幽门螺杆菌依赖蛋白酪氨酸激酶途径诱导胃上皮细胞分泌IL-8

目的 分析中国临床分离的幽门螺杆菌的cag致病岛的差异和不同激酶抑制剂对幽门螺杆菌诱导中国人胃上皮细胞IL-8分泌的影响。方法 在体外分别用中国临床分离的cagA^ cagE^ ,cagA^ cagE^-0、cagA^-cagE^ 、cagA^-cagE^-的幽门螺杆菌与中国人胃上皮细胞MGC-803共培养,分泌的IL-8用ELISA进行检测,比较蛋白激酶A、C、G和蛋白酪氨酸激酶的抑制剂对幽门螺杆菌诱导分泌的IL-8用ELISA进行检测,比较蛋白激酶A、C、G和蛋白酪氨酸激酶的抑制剂对幽门螺杆菌诱导胃上皮细胞IL-8分泌的影响。结果 cagA^ cagE^ 幽门螺杆菌显著增加了胃上皮细胞IL-8的分泌,cagA^ cagE^-,cagA^-cagE^ 幽门螺杆菌作用次之,而cagA^-cagE^-幽门螺杆菌不能增加胃上皮细胞IL-8的分泌,蛋白激酶A、C、G的抑制剂不能阻断幽门螺杆菌增加胃上皮细胞IL-8的分泌,而蛋白酪氨酸激酶的抑制剂阻断了幽门螺杆菌增加胃上皮细胞IL-8的分泌。结论 cagA^ cagE^ 幽门螺杆菌显著增加了胃上皮细胞IL-8的分泌并且依赖于蛋白酪氨酸激酶的活性。     

幽门螺杆菌毒素相关蛋白CagA上调胃泌素基因表达

目的 对幽门螺杆菌分泌的cagA蛋白能否调控胃泌素基因表达及作用机制进行研究.方法 用含有cagA基因的真核表达载体——pcDNA3.1ZEO(-)/cagA7转染AGS和SGC-7901细胞;同时培养幽门螺杆菌NCTC11637后感染AGS和SGC-7901细胞;加入JAK2信号通路抑制剂AG490和ERK信号通路抑制剂U0126,实时荧光定量PCR检测转染和感染细胞中胃泌素mRNA的表达.结果 用PeDNA3.1ZEO(-)/eagA7转染和NCTC11637感染胃癌细胞AGS和SGC-7901后胃泌素mRNA表达最显著增加(P＜0.05),但加入AG490和U0126后胃泌素mRNA的表达量显著降低(P＜O.05).结论 cagA上调胃泌素基因的表达,ERK/MAPK和JAK/STAT信号通路参与了CagA对胃泌素表达的调控.     

胃十二指肠疾病时胃液表皮生长因子及CagA幽门螺杆菌感染的关系

为探讨胃液表皮生长因子（EGF）含量变化及幽门螺杆菌（Hp）感染与胃十二指肠疾病的相关性。以幽门螺杆菌感染阳性的78例消化性溃疡和61例慢性胃炎患者为研究对象,健康人20例作对照,观察胃液EGF含量变化与胃十二指肠疾病的相关性。结果发现,消化性溃疡和慢性胃炎患者胃液EGF含量分别为175．1±103．5ng／L和240．9±182．1ng／L,明显低于正常人,511.9±54．1ng／L,（均P＜0.01）。消化性溃疡患者EGF含量明显低于慢性胃炎患者,P＜0．01。具有CagA基因的Hp感染的消化性溃疡患者EGF含量明显低于慢性胃炎组,P＜0．05。表明胃液EGF含量与Hp感染有关,尤其是具有CagA基因的幽门螺杆菌感染。     

