红霉素对支气管上皮细胞细胞外信号调节蛋白激酶1及转录激活蛋白-1的影响

目的探讨红霉素对支气管上皮细胞细胞外信号调节蛋白激酶1(ERK1)及转录激活蛋白-1(AP-1)活性的影响。方法将人支气管上皮细胞(16-HBE)分为4-羟基壬烯醛(4-HNE)组(10μmol/L)和对照组,4-HNE刺激细胞0.5h、2h、4h、8h及12h后,检测磷酸化c-Jun氨基末端激酶(JNK)、p38丝裂素活化蛋白激酶(MAPK)、ERK1/2及AP-1活性的变化,观察MAP激酶1(MEK1)抑制剂PD98059对4-HNE引起的AP-1结合活性的影响;观察红霉素对磷酸化ERK1、AP-1结合活性的影响。结果 4-HNE组在2h、4h、8h及12h各时间段ERK1/β-actin分别为2.1依0.4、1.8依0.4、1.6依0.6、1.3依0.8,对照组分别为4.2依0.2、4.8依0.7、4.4依0.5、4.3依0.6,两组比较差异有统计学意义(均P约0.05)。4-HNE组各时间段AP-1结合活性表达分别为90.6依2.0、85.7依2.2、78.2依2.6、70.6依1.8和64.9依4.8,对照组分别为98.6依2.1、98.7依3.4、100.1依3.8、101.3依4.2和97.4依3.6,两组比较差异有统计学意义(均P约0.05)。PD98059和红霉素可降低4-HNE引起的AP-1结合活性,红霉素增加4-HNE引起的磷酸化ERK1的表达。结论红霉素引起支气管上皮细胞ERK1磷酸化的增加,但可以抑制其AP-1的结合活性,可能与对ERK1/2下游信号传导通路的阻断有关。     

同型半胱氨酸对谷胱甘肽合成的抑制作用

目的：探讨同型半胱氨酸对谷胱甘肽合成的影响及其发生的可能原因.方法：实验于2004-08/2005-02在新乡医学院生物化学与分子生物学实验室进行.选用健康家兔20只,取抗凝血,离心收集红细胞,制备含有γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶体系的红细胞裂解液,设置对照组,高、低剂量同型半胱氨酸处理组,每组均设8个平行管.各组均加入100mmol/L的Tris-Hcl缓冲液（pH 8.0）60 μL,1 mol/L MgCl2 8 μL,10 mmol/L ATP 40μL,50 mmol/L谷氨酸（调pH为中性）40 μL,红细胞裂解液40μL.低、高浓度同型半胱氨酸处理组再分别加入100 mmol/L同型半胱氨酸20μL和60μL,其余实验条件相同.以反应中生成无机磷的mmol数表示γ-谷氨酰半胱氨酸合成酶活性大小.分别用紫外分光光度法和荧光法检测各组无机磷和谷胱甘肽含量.结果：[1]无机磷的生成量：低浓度同型半胱氨酸组和高浓度同型半胱氨酸组明显低于对照组[（1.476&;#177;0.240）,（0.751&;#177;0.085）,（3.279&;#177;0.470）mmol/L,P＜0.01],高浓度同型半胱氨酸组明显低于低浓度同型半胱氨酸组（P＜0.05）.[2]还原型谷胱甘肽含量：低同型半胱氨酸组和高同型半胱氨酸组明显低于对照组[（3.058&;#177;0.264）,（1.908&;#177;0.301）,（4.476&;#177;0.462）μmol/L,＜0.01],高浓度同型半胱氨酸组明显低于低浓度同型半胱氨酸组（P＜0.01）.结论：同型半胱氨酸有可能通过竞争性抑制γ-谷氨酰半胱氨酸合成酶的活性,减少细胞内还原型谷胱甘肽的生成.     

