烧伤后血清触珠蛋白对T淋巴细胞功能的影响

采用亲和层析法从烧伤血清纯化触珠蛋白（Ｈａｐｔｏｇｌｏｂｉｎ，ＨＰ）后，体外观察了纯化ＨＰ对正常小鼠胸腺细胞在ｃｏｎＡ刺激下增殖与ＩＬ－２产生及对ＩＬ－２诱导的ＩＬ－２依赖细胞株（ＣＴＬＬ－２）增殖的影响。结果表明，纯化ＨＰ在相当于烧伤血清ＨＰ浓度时，对小鼠胸腺细胞增殖、ＩＬ－２产生及ＣＴＬＬ－２增殖具有非常明显的抑制作用；但在相当于正常血清ＨＰ浓度时纯化ＨＰ则无抑制作用。提示烧伤血清高浓度的ＨＰ可能是烧伤后Ｔ淋巴细胞免疫功能低下的一个重要原因。     

Haptoglobin genotypes polymorphism as a risk factor for subclinical atherosclerosis in beta-thalassemia major children; a single center Egyptian study

Abstract

Background/Objectives

Haptoglobin (Hp) is an antioxidant protein. Its genotypic polymorphism had been proposed to influence vascular complications among diabetics, but no data are available about this association among thalassemia patients so far. We have investigated the assumption of an association between Hp genotypes and subclinical atherosclerosis among beta-thalassemia major (TM) children.

Methods

One hundred beta-TM children and 70 matched healthy controls were included. Serum ferritin level and fasting lipid profile were assayed. Haptoglobin genotyping was determined by amplification gel electrophoresis. Carotid intima media thickness (cIMT) was measured using high resolution ultrasound.

Results

The relative distribution of the three Hp genotypes among thalassemia group and the control group were 18 and 14.3% for Hp1-1; 38 and 37.1% for Hp2-1; and 44 and 48.6% for Hp2-2 respectively. There was no significant difference between patients and controls regarding Hp genotypes distribution. Hp2-2 genotype TM children had significantly higher cIMT compared to other genotypes (P < 0.0001). Elevated cIMT was significantly represented in Hp2-2 genotype patients (P < 0.0001) who had higher serum ferritin compared to their counterparts (P < 0.05). Hp2-2 patients were five times more likely to suffer from subclinical atherosclerosis than Hp1-1 and six times than Hp2-1 genotype patients (P = 0.008 and 0.001, respectively); a difference that persisted significant after adjustment for some risk factors compared to Hp2-1 patients (OR 3.96; P = 0.02).

Conclusions

Hp2-2 genotype is a significant predictor for premature atherosclerosis in TM children and confers them an increased risk for iron overload.     

卵巢癌患者血清中肿瘤特异标志物的鉴定研究

目的:通过分析卵巢癌患者血清中的蛋白质组分,寻找可能的肿瘤相关蛋白并对该蛋白鉴定,以期确定其作为卵巢癌血清学诊断的特异标志物。方法:采用双向凝胶电泳方法分离6例卵巢癌患者及2例正常健康妇女血清总蛋白,经考马斯亮蓝染色后对比找出差异蛋白斑点,该蛋白斑点经蛋白酶水解后进行液质联用串联质谱(LC-MS/MS)分析,用SEQUEST搜索软件查询Finngan公司提供的FASTA格式蛋白序列数据库。采用WesternBlotting、ELISA、斑点印迹及亲和层析等方法对目标蛋白作进一步的分析。结果:经双向电泳分离,卵巢癌患者血清中有3-4个组分显示含量高于正常健康妇女血清,选其中最明显的一个差异斑点进行质谱分析,对库检索确定为结合珠蛋白。进一步的分析证实,卵巢癌患者血清中结合珠蛋白总含量有高于正常健康妇女的趋势。此外,通过凝集素斑点印迹法检测发现,岩藻糖基化结合珠蛋白成分明显增加是该蛋白总量增高的主要特性。结论:检测血清结合珠蛋白总量以及特异的岩藻糖基化组分可能成为卵巢癌的血清诊断指标之一,可与其它检测方法相结合,提高卵巢癌早期诊断的有效性。     

