Increased serum levels of pigment epithelium-derived factor by excessive alcohol consumption-detection and identification by a three-step serum proteome analysis

Background: The search for biological markers of alcohol abuse is of continual interest in experimental and clinical alcohol research. We previously used gel‐free proteome analysis methods such as the ProteinChip^® system and the ClinProt? system to search for new serum markers for alcoholism and found several novel marker candidates. As serum contains thousands of proteins and peptides that are present in a large dynamic concentration, depletion of the abundant proteins and further fractionation of the remainder is necessary to get into the deep proteome. We recently described a simple and highly reproducible three‐step method for identifying potential disease‐marker candidates among the low‐abundance serum proteins. Methods: Two serum samples—one on admission and one after 8 weeks of abstinence—were obtained from 8 patients with alcohol dependency. The samples were subjected to a three‐step serum proteome analysis. The steps were the following: first, immunodepletion of the 6 most abundant proteins; second, fractionation using reverse‐phase high‐performance liquid chromatography; and third, separation using one‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Differences revealed by protein staining were further confirmed by Western blotting and by enzyme‐linked immunosorbent assays (ELISA). Results: Three‐step serum proteome analysis revealed that the serum levels of 5 proteins, alpha2‐HS glycoprotein, apolipoprotein A‐I, glutathione peroxidase 3, heparin cofactor II, and pigment epithelial‐derived factor (PEDF), were significantly greater on admission than after 8 weeks of abstinence. We focused on PEDF because alterations in its levels in alcoholic subjects are not well known. Western blotting and ELISA confirmed the upregulation of PEDF. Serum PEDF levels were significantly greater in moderate to heavy habitual drinkers (14.2 ± 7.7 μg/ml) than in healthy subjects without a drinking history (5.5 ± 3.0 μg/ml) (p < 0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects. Conclusion: Three‐step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.     

Cumulative glycemia and microangiopathy in subjects with impaired glucose regulation in the Inter99 study

Aims

To assess cumulative glycemia, microvascular characteristics, and associated risk factors for diabetes in subjects with impaired glucose regulation.

Methods

Cross-sectional, population-based study comprising systemic characteristics in 6487 participants and ocular characteristics in 970 participants.

Results

Lens fluorescence, a quantitative index of life-long cumulative glycemia, was increased by 7.5% (CI₉₅ 0.37-15.1%) in subjects with impaired fasting glucose, by 13.0% (CI₉₅ 5.5-21%) in subjects with combined impaired fasting glucose and impaired glucose tolerance (IFG + IGT), and by 11.8% (CI₉₅ 6.8-17.1%) in subjects with screen-detected diabetes compared to normoglycemic subjects, adjusted for age, sex, and smoking. The prevalences of microalbuminuria and retinopathy were significantly increased in subjects with screen-detected diabetes after adjusting for age, sex and systolic blood pressure. The prevalences of associated risk factors for diabetes were elevated in all categories of abnormal glucose regulation compared to normoglycemic subjects.

Conclusions

Life-long cumulative glycemia, microangiopathy, and associated risk factors for diabetes were significantly elevated in subjects with abnormal glucose metabolism, most prominently in subjects with IFG + IGT and in subjects with screen-detected diabetes. These results provide the first objective evidence that cumulative glycemic load is increased at the earliest stage of impaired glucose regulation.     

TNF-α对缺氧复氧损伤的肾小管上皮HK-2细胞NGAL表达的影响

目的研究TNF-α对缺氧复氧损伤的HK-2细胞中NGAL表达的影响,探讨NGAL作为早期AKI标志的意义。方法模拟缺氧复氧模型,RT-PCR法检测不同缺氧时间和不同浓度TNF-α作用后NGAL表达水平。结果缺氧复氧后NGAL表达上调,1、2、4 h组均高于0.5 h组。随TNF-α浓度增高NGAL表达增加。缺氧1 h,同时200μg/L TNF-α刺激后,NGAL明显提高。结论缺氧复氧后,NGAL表达明显增强,TNF-α的大量产生,可能是NGAL表达增加的直接原因之一。     

HSP70-抗原肽负载的DCs对肺癌细胞杀伤作用的实验研究

目的 探讨肿瘤热休克蛋白70-多肽复合物（heat shock protein 70-peptide complexes,HSP70-PCs）负载的树突状细胞（dendritic cells,DCs）对肺癌细胞（SPC-A-1）的杀伤效果,并与未加抗原的DCs进行比较.方法 用加热、ADP亲和层析法提取肺癌细胞HSP70-PCs作为抗原,使之与DCs共同培养,检测其对肺癌细胞的杀伤作用.结果 与未加抗原的DCs比较,加入HSP70-PCs的DCs对肺腺癌细胞SPC-A-1的杀伤效果明显升高.结论 负载HSP70-PCs的DCs对肺腺癌细胞SPC-A-1的杀伤效果明显高于未加抗原的DCs.     