同型半胱氨酸对谷胱甘肽合成的抑制作用

目的:探讨同型半胱氨酸对谷胱甘肽合成的影响及其发生的可能原因。方法:实验于2004-08/2005-02在新乡医学院生物化学与分子生物学实验室进行。选用健康家兔20只,取抗凝血,离心收集红细胞,制备含有γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶体系的红细胞裂解液,设置对照组,高、低剂量同型半胱氨酸处理组,每组均设8个平行管。各组均加入100mmol/L的Tris-Hcl缓冲液(pH8.0)60μL,1mol/LMgCl28μL,10mmol/LATP40μL,50mmol/L谷氨酸(调pH为中性)40μL,红细胞裂解液40μL。低、高浓度同型半胱氨酸处理组再分别加入100mmol/L同型半胱氨酸20μL和60μL,其余实验条件相同。以反应中生成无机磷的mmol数表示γ-谷氨酰半胱氨酸合成酶活性大小。分别用紫外分光光度法和荧光法检测各组无机磷和谷胱甘肽含量。结果:①无机磷的生成量:低浓度同型半胱氨酸组和高浓度同型半胱氨酸组明显低于对照组犤(1.476±0.240),(0.751±0.085),(3.279±0.470)mmol/L,P<0.01犦,高浓度同型半胱氨酸组明显低于低浓度同型半胱氨酸组(P<0.05)。②还原型谷胱甘肽含量:低同型半胱氨酸组和高同型半胱氨酸组明显低于对照组犤(3.058±0.264),(1.908±0.301),(4.476±0.462)μmol/L,P<0.01犦,高浓度同型半胱氨酸组明显低于低浓度同型半胱氨酸组(P<0.01)。结论:同型半胱氨酸有可能通过竞争性抑制γ-谷氨酰半胱氨酸合成酶的活性,减少细胞内还原型谷胱甘肽的生成。     

吸烟对人气道上皮细胞γ-谷氨酰半胱氨酸合成酶表达的影响

目的 通过观察吸烟对人气道上皮细胞γ-谷氨酰半胱氨酸合成酶(γ-GCS)表达的影响,探讨γ-GCS在吸烟所致慢性气道炎症及氧化/抗氧化失衡中的作用.方法 选取行支气管镜检查的患者40例,分为4组:吸烟慢支组、吸烟非慢支组、不吸烟慢支组、不吸烟非慢支组,每组各10例.通过支气管镜刷检获取支气管上皮细胞,采用免疫细胞化学法半定量检测支气管上皮细胞中γ-GCSh蛋白的表达水平.结果 ①吸烟慢支组气道上皮细胞γ-GCSh的表达水平(0.216±0.120)低于不吸烟慢支组(0.426±0.111)、不吸烟非慢支组(0.589±0.220)及吸烟非慢支组(0.386±0.175),差异均有统计学意义(P均＜0.05);②吸烟非慢支组气道上皮细胞γ-GCSh的表达水平(0.386±0.175)低于不吸烟非慢支组(0.589±0.220),差异有统计学意义(P＜0.05);③不吸烟慢支组气道上皮细胞γ-GCSh的表达水平(0.426±0.111)低于不吸烟非慢支组(0.589±0.220),差异有统计学意义(P＜0.05).结论 γ-GCSh在不吸烟非慢支者表达最强,而在吸烟慢支者的气道内表达降低.提示γ-GCS在香烟所致气道炎症、氧化应激及氧化/抗氧化失衡中发挥一定的作用.     

左卡尼汀对肾脏缺血再灌注损伤中Nrf2和7-GCS表达影响

目的研究左卡尼汀对大鼠肾脏缺血再灌注损伤中核因子相关因子2（Nrf2）、γ-谷氨酰半胱氨酸合成酶（3＇-GCS）表达的影响。方法将大鼠随机分为假手术组、缺血再灌注组及治疗组。缺血再灌注组及治疗组建立肾脏缺血再灌注损伤模型,治疗组在缺血再灌注损伤模型建立前后尾静脉注射左卡尼汀2mL。假手术组不行缺血再灌注处理。再灌注6h后处死大鼠,取血检测血清肌酐（cr）、尿素氮（BUN）及胱抑素C（Cysc）的水平,并测定血清超氧化物歧化酶（SOD）活性及丙二醛（MDA）含量。应用RT—PCR及Western—blot方法检测肾组织中Nrf2和7-GCS的表达水平。结果治疗组Cr、BUN、CysC及MDA水平显著低于缺血再灌注组,SOD活性显著高于缺血再灌注组（F=8．58～57．42,q=4．06～11．26,P〈0．05）;与假手术组相比,缺血再灌注组肾组织中Nrf2、3＇-GCSmRNA及蛋白表达水平显著升高,而治疗组肾组织中上述指标较缺血再灌注组显著增高（F=143．53～241．64,q=3．76～13．51,P〈0．05）。结论左卡尼汀可减轻肾脏缺血再灌注损伤,其机制可能为通过激活Nrf2-ARE通路进而诱导3＇-GCS的表达而实现的。     