Haptoglobin measurements in a number of laboratory animal species

The ability to measure haptoglobin levels is an invaluable tool for the detection of low-grade intravascular haemolysis.The suitability of two methods, radial immunodiffusion (Mancini et al. 1965) and photometric (Batchelor et al. 1989), for toxicological studies were investigated. These methods were selected for assessment due to their suitability for analysing a large number of samples. A study was initiated using three laboratory species, the cynomolgus monkey, the dog and the rat, to find the method of choice for each species. These animals were subjected to a simulated haemolytic episode and their haptoglobin levels were monitored by both analytical methods over a period of time.We found that the photometric method was suitably sensitive in detecting haptoglobin levels in the cynomolgus monkey and the dog but not in the rat. The RID method, however, worked well in rats.A comparison between the photometric and Sephadex Gel 100 method (Ratcliff and Hardwick 1964) was also carried out on approximately 250 dog samples. A close correlation between the two methods was found.     

Haptoglobin: a review of the major allele frequencies worldwide and their association with diseases

Haptoglobin (Hp) is a plasma alpha(2)-glycoprotein which binds free haemoglobin, thus preventing oxidative damage. The complex is rapidly removed from the circulation by a specific receptor (CD163) found on macrophages. Three major subtypes, Hp1-1, Hp2-1 and Hp2-2 are the product of two closely related genes HP(1) and HP(2). The frequency of the HP(1) and HP(2) genes varies worldwide depending on racial origin: the HP(1)frequency varying from about 0.07 in parts of India to over 0.7 in parts of West Africa and South America. Both HP(1) and HP(2) have been linked to susceptibility to various diseases. Such associations may be explained by functional differences between the subtypes in the binding of Hb and its rate of clearance from the plasma. However, there are also corresponding negative reports for disease associations. The conflicting evidence on disease association and the lack of association between disease and particular populations, despite the wide range of HP(1) and HP(2) gene frequencies across the world, may indicate that any associations are marginal.     

Pharmacological intervention for renal protection during cardiopulmonary bypass

Summary The possibility of minimizing organ damage following cardiopulmonary bypass (CPB) was examined. In the control group,n = 21, upon completion of CPB, elevation of the lysosomal enzyme -glucuronidase, which is a sensitive indicator of cellular damage, was affected by the concentration of granulocyte elastase (r = 0.59) or the endothelial-derived constricting factor, endothelin, (r = 0.8). Renal damage, which was detected by an increase in renal tubular enzymes (N-acetyl--D-glucosaminidase and -glutamyltranspeptidase) in urine, was also affected by endothelin (r = 0.79, r = 0.56), elastase (r = 0.6, r = 0.71), and by free hemoglobin levels (r = 0.76, r = 0.82). Next, the efficacy of pharmacological intervention for the prevention of renal damage was evaluated. During CPB, the administration of an elastase inhibitor (ulinastatin, 3 × 10⁵IU),n = 8, or a calcium antagonist (nicaldipine HCl, elastase release inhibitor; 5 /kg per min),n = 8, significantly reduced the elevation of -glucuronidase and renal tubular enzymes (p < 0.05). Although the ulinastatin and nicardipine groups demonstrated low values of elastase in the Intensive Care Unit (ICU), only the values of the nicardipine group reached statistical significance (p < 0.05). A reduction in endothelin levels compared to the control group was observed in the nicardipine group. However, preventive and counteractive effects of nicardipine against vasoconstriction caused by endothelin were also considered to play an important role in the prevention of renal damage. The addition of haptoglobin (4,000 IU) to the priming solution of the CPB also reduced levels of renal tubular enzymes (p < 0.05). We concluded that elastase, endothelin, and free hemoglobin were causes of renal damage during CPB. The administration of ulinastatin, nicardipine, or haptoglobin possibly prevent apparent renal dysfunction after CPB.     