ET-1介导损伤气道上皮诱导成纤维细胞活化的信号机制

目的： 探讨p38丝裂原活化蛋白激酶(MAPK)、磷脂酰肌醇3-激酶(PI3K/Akt)在内皮素(ET)-1介导损伤气道上皮诱导上皮下成纤维细胞活化过程中的作用及对白细胞介素(IL)-6的影响。方法：将正常或经多聚左旋精氨酸(PLA)刺激的人气道上皮细胞与原代气道成纤维细胞共培养并分别加入p38 MAPK、PI3K特异性抑制剂SB203580、LY294002或ET受体A阻断剂BQ123,应用免疫组化、免疫印迹技术或ELISA检测成纤维细胞α-平滑肌肌动蛋白（α-SMA）表达、p38 MAPK、Akt的活化及成纤维细胞上清IL-6水平;制备成纤维细胞胶原凝胶并与不同方法处理的上皮细胞共培养,测量各组凝胶面积变化以了解上皮细胞对成纤维细胞收缩反应的诱导及其上述处理因素的影响。结果：与损伤上皮细胞共培养的成纤维细胞上清中ET-1、IL-6水平［（13.69±1.36） ng/L、(56.7±10.7) ng/L］明显高于与正常上皮细胞共培养的成纤维细胞上清［（3.79±0.64） ng/L、（15.5±3.2）ng/L］,BQ123、SB203580或LY294002皆不同程度减弱损伤上皮细胞诱导的IL-6释放［分别为(27.2±3.1) ng/L、(31.5±3.6) ng/L、(41.3±3.2) ng/L］;成纤维细胞与损伤气道上皮细胞共培养后p38 MAPK、Akt先后激活,BQ123减弱磷酸化p38 MAPK、Akt水平,SB203580浓度依赖性减弱Akt磷酸化水平,而LY294002对磷酸化p38 MAPK水平影响很小。与损伤气道上皮细胞共培养后成纤维细胞α-SMA表达增加,并且胶原收缩百分比明显大于与正常气道上皮共培养的成纤维细胞［(61.2±2.7)% vs (15.4±7.3)%］;BQ123、SB203580及LY294002皆不同程度减弱成纤维细胞α-SMA表达与凝胶收缩且BQ123、SB203580抑制凝胶收缩作用较LY294002更明显。结论：ET-1通过激活p38 MAPK、PI3K/Akt信号通路并促进IL-6分泌在损伤气道上皮诱导成纤维细胞活化过程中发挥关键作用。     

幽门螺杆菌及其L型与表面蛋白-D的结合力的检测与分析

目的 研究幽门螺杆菌（Hp）及其L型与表面活性蛋白-D（SP-D）的结合力，探讨SP-D在Hp感染和免疫中的意义。方法 用Tris-HCl－EDTA法和麦芽糖-琼脂糖亲和柱层析法制备大鼠肺SP-D。用Hp与SP-D进行玻片和试管凝集试验，加入SP-D家兔抗血清进行凝集抑制试验。以抗生素诱导法获得Hp L型，通过Hp 16SrDNA聚合酶链反应和扩增产物的序列测定鉴定L型菌种。以玻片凝集试验检测Hp L型与SP-D的结合力。结果Hp NCTC11637可与SP-D发生肉眼可见的玻片凝集，而Hp SS1、NCTC11639及L型与SP-D未发生凝集。Hp NCTC11637与SP-D在反应45min时出现明显凝集。SP-D抗血清抑制Hp与SP-D的凝集。结论 Hp不同菌株与SP-D的结合力不同；发生细胞壁缺陷变异后Hp与SP-D不再发生明显的凝集。     

幽门螺杆菌感染在肝硬化、肝癌高动力循环及胃黏膜病变中的作用

目的：为探讨幽门螺杆菌感染在肝硬化、肝硬化并发肝癌患者高动力循环及胃黏膜病变中的作用.方法：应用快速尿素酶试验、14C-尿素呼气试验、鲎血试验定量法、免疫印迹试验、亚硝酸盐比色法及ELISA法检测肝硬化、肝硬化并发肝癌患者胃黏膜标本HP感染;胃黏膜组织、外周血IL-1、IL-8、TNF-α、内毒素、一氧化氮（NO）、内皮素（ET）及VacA和CagA抗体水平;应用彩色多普勒超声检测门静脉系统血流量参数.结果：肝硬化患者HP感染率77.1%,VacA和CagA抗体同时阳性率为60.4%;VacA和CagA抗体阳性HP感染的肝硬化患者胃黏膜组织、外周血IL-1、IL-8、TNF-α、内毒素、NO及ET水平明显高于VacA和CagA抗体阴性HP感染的肝硬化患者（P＜0.05,P＜0.01）,VacA和CagA抗体阳性HP感染的肝硬化患者其脾静脉血流量（SVF）与肠系膜上静脉血流量（SMVF）之和显著大于、门静脉血流量（PVF）显著小于VacA和CagA抗体阴性HP感染的肝硬化患者SVF与SMVF之和及PVF.结论：产毒素幽门螺杆菌感染在肝硬化、肝癌高动力循环及其胃黏膜病变中具有重要作用.     