血管紧张素Ⅱ对大鼠肺泡上皮钠通道表达的影响

目的 探讨外源性血管紧张索Ⅱ (Ang Ⅱ)对大鼠肺泡液体清除及肺泡上皮钠通道(ENaC)表达的影响。方法 按随机数字表法将15只SD大鼠分为对照组、Ang Ⅱ组及Ang Ⅱ 1型受体阻滞剂ZD7155干预组,每组5只。Ang Ⅱ组和ZD7155干预组大鼠经左侧颈静脉置管后,微量泵持续泵入外源性Ang Ⅱ10 μg．kg-1．min-1;对照组泵入等量生理盐水。ZD7155干预组于泵入Ang Ⅱ前30 min经腹腔注射10 mg/kg ZD7155。6h后观察肺组织病理学改变;用伊文思蓝标记5％白蛋白法测定离体肺组织肺泡液体清除率(AFC);用逆转录-聚合酶链反应(RT-PCR)测定ENaC mRNA表达,用蛋白质免疫印迹法(Western blotting)测定ENaC蛋白表达。结果 Ang Ⅱ组AFC较对照组显著降低[（6.16±3.01）％比(16.10±3.46)％,P＜0.01],ZD7155干预组AFC较Ang Ⅱ组显著升高[（10.60±2.05）％比(6.16±3.01)％,P＜0.05]。Ang Ⅱ组α-ENaC mRNA表达较对照组显著升高（0.663±0.068比0.236±0.030,P＜0.01）,ZD7155干预组α-ENaC mRNA表达较Ang Ⅱ组显著降低（0.386±0.061比0.663±0.068,P＜0.01）;β-ENaC及γ-ENaC的mRNA表达无明显差异。与对照组比较,Ang Ⅱ组α-ENaC蛋白表达显著增加(0.343±0.053比0.145±0.030,P＜0.01),β-ENaC和γ-ENaC的蛋白表达显著降低（0.286±0.038比0.512±0.055,0.144±0.040比0.460±0.066,均P＜0.01）;与Ang Ⅱ组比较,ZD7155干预组α-ENaC蛋白表达显著降低(0.228±0.045比0.343±0.053,P＜0.01),β-ENaC和γ-ENaC的蛋白表达显著升高(0.358±0.043比0.286±0.038,0.220±0.033比0.144±0.040,均P＜0.05)。结论 外源性Ang Ⅱ通过其1型受体途径调节ENaC基因及蛋白表达,减弱大鼠肺泡液体清除,加重肺水肿。     

丙型肝炎病毒全长基因嵌合萤光素酶重组质粒转染细胞系的建立与初步应用

目的 构建能产生较高效价重组病毒的HCV细胞感染模型,为HCV致病机制的研究和抗病毒药物的筛选提供一个有效的体外细胞培养系统.方法 利用PCR技术在HCV NS5A C端插入海肾萤光素酶( Renilla Luciferase,Rluc)报告基因,并引入能提高HCV效价的突变,酶切鉴定重组基因序列构建成功后,转染入...     

树突细胞疫苗与恶性黑色素瘤免疫治疗研究进展

以免疫治疗为代表的生物治疗日益受到重视。树突细胞（DC）是体内抗原提呈功能最强的细胞,在肿瘤免疫治疗中占重要地位。DC具有加工、提呈抗原的功能,同时DC在启动特异性抗肿瘤免疫反应中发挥着关键性作用。通过不同形式的抗原修饰的DC可增强其抗肿瘤的免疫原性。就DC疫苗与恶性黑色素瘤的免疫治疗研究进展做一综述。     

囊袋张力环在晶状体半脱位超声乳化术中的应用

目的评价在晶状体半脱位超声乳化术中植入囊袋张力环（CTR）的临床应用效果。方法对13例（13眼）晶状体半脱位患者行晶状体超声乳化手术,术中连续环形撕囊后植入囊袋张力环,对晶状体半脱位范围〉1／2圆周者,将囊袋张力环用缝线固定在悬韧带离断一侧巩膜壁上,随后行晶状体超声乳化术,囊袋内植入折叠式人工晶状体。结果13眼晶状体半脱位患者手术均顺利植入了囊袋张力环及后房型人工晶状体,术后患者视力均得到提高,人工晶状体位正,无明显手术并发症。结论囊袋张力环可提高晶状体半脱位超声乳化术的安全性,防止手术并发症,提高手术效果。     

阿糖胞苷对人肺腺癌细胞A549凋亡和脂质过氧化的影响

目的探讨阿糖胞苷（Ara-C）对人肺腺癌细胞A549凋亡和脂质过氧化的影响。方法采用四唑氮蓝（MTT）法测定不同浓度和时间阿糖胞苷对A549细胞的生长抑制作用;采用羟胺比色法对超氧化物歧化酶（SOD）活性进行测定,硫代巴比妥酸比色法进行丙二醛（MDA）含量测定,5.5-二硫代双（2-硝基苯甲酸）比色法进行还原性谷胱甘肽（GSH）含量、谷胱甘肽过氧化物酶（GSH-Px）活力测定。吖啶橙（AO）/溴乙锭（EB）混合染色检测A549细胞凋亡。结果 Ara-C对A549细胞的增殖有明显抑制作用,并具有明显的时效和量效关系。随着干预剂量的增加,细胞内MDA含量水平逐渐升高,与对照组比较（P〈0.05,P〈0.01）;GSH含量,SOD、GSH-Px活性则逐渐下降,与对照组比较（P〈0.05,P〈0.01）;阿糖胞苷作用A549细胞后可以出现凋亡增高和形态学改变。结论阿糖胞苷对人肺腺癌A549细胞有生长抑制作用,并引起脂质过氧化水平改变,其机制可能与诱导A549细胞周期阻滞和凋亡有关。