线粒体ALDH2在心血管疾病中作用机制的研究进展

心血管疾病是一种由多因素引起的具有高发病率、高死亡率的慢性非传染性疾病。目前已成为人类的首要死亡原因。线粒体乙醛脱氢酶2(ALDH2)是一种广泛存在于哺乳动物体内的醛类氧化还原酶,它可以通过代谢有毒醛类[如乙醛、4-羟基壬烯醛(4-HNE)、丙二醛(MDA)等]、抗氧化应激反应、调控细胞凋亡、保护线粒体功能等途径减轻对心肌细胞的损伤。文章主要对目前有关线粒体ALDH2在心血管疾病中作用机制的研究进行综述,希望为心血管疾病未来的研究提供新的思路和方向。     

阳和平喘颗粒对哮喘模型大鼠IgE、IL-4及AP-1表达的影响

目的:通过观察阳和平喘颗粒对哮喘大鼠模型肺组织活化蛋白-1(activatorprotein-1,AP-1)及免疫球蛋白E(Immunoglobulin E,Ig E)、白介素-4(Interleukin4,IL-4)的影响,探讨阳和平喘颗粒防治哮喘的可能作用机制。方法:选择50只健康雄性大鼠,随机分为正常组、模型组、阳和平喘高、中、低剂量组。采用卵清白蛋白(Oalbumin,OVA)致敏的方法复制哮喘大鼠模型。各给药组分别给予相应剂量的药物,连续灌胃14天。正常组和模型组给予等体积生理盐水。给药结束后取主动脉血和肺组织,检测血清IL-4和Ig-E含量,肺组织匀浆采用RT-PCR测定AP-1m RNA。结果:与正常组相比,模型组大鼠的血清IL-4和Ig-E含量明显升高(P0.01),AP-1m RNA的c-Fos和c-Jun表达量明显增高(P0.01);与模型组相比,各给药组IL-4和Ig-E含量显著低于模型组(P0.01),且呈量效关系,阳和平喘高、中剂量组AP-1m RNA蛋白表达量明显降低(P0.01),阳和平喘低剂量组的蛋白表达也有一定降低,但无统计学意义。结论:阳和平喘颗粒可通过下调AP-1m RNA的表达量,阻碍细胞下游因子IL-4、Ig-E的合成,推断其为防治支气管哮喘的作用机制。     

不同潮气量机械通气对大鼠肺组织γ-谷氨酰半胱氨酸合成酶和转化生长因子β1的影响

目的 观察不同潮气量机械通气大鼠肺组织γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase,γ-GCS)和转化牛长因子β_1(transforming growth factor-β_1,TGF-β_1)mRNA及其蛋白表达水平,探讨氧化/抗氧化失衡在呼吸机相关性肺损伤(ventilator associated lung injury,Vau)中的作用.方法 24只雄性Wistar大鼠随机(随机数字法)分为对照组、小潮气量组和大潮气量组(各组8只),测定其肺组织和血浆中MDA含量,并分别采用原位分子杂交技术和免疫组织化学染色法检测肺组织γ-GCS,TGF-β_1,mRNA及其蛋白表达水平,并进行相关性分析.组间差异比较采用方差分析法,各组均数间两两比较采用SNK-q检验,以P＜0.05表示差异有统计学意义,相关性分析采用Pearson直线相关分析法.结果 与对照组和小潮气量组比较,大潮气量组大鼠肺组织和血浆MDA含量、肺组织TGF-β_1mRNA及其蛋白表达水平明显升高(P＜0.01),而γ-GCS mRNA及其蛋白表达水平则明显低于对照组和小潮气量组(P＜0.01);小潮气量组与对照组各项指标比较,差异无统计学意义(P＞0.05).相关分析结果表明大潮气量组大鼠肺气道卜皮细胞γ-GCS和TGF-β_1 mRNA及其蛋白表达均呈负相关(r值分别为-0.96,-0.85,P＜0.01).结论 大潮气量机械通气町诱导肺组织TGF-β_1 mRNA及其蛋白高表达,使γ-GCS表达降低,谷胱甘肽合成减少,导致局部肺组织氧化/抗氧化系统失衡,是VALI发生的重要因素之一.     