急性呼吸窘迫综合征患者血浆TNF-α、IL-6、IL-10和IL-13水平变化

目的：通过检测分析急性呼吸窘迫综合征（ARDS）和全身炎症反应综合征（SIRS）病人血浆促炎症细胞因子肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）和抗炎细胞因子白细胞介素10（IL-10）、白细胞介素13（IL-13）水平的变化，探讨这些细胞因子在ARDS炎症发展机制中的作用。 方法： 选择临床诊断ARDS病例22例和SIRS 8例，以及正常对照10例，收集血样品，采用酶联免疫法（ELISA）检测TNF-α、IL-6和IL-10、IL-13蛋白含量。 结果： ARDS病人血浆TNF-α、IL-6、IL-10、IL-13含量分别为(629.30±187.00)ng/L、(261.10±71.30)ng/L、(458.10±111.93)ng/L、(5.21±2.02) ng/L，SIRS病人则分别为(206.10±85.90) ng/L、(141.40±41.50)ng/L、(259.60±54.34) ng/L、(1.69±0.39) ng/L，两者血浆细胞因子水平比较有显著差异(P<0.01)；但SIRS和ARDS病人的细胞因子水平均显著高于正常对照组(P<0.01)。 结论： TNFα、IL-6是SIRS和ARDS演变中的重要促炎细胞因子，抗炎细胞因子IL-10和IL-13的过度释放在促进炎症反应失控和ARDS发展中发挥一定作用。     

白细胞介素-18基因多态性与其表达量关系的研究

目的:探讨IL-18基因启动子-607C/A、-137G/C位点多态性是否影响其表达量。方法:选择80例体检健康个体,确定IL-18基因启动子区-137G/C、-607C/A位点基因型,分离上述研究对象外周血单个核细胞(PeripheralBloodMonocytes,PBMC),统一浓度培养24h后,收集培养细胞和上清液。采用ELISA检测上清液中IL-18的含量,并用RT-PCR检测培养细胞中IL-18mRNA的水平。结果:IL-18基因启动子-607位点CC、CA和AA基因型个体PBMC分泌IL-18的浓度分别为(11.54±6.48)ng/ml、(10.92±5.16)ng/ml和(11.79±3.18)ng/ml,表达IL-18mRNA水平分别为0.878±0.633、0.877±0.521和0.881±0.400;IL-18基因启动子-137位点GG、GC或CC基因型个体PBMC分泌IL-18的浓度分别为(11.27±5.42)ng/ml和(11.31±4.62)ng/ml,表达IL-18mRNA水平分别为0.835±0.485和0.984±0.613。两个位点各基因型间个体PBMC分泌和表达IL-18水平比较,差异无显著性(P>0.05)。结论:IL-18基因外显子1上游启动子-607C/A、-137G/C位点基因多态性对IL-18分泌和表达量没有明显影响。     

幽门螺杆菌对大鼠胃上皮细胞环氧合酶-2表达的影响

目的： 探讨幽门螺杆菌（Hp）水提取物对大鼠胃上皮细胞环氧合酶-2（COX-2）表达及前列腺素E₂（PGE₂）合成的影响。方法：大鼠胃上皮细胞株RGM1体外常规培养，Hp组细胞培养液内Hp水提取物终浓度为2.5 mg/L、5 mg/L、10 mg/L，大肠埃希菌组大肠埃希菌水提取物终浓度10 mg/L。培养 24 h 后收集细胞和上清液分别用于Western blotting分析COX-1、COX-2表达和酶免疫方法测定前列腺素E₂（PGE₂）含量。结果：Hp水提取物剂量依赖地增加RGM1细胞COX-2表达但不影响COX-1表达，而大肠埃希菌水提取物对COX-1和COX-2表达均无明显影响。RGM1细胞培养上清液中PGE₂水平在Hp组（2.5、5、10 mg/L）和大肠埃希菌组分别为（230.70±48.55）ng/g protein、（291.82±33.49）ng/g protein、（342.94±28.70）ng/g protein和（130.54±42.81）ng/g protein。结论：Hp体外诱导大鼠胃上皮细胞COX-2表达而增加PGE₂合成，Hp感染的致癌机制可能与其诱导的COX-2表达有关。     

