B-AL...     

姜黄素抑制肺上皮细胞BEAS-2B真菌产生炎症细胞因子的作用

目的探讨姜黄素(Cur)抑制肺上皮细胞BEAS-2B真菌产生炎症细胞因子的作用。方法黄曲霉菌(AFT)感染肺上皮细胞BEAS-2B后,用不同浓度的姜黄素(10μmol/L、25μmol/L、50μmol/L)进行抗炎抑制作用。采用ELISA法检测细胞上清液中炎症因子TNF-α、IL-Iβ、IL-4、IL-6、IL-8、IL-10、TLR4和NF-κB的表达水平;采用Western blot和Real-time PCR检测重组高迁移率族蛋白B1(high mobility group box 1,HMGB1)、TLR4和NF-κB蛋白和m RNA表达。结果随着姜黄素剂量的升高,细胞上清液中炎症因子TNF-α、IL-Iβ、IL-4、IL-6、IL-8、IL-10、TLR4和NF-κB的表达显著降低(P0.05);HMGB1、TLR4和NF-κB蛋白和m RNA表达明显降低(P0.05)。结论姜黄素调节TLR4/NF-κB信号通路来产生抗炎作用,可以抑制真菌刺激肺上皮细胞BEAS-2B产生炎症细胞因子。     

Role of Interleukin-32 in Helicobacter pylori-Induced Gastric Inflammation

Helicobacter pylori infection is associated with gastritis and gastric cancer. An H. pylori virulence factor, the cag pathogenicity island (PAI), is related to host cell cytokine induction and gastric inflammation. Since elucidation of the mechanisms of inflammation is important for therapy, the associations between cytokines and inflammatory diseases have been investigated vigorously. Levels of interleukin-32 (IL-32), a recently described inflammatory cytokine, are increased in various inflammatory diseases, such as rheumatoid arthritis and Crohn''s disease, and in malignancies, including gastric cancer. In this report, we examined IL-32 expression in human gastric disease. We also investigated the function of IL-32 in activation of the inflammatory cytokines in gastritis. IL-32 expression paralleled human gastric tissue pathology, with low IL-32 expression in H. pylori-uninfected gastric mucosa and higher expression levels in gastritis and gastric cancer tissues. H. pylori infection increased IL-32 expression in human gastric epithelial cell lines. H. pylori-induced IL-32 expression was dependent on the bacterial cagPAI genes and on activation of nuclear factor κB (NF-κB). IL-32 expression induced by H. pylori was not detected in the supernatant of AGS cells but was found in the cytosol. Expression of the H. pylori-induced cytokines CXCL1, CXCL2, and IL-8 was decreased in IL-32-knockdown AGS cell lines compared to a control AGS cell line. We also found that NF-κB activation was decreased in H. pylori-infected IL-32-knockdown cells. These results suggest that IL-32 has important functions in the regulation of cytokine expression in H. pylori-infected gastric mucosa.     

白介素32在胃癌细胞中的表达及意义

目的观察白介素32（IL-32）在胃癌细胞株中的表达情况,探讨其在胃癌发生、发展中的意义。方法采用逆转录PCR、real-time PCR及Western blot法分别从基因和蛋白水平上检测IL-32在正常胃上皮细胞GES-1以及胃癌细胞株MKN-45、KATO中的表达情况。结果逆转录PCR及real-time PCR结果均显示胃癌细胞IL-32 mRNA表达水平明显高于正常胃上皮细胞,逆转录PCR灰度比值（目的 /内参）为：GES-1（0.34±0.09）、MKN-45（0.79±0.11）、KATO（0.90±0.17）,差异有统计学意义（P〈0.05）。real-time PCR Ct值显示：MKN-45的IL-32基因表达量是GES-1的（5.34±1.09）倍,KATO是GES-1的（4.07±1.69）倍,差异均有统计学意义（P〈0.05）。Western blot结果显示IL-32在蛋白表达水平的结果与前一致,灰度比值（目的 /内参）为：GES-1（0.30±0.10）、MKN-45（0.92±0.32）、KATO（0.86±0.15）,差异有统计学意义（P〈0.05）。结论IL-32在胃癌细胞株中呈高表达,推测其可能在胃癌的发生、发展中起一定作用。     

Induction of interleukin-8 secretion from gastric epithelial cells by a cagA negative isogenic mutant of Helicobacter pylori.

The ability of Helicobacter pylori strains to induce interleukin-8 (IL-8) gene expression and protein secretion from gastric epithelial cell lines in vitro is variable. This cellular response is associated with bacterial expression of the CagA protein present in type I H pylori strains. To determine the role of CagA in this host cell response, an isogenic cagA negative mutant, N6.XA3, was constructed. The cagA negative isogenic mutant and the wild-type parental cagA positive strain, N6, were cocultured with AGS, ST-42 and KATO-3 gastric epithelial cell lines and secreted interleukin-8 assayed by enzyme linked immunosorbent assay. In all three cell lines there was no significant difference in the IL-8 secretion induced by the cagA negative isogenic mutant, N6.XA3, and the wild-type parent strain, N6. These studies show that CagA is not the inducer of IL-8 secretion from gastric epithelial cells. As all wild-type CagA positive strains studied to date induce IL-8, the bacterial factor(s) inducing this inflammatory response is closely associated with the expression of CagA.     