双表达外源性4—1BBL／RANTES的K562细胞活化外周血淋巴细胞的研究

目的探讨K562细胞表达外源性蛋白4-1BBL／RANTES后，对T细胞、NK细胞有无活化作用，为进一步构建人工抗原提呈细胞提供前提。方法采用分子克隆技术，分别将4-IBBL和RANTES基因插入双表达载体pVITRO-2，命名为pV4-1BBL-RANTES。经测序鉴定后，利用脂质体介导的转染及潮霉素筛选，获得稳定双表达4-1BBL、RANTES分子的K562细胞（K562／4—1BBL/RANTES）。经流式细胞仪（FACS）分选后，K562／4—1BBL／RANTES细胞用丝裂霉素C处理，与外周血淋巴细胞孵育24，FACS检测淋巴细胞表面活化性受体CD69的表达；对NK细胞同时检测了活化性受体NKG2D的表达情况。结果受K562／4-1BBL／RANTES细胞刺激后，CD3^＋T细胞、CD4^＋T细胞、γδT细胞和NK细胞的活化性受体CD69的表达与未受刺激前相比，均有明显上调；但与单纯K562细胞刺激组相比，无显著差异。另外，CD8^＋T细胞CD69的表达及NK细胞NKG2D的表达无明显变化。结论将4-1BBL和RANTES共表达于K562细胞，不具备活化淋巴细胞的效应。     

肝X受体对支气管上皮细胞中TGF-β1生物功能的影响

目的观察肝X受体(liver-X-receptors,LXRs)对转化生长因子β1(TGF-β1)生物功能的影响,并探讨其可能的机制。方法将培养的人气道上皮细胞分为对照组、TGF-β1组、LXRs低剂量组、LXRs中剂量组和LXRs高剂量组,LXRs组分别予2.5 mol/L、5 mol/L和10 mol/L的LXRs共孵育2 h后,与TGF-β1组一起加TGF-β1反应。2 h后用Real-time PCR方法检测各组细胞纤溶酶原激活物抑制剂1(PAI-1)的mRNA表达,在反应30 min时用免疫荧光法检测P-Smad2的核转位情况。结果 TGF-β1组PAI-1 mRNA表达较对照组明显升高,差异有统计学意义(P<0.05),LXRs组PAI-1 mRNA表达较TGF-β1组明显降低,差异有统计学意义(P<0.05),LXRs剂量越大,降低越明显,予LXRs 10 mol/L干预PAI-1的mRNA下降最明显。对照组、TGF-β1组、LXRs高剂量组P-Smad2阳性细胞百分比分别为(1.752±0.423)%、(95.060±1.854)%、(1.941±0.409)%,对照组与TGF-β1组比较差异有统计学意义(P<0.05),TGF-β1组与LXRs高剂量组比较差异有统计学意义(P<0.05)。结论 LXRs可能通过减少核内P-Smad2含量来影响TGF-β1的生物学功能。     

氧化损伤产物8-OHdG、4-HNE、NTY在成年小鼠卵巢组织中的增龄变化

目的:探讨DNA、脂质和蛋白质氧化损伤产物在成年小鼠卵巢组织中的增龄变化.方法:选用12、24、48、75周龄自然衰老C57BL/6小鼠为模型.采用免疫组织化学方法和蛋白印迹技术检测各年龄组小鼠卵巢中8羟基脱氧鸟苷(8-hydroxy-2-deoxyguanosine,8-OHdG)、4羟基壬烯醛(4-hydroxynonenal,4-HNE)、硝基酪氨酸(nitrotyrosine,NTY)的表达变化情况.结果:8-OHdG、4-HNE、NTY在卵巢间质细胞随增龄表达增加(P＜0.05).12周龄组3种物质的表达均明显低于其他各组,差异有显著统计学意义(P＜ 0.01);24周龄含量低于48、75周龄组,差异有显著统计学意义(P＜ 0.05);48周龄组与75周龄组比较,差异无统计学意义(P＞0.05).而各年龄组卵泡膜细胞、颗粒细胞及卵母细胞中未见明显变化.结论:氧化损伤在卵巢间质细胞中随增龄表现明显,间质细胞氧化损伤可能是导致卵巢衰老的原因之一.     