幽门螺杆菌作用巨噬细胞后诱导的炎症反应研究

目的:探讨幽门螺杆菌(Helicobacter pylori,H.pylori)作用巨噬细胞后导致的细胞炎症反应机制及对宿主巨噬细胞的损伤作用。方法:ELISA方法检测幽门螺杆菌感染巨噬细胞后细胞培养上清液中细胞因子IL-23、IL-10、TNF-α及IL-8的含量,Western blot分析幽门螺杆菌作用巨噬细胞后细胞胞内蛋白NOS2、COX2的表达水平;同时流式细胞技术分析幽门螺杆菌处理巨噬细胞后对细胞的凋亡作用。结果:细胞因子IL-23、IL-10、TNF-α、IL-8在幽门螺杆菌作用巨噬细胞后细胞培养上清中分泌显著增多(P0.05),且NOS2、COX2蛋白表达显著增强(P0.03);幽门螺杆菌处理巨噬细胞后巨噬细胞凋亡显著增多(P0.05)。结论:幽门螺杆菌作用巨噬细胞后诱导巨噬细胞增强炎症反应而抑制或杀伤幽门螺杆菌,同时长时间作用能诱导宿主巨噬细胞凋亡。     

Production of a monoclonal antibody against the 128 kDa (CagA) protein of Helicobacter pylori

Helicobacter pylori is recognised as an important factor in gastroduodenal pathology. The 128 kDa CagA protein has been established as a useful marker of H. pylori strains associated with more severe forms of disease. A mouse monoclonal antibody raised against the CagA protein has been produced and characterised as belonging to the IgG1 subtype. It identified the protein in all clinical isolates (10/10) from this laboratory and in two NCTC reference strains (NCTC 11637 and NCTC 11961). No cross-reacting proteins were detected in H. pylori L2, a well characterised strain known not to contain the cagA gene, or in four Helicobacter sp. from non-human sources (H. canis, H. mustelidae, H. muridarum and H. acinonyx). The monoclonal antibody was used to develop an antigen capture ELISA system for detecting the presence of antibodies to the CagA protein in human serum samples.     

标准菌株和临床菌株oipA基因的检测及其核苷酸序列比对

目的：检测幽门螺杆菌标准菌株NCTC11637及临床分离菌株Hp1和Hp2的oipA基因,分析其核苷酸序列,比对其与国际标准菌株Hp 26695的同源性.方法：常规方法培养幽门螺杆菌,提取DNA,PCR法扩增oipA基因,检测其核苷酸序列,并比较其与Hp 26695的同源性.结果：NCTC11637及Hp1、Hp2均表达oipA基因.其核苷酸序列与Hp 26695比对,NCTC11637有48个突变位点、Hp1有48个突变位点、Hp2有50个突变位点,同源性均为94%.NCTC11637与Hp1的同源性为100%、与Hp2的同源性为97%.结论：NCTC11637、Hp1、Hp2均表达oipA基因,但不同菌株oipA基因的核苷酸序列有所不同.     

Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease.

The pathogenic role of Helicobacter pylori virulence factors has been studied with a mouse model of gastric disease. BALB/c mice were treated orally with different amounts of sonic extracts of cytotoxic H. pylori strains (NCTC 11637, 60190, 84-183, and 87A300 [CagA+/Tox+]). The pathological effects on histological sections of gastric mucosae were assessed and were compared with the effects of treatments with extracts from noncytotoxic strains (G21 and G50 [CagA-/Tox-]) and from strains that express either CagA alone (D931 [CagA+/Tox-]) or the cytotoxin alone (G104 [CagA-/Tox+]). The treatment with extracts from cytotoxic strains induced various epithelial lesions (vacuolation, erosions, and ulcerations), recruitment of inflammatory cells in the lamina propria, and a marked reduction of the mucin layer. Extracts of noncytotoxic strains induced mucin depletion but no other significant pathology. Crude extracts of strain D931, expressing CagA alone, caused only mild infiltration of inflammatory cells, whereas extracts of strain G104, expressing cytotoxin alone, induced extensive epithelial damage but little inflammatory reaction. Loss of the mucin layer was not associated with a cytotoxic phenotype, since this loss was observed in mice treated with crude extracts of all strains. The pathogenic roles of CagA, cytotoxin, and urease were further assessed by using extracts of mutant strains of H. pylori defective in the expression of each of these virulence factors. The results obtained suggest that (i) urease activity does not play a significant role in inducing the observed gastric damage, (ii) cytotoxin has an important role in the induction of gastric epithelial cell lesions but not in eliciting inflammation, and (iii) other components present in strains which carry the cagA gene, but distinct from CagA itself, are involved in eliciting the inflammatory response.     