Methylation-silencing RCC1 expression is associated with tumorigenesis and depth of invasion in gastric cancer

Introduction: Regulator of chromosome condensation 1 (RCC1) is a critical cell cycle regulator. We firstly identified RCC1 gene hypermethylation in gastric tumor tissues using the differential methylation hybridization (DMH) microarray, but the role of RCC1 in the pathogenesis of gastric carcinoma is largely unknown. Methods: Three gastric cancer cell lines (AGS, MKN45, and TSGH9201) were used to analyze RCC1 gene methylation, mRNA and protein expressions. Furthermore, 85 pairs of matched human gastric carcinoma samples in a tissue microarray were used to analyze RCC1 expression by immunohistochemistry staining. Results: A differential methylation pattern was found in TSGH9201 (100%), MKN45 (87%), and AGS (62%) cell lines at the 9th CpG site of RCC1 exon 1. RCC1 mRNA and protein expressions in AGS cells were significantly higher than in TSGH9201 and MKN45 cell lines (P < 0.05). Tissue array data showed that RCC1 expression was detected in 21% (18/85) of gastric carcinoma tissues and in 80% (76/95) of adjacent non-tumor tissues. The expression of RCC1 in gastric carcinoma tissues was significantly lower than in adjacent non-tumor tissues (P < 0.001). Furthermore, an association between RCC1 expression and clinicopathological features showed that RCC1 expression was closely correlated with tumor differentiation and depth of invasion (P < 0.05). Conclusions: Our data indicate that RCC1 expression is frequently lost in poorly differentiated gastric cell lines and gastric carcinoma tissues. Loss of RCC1 expression is correlated with tumor differentiation and depth of invasion. These findings suggest that RCC1 may play a tumor suppressor role in gastric carcinoma.     

IFN-gamma synergizes with TNF-alpha but not with viable H. pylori in up-regulating CXC chemokine secretion in gastric epithelial cells.

Helicobacter pylori colonizes the gastric epithelial surface and induces epithelial cells to increase production of the neutrophil attractant IL-8. Little is known about the role of the gastric epithelium in regulating mucosal T cell trafficking. We therefore characterized constitutive and regulated epithelial expression of the CXC chemokines IP-10, I-TAC and Mig, which specifically attract CXCR3 expressing CD4(+) T cells. Human gastric epithelial cell lines (AGS, Kato III, NCI) were used to characterize the constitutive and regulated expression of three CXC chemokines in response to IFN-gamma, TNF-alpha and different H. pylori preparations. Chemokine mRNA and protein production were measured by RT-PCR and ELISA. Gastric epithelial cells constitutively expressed mRNA for IP-10, Mig and I-TAC. IFN-gamma in combination with TNF-alpha strongly induced secretion of those chemokines. Soluble or membranous fractions of H. pylori significantly inhibited IFN-gamma/TNF-alpha induced epithelial cell IP-10 and Mig production. Gastric epithelial cells may contribute to mucosal T cell trafficking. The capacity of H. pylori products to inhibit IP-10 and Mig secretion may explain, at least in part, the failure to induce protective immunity against this bacterium and the ability of H. pylori to affect the presentation of the local inflammation.     

The scavenger receptor,cysteine-rich domain-containing molecule gp-340 is differentially regulated in epithelial cell lines by phorbol ester

Gp-340 is a glycoprotein belonging to the scavenger receptor cysteine rich (SRCR) group B family. It binds to host immune components such as lung surfactant protein D (SP-D). Recent studies found that gp-340 interacts directly with pathogenic microorganisms and induces their aggregation, suggesting its involvement in innate immunity. In order to investigate further its potential immune functions in the appropriate cell lines, the expression of gp-340 in four conventional immune cell lines (U937, HL60, Jurkat, Raji), and two innate immune-related epithelial cell lines (A549 derived from lung and AGS from stomach), was examined by RT-PCR and immunohistochemistry. The resting immune cell lines showed weak or no gp-340 mRNA expression; while the two epithelial cell lines expressed gp-340 at much higher level, which was differentially regulated by phorbol myristate acetate (PMA) treatment. In the A549 cells, gp-340 was up-regulated along with the PMA-induced proinflammatory expression of both IL-6 and IL-8. In AGS cells, PMA down-regulation of gp-340 was seen in parallel with an up-regulation of the two mature gastric epithelial specific proteins TFF1 (trefoil factor 1) and TFF2, which are implicated as markers of terminal differentiation. Analysis of the distribution of gp-340, together with the TFFs and SP-D in normal lung and gastric mucosa, supported further our in vitro data. We conclude that the differential regulation of gp-340 in the two epithelial cell lines by PMA indicates that gp-340 s involvement in mucosal defence and growth of epithelial cells may vary at different body locations and during different stages of epithelial differentiation.     

IL-22 promotes the migration and invasion of gastric cancer cells via IL-22R1/AKT/MMP-9 signaling

IL-22, one important inflammatory cytokine of the IL-10 family, exerts its functions via IL-22 receptor that is composed of IL-22R1 and IL-10R2 subunits. Although IL-22 expression is reported to be elevated in many cancers, and increased IL-22 expression correlates with tumor progression and poor prognosis, little is known about the role of IL-22 in gastric cancer. In our study, we found that IL-22 stimulation promoted the migration and invasion of SGC-7901 cells. Furthermore, IL-22 increased AKT activation and MMP-9 production in a time- and dose-dependent manner, while knockdown of IL-22R1 attenuated the effect of IL-22 on gastric cancer cells. In addition, blocking of AKT activation suppressed the expression and secretion of MMP-9. Taken together, this present study suggests that IL-22 stimulation enhances the migration and invasion of gastric cancer cells by regulating IL-22R1/AKT/MMP-9 signaling axis.     

GKN2在胃癌细胞增殖、转移中的作用及机制

目的 探讨胃动蛋白2(gastrokine 2,GKN2)对胃癌细胞的生长增殖、侵袭、转移的影响及分子机制.方法 利用qRT-pCR和Western blot法检测GKN2在胃癌组织中的表达;构建对照细胞株AGS-CON、SGC-7901-CON与GKN2过表达细胞株AGS-GKN2、SGC-7901-GKN2,应用q...