Estrogen increases nitric-oxide production in human bronchial epithelium

Although sex differences in asthma severity are recognized, the mechanisms by which sex steroids such as estrogen influence the airway are still under investigation. Airway tone, a key aspect of asthma, represents a balance between bronchoconstriction and dilation. Nitric oxide (NO) from the bronchial epithelium is an endogenous bronchodilator. We hypothesized that estrogens facilitate bronchodilation by generating NO in bronchial epithelium. In acutely dissociated human bronchial epithelial cells from female patients exposure to 17β-estradiol (E(2); 10 pM-100 nM) resulted in rapid increase of diaminofluorescein fluorescence (NO indicator) within minutes, comparable with that induced by ATP (20 μM). Estrogen receptor (ER) isoform-specific agonists (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC) (ERα) and diaryl-propionitrile (DPN) (ERβ) stimulated NO production to comparable levels and at comparable rates, whereas the ER antagonist 7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780) (1 μM) was inhibitory. Estrogen effects on NO were mediated via caveolin-1 (blocked using the caveolin-1 scaffolding domain peptide) and by increased intracellular calcium concentration [prevented by 20 μM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester but not by blocking Ca(2+) influx using LaCl(3)]. Estrogen increased endothelial NO synthase activation (inhibited by 100 μM N(G)-nitro-l-arginine methyl ester) and phosphorylated Akt. In epithelium-intact human bronchial rings contracted with acetylcholine (1 μM), E(2), THC, and DPN all produced acute bronchodilation in a dose-dependent fashion. Such bronchodilatory effects were substantially reduced by epithelial denudation. Overall, these data indicate that estrogens, acting via ERα or ERβ, can acutely produce NO in airway epithelium (akin to vascular endothelium). Estrogen-induced NO and its impairment may contribute to altered bronchodilation in women with asthma.     

Differential diagnostic cytochemical signs of dysplasia of the bronchial epithelium and squamous cell carcinoma of the lung

The cytograms of smears, obtained in bronchoscopic examinations of 59 patients with bronchial epithelium dysplasia and 44 patients with initial forms of squamous-cell carcinoma of the lung are analyzed. A histologic control has been made in all the cases. The authors classify the cytomorphologic criteria, they have statistically processed 140 signs, minimizing their number and selecting their combinations for the differential diagnosis between dysplasia and carcinoma of the lung. Cytologically dysplasia in squamous-cell metaplasia of the bronchial epithelium and dysplasia of the cylindrical epithelium have been distinguished. Three degrees of dysplasia have been singled out: (I) weak, (II) moderate, (III) grave.     

New aspects of studying the morphofunctional insufficiency of bronchial epithelium in chronic bronchitis

The paper is concerned with some data on the state of the epithelium in chronic bronchitis from a view-point of its quantitative assessment with plotting models of the epithelium--morphograms reflecting its morphofunctional insufficiency. A total of 137 patients with chronic bronchitis were investigated. Morphofunctional epithelial insufficiency was shown to be an obligatory symptom of chronic bronchitis. Biopsy of the central, lobar bronchi was sufficient for the detection of morphological (quantitative) signs of chronic bronchitis. Results of morphometric investigations can be applied to differential diagnosis of chronic nonobstructive and obstructive bronchitis as well as chronic bronchitis and other types of pulmonary pathology. Morphometry of biopsy specimens is an indispensable and adequate method of therapeutic control.     

Ultrastructure and mucociliary transport of bronchial respiratory epithelium in intubated patients

Objective The objective of this study was to investigate whether reduced bronchial mucus transport velocity (BTV) is associated with a loss of cilia or ultrastructural abnormalities of cilia in intubated patients.Design The patients were studied prospectively in a convenience sample trial.Setting The study took place in a university hospital.Patients and participants 29 orally intubated patients in a surgical ICU.Interventions BTV was measured with radiolabeled microspheres in the right and left primary bronchus. Following these measurements, biopsy samples were taken from the bronchi for scanning (SEM) and transmission (TEM) electron-microscopic investigations.Measurements and results SEM: Patients with normal or slight impaired BTV (group 1,n=14: BTV: 8.5 mm/min (3.8–11.5); median with range) showed more cilia on the luminal surface than patients with markedly depressed BTV (p<0.05) (group 2,n=15: BTV: 0(0–2.1)). The difference was statistically significant. The BTV values correlated moderately with the number of cilia on the luminal surface (r=0.46;p=0.02). TEM: In group 1, 6.5% (3.9–14.9) of cilia were abnormal (median with range) vs 9.3% (4.9–13.7) in group 2; these differences were not statistically significant. Neither was there any significant correlation between BTV and the frequency of abnormal cilia.Conclusions Impaired mucociliary transport in intubated patients is associated with a loss of cilia rather than ultrastructural abnormalities of cilia, which are less relevant.