DNA-DNA hybridization incompatibility of Campylobacter pylori with other Campylobacter and Wolinella species

DNA-DNA hybridization in solution was used to characterize 23 human isolates of Campylobacter pylori. The 23 isolates showed DNA affinity with the type strain (NCTC 11637). The relative binding ratios varied between 0.83 and 1. Type strains of C. coli (NCTC 11366), C. jejuni (NCTC 11351), C. laridis (NCTC 11352), C. sputorum subsp. sputorum (ATCC 35980), Wolinella recta (NCTC 11489) and W. succinogenes (ATCC 29543) showed relative binding ratios less than 0.01 compared to the C. pylori type strain. The results suggest that C. pylori is a homogenous taxonomic unit distinctly separated from these other species.     

霉酚酸影响T细胞增殖及细胞因子表达的体外实验

目的： 探讨霉酚酸（MPA）或与抗B7-1单克隆抗体联用对T细胞增殖的影响及其机制。 方法： 体外建立T细胞反应系统，BrdU掺入法检测T细胞增殖，RT-PCR及ELISA法检测细胞因子IL-2、IFN-γ、IL-10的mRNA及蛋白表达水平。 结果： （1）50 μmol/L MPA能明显抑制T细胞增殖，与对照组相比，差异显著（P＜0.01)。（2）MPA可抑制IL-2、IFN-γ表达，增强IL-10表达。IL-2蛋白表达水平在MPA 组明显低于对照组(42.73 ng/L±14.64 ng/L vs 99.70 ng/L±9.15 ng/L，P＜0.01)；IFN-γ蛋白表达水平在MPA组明显低于对照组（7.87 ng/L±4.22 ng/L vs 82.42 ng/L±25.55 ng/L，P＜0.05)；与对照组比较，MPA明显增强IL-10表达水平（770.95 ng/L±126.85 ng/L vs 545.71 ng/L±22.45 ng/L，P＜0.05）；联用抗B7-1单抗能加强此效应（941.90 ng/L±56.61 ng/L vs 770.95 ng/L±126.85 ng/L，P＜0.05)。（3）IL-2、IFN-γ mRNA表达量在MPA组均明显低于对照组（0.74±0.10 vs 1.17±0.15，0.52±0.05 vs 0.75±0.12，P＜0.01）；IL-10 mRNA表达水平在MPA处理组与对照组间差异不明显，但与抗B7-1单抗联用后，其表达水平高于单用MPA组（1.28±0.06 vs 0.84±0.09，P＜0.01）。 结论： MPA可改变细胞因子表达谱，促使Th1亚群向Th2亚群偏移可能是其发挥免疫负调节作用的机制之一；联用抗B7-1单抗可能有一定协同效应。     

Endocrinology and paracrinology: Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells

lnterleukin-8 (IL-8) is a chemoattractant and activating factorfor human neutrophils and a potent angiogenic agent. The peritonealfluid of women with endometriosis has been shown to have increasedneutrophil chemotactic activity. We postulate that IL-8 maybe an important modulator in the pathogenesis of endometriosisand adhesion formation. We first investigated IL-8 concentrationsin the peritoneal fluid of women with or without endometriosis,then assessed peritoneal mesothelial cells as a potential sourceof peritoneal fluid IL-8. Northern blot analysis and enzyme-linkedimmunosorbent assay (ELISA) were used to investigate IL-8 mRNAand protein modulation. The mean concentration of IL-8 in samplesobtained from control patients (n = 28) was 4.8 ± 0.5pg/ml; from patients with minimal-mild endometriosis (n = 24)was 27.5 ± 2.6 pg/ml; and from patients with moderate-severeendometriosis (n = 21) was 530.2 ± 65.1 pg/ml. Confluentmesothelial cells were incubated with human recombinant IL-1a(0.01–100 IU/ml) or tumour necrosis factor (TNF)-a (0.01to 100 ng/ml) for 2–24 h. IL-8 mRNA was detectable innon-treated cells, however both IL-1a and TNF-