冠心病合并糖尿病患者结合珠蛋白基因多态性研究

目的 检测冠心病合并糖尿病患者结合珠蛋白（Hp）基因型的分布,探讨其易感性及与冠状动脉病变严重程度的关系.方法 选择92例冠心病患者（其中45例合并糖尿病）及74名健康对照者,应用PCR-SSP技术检测Hp基因;采用Gensini冠状动脉评分系统（Gensini积分）对冠状动脉的狭窄程度进行评分.结果 冠心病组与对照组间的Hp2-2基因型频率分别为43.48％和54.05％,差异无统计学意义（P=0.376）.冠心病合并糖尿病组、单纯冠心病组的Hp2-2基因型频率分别为77.78％和42.55％,冠心病合并糖尿病组Hp2-2基因频率明显高于单纯冠心病组和对照组（P,＜0.01）.冠心病合并糖尿病组Hp1-1、Hp1-2、Hp2-2基因型Gensini积分分别为25.16±11.13、42.05±20.04和65.32±32.78,Hp2-2基因型积分显著高于Hp1-1基因型（P＜0.01）.结论 Hp2-2基因型可能是冠心病合并糖尿病的独立危险因素且与冠心病冠状动脉狭窄程度相关.     

Soluble CD163: a marker molecule for monocyte/macrophage activity in disease

By immunoprecipitation we have identified a soluble plasma form of CD163 (sCD163), the IL-6 inducible macrophage-receptor for clearing haptoglobinhaemoglobin complexes. A sandwich ELISA for measuring sCD163 was established and used to determine the sCD163 levels in normal subjects and patients with inflammatory and myeloproliferative diseases. In normal subjects, the concentration of sCD163 was high (median 1.9 mg/l) with low intraindividual variation. Highly increased levels were seen in patients with sepsis, myeloid leukaemia and in patients with Gaucher disease characterized by accumulation of tissue macrophages. Although the physiological role of sCD163 remains unknown, our present data suggest that sCD163 might prove to be a valuable marker molecule in infectious and myeloproliferative diseases.     

电离辐射后小鼠骨髓组成中结合珠蛋白的表达规律研究

目的研究照射前后结合珠蛋白在小鼠骨髓中的表达和分布，及其与骨髓细胞周期进程改变及凋亡的可能关联。方法用RT-PCR，Western blotting及流式细胞术的方法检测骨髓细胞Hp mRNA及细胞内外Hp蛋白的存在，及其辐射后的变化。用流式细胞术的方法分析了骨髓细胞中Hp阴性及阳性细胞群周期分布及凋亡率的差异。结果在骨髓细胞中检测到Hp mRNA的存在，而且8Gy照射后24h较正常小鼠有明显升高。骨髓细胞内及外液中检测到Hp蛋白的存在，同样8Gy照射后24h较正常小鼠亦呈明显升高。流式细胞术检测发现小鼠骨髓细胞中部分细胞含有Hp蛋白，照射后Hp阳性细胞的比例逐渐增加，并有着较好的时效关系和剂量依赖关系。骨髓Hp阳性细胞与阴性细胞的细胞周期分布存在显著的差异。在照射后6h骨髓细胞凋亡率随照射剂量增加而明显增加，而Hp^ 细胞凋亡率增加不明显。结论小鼠骨髓组织中可以检测到结合珠蛋白的存在，并且在辐射后有明显的增加，骨髓细胞中Hp阴、阳性细胞的周期分布和凋亡存在明显的差异。推测Hp可能参与辐射诱导骨髓损伤的修复过程。     

烧伤后血清类触珠蛋白物质性质的确定

为进一步明确类触珠蛋白物质的性质，采用十二烷基硫酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）分析，结果表明，过去发现的烧伤血清异常蛋白带是触珠蛋白的不同亚单位，即β、α２和α１链，提示类触珠蛋白物质可能即是血清触珠蛋白。进一步采用ＳＤＳ－ＰＡＧＥ、等电聚焦、免疫化学及生化等方法，对从正常血清及烧伤血清提取的触珠蛋白进行比较，发现烧伤血清触珠蛋白的分子量、等电点、结合血红蛋白的性质与能力以及免疫化学性质均与正常血清触珠蛋白相同。因此，认为过去发现的类触珠蛋白可能是血清触珠蛋白，烧伤后血清触珠蛋白的性质未发生改变。