上皮钙黏素及埃兹蛋白在胃癌和淋巴结转移灶中的表达及其意义

目的 检测胃腺癌组织和淋巴结转移灶中细胞骨架相关蛋白上皮钙黏素(E-cad)及埃兹蛋白(Ezrin)的表达,观察其与胃腺癌侵袭和转移的关系,探讨胃腺癌的浸润和转移机制.方法 用SP免疫组织化学法检测80例胃癌和40例淋巴结转移灶中E-cad和Ezrin的表达.结果 E-cad和Ezrin在癌旁正常胃黏膜上皮为胞膜表达,而在胃腺癌组织中E-cad出现胞膜、胞质两种表达形式,Ezrin在肿瘤细胞中则为胞质表达.在胃腺癌原发灶中,E-cad、Ezrin的表达与胃腺癌的分化程度相关.E-cad的膜表达及Ezrin的表达随分化程度降低而下降(P＜0.01;P＜0.05),E-cad的浆表达随分化程度降低而升高(P＜0.01).E-cad浆表达的升高与浸润深度、淋巴结状态相关且在原发灶显著高于转移灶(P＜0.01;P＜0.05;P＜0.01).在淋巴结转移灶中,E-cad的膜表达在淋巴结转移的早期阶段高于晚期阶段(P＜0.05).另外,E-cad膜表达及Ezrin的表达呈正相关(P＜0.01).结论 胃腺癌中E-cad的表达异常导致了细胞之间的黏附力下降,从而在胃腺癌的侵袭和转移中发挥重要作用.埃兹蛋白参与调节胃腺癌细胞的分化,可能通过与E-cadherin/catenin作用来调控肿瘤细胞的黏附和侵袭.     

Fabrication of protein chips based on 3-aminopropyltriethoxysilane as a monolayer

Although 3-aminopropyltriethoxysilane (APTES) is widely adopted as a monolayer in biosensors, experimental silanization takes at least 1 h at high temperature. Therefore, the feasibility of the silanization with APTES in a short reaction time and at room temperature was investigated. The surface modification of glass slides using a self-assembled monolayer of APTES with a concentration of 10% was studied by immobilizing FITC. APTES was successfully immobilized on the glass slide. The effect of reaction temperature and time of silanization were investigated. Various silanization conditions of APTES were examined by contact angle measurement and fluorescence microscopy. The surface of glass patterns with a gold thin film as background was characterized by determining the fluorescent intensities following the immobilization of fluorescein isothiocyanate (FITC), protein A-FITC, antimouse IgG-FITC and sheep anti-bovine albumin-FITC. The normalized fluorescent intensity indicated that a short period (4 min) of silanization at 25°C suffices to form an APTES thin film by the immobilization of protein A on a glass surface. Such a condition does not require microheaters and temperature sensors in a microfluidic system, which will significantly reduce the manufacturing process, cost, and reaction time in the future.     

A novel strategy based on histological protein profiling in‐silico for identifying potential biomarkers in urinary bladder cancer

OBJECTIVE

To screen a publicly available immunohistochemistry (IHC) based web‐atlas, to identify key proteins in bladder cancer that might serve as potential biomarkers.

MATERIALS AND METHODS

The first version of the Human Protein Atlas (HPA 1.0), with 660 proteins, was visually examined to identify proteins with a variable staining pattern among the 12 tissue samples representing bladder cancer. None or limited previous characterization in bladder cancer, as well as a supportive Western blot, were also required. The selected proteins were then evaluated in an independent set of patient samples (106 tumour samples of differing stage and grade) represented in a tissue microarray (TMAi). The IHC expression of the identified proteins in the TMAi was scored and related to tumour stage and grade.

RESULTS

The expression profiles of the 13 proteins selected from the web‐atlas were confirmed in the TMAi. Expression patterns for seven proteins were significantly altered (P < 0.05) with higher stage and/or grade. Three of those (CN130, DSG3, PHF6) lack characterization in bladder cancer, whereas the remaining four proteins have previously been suggested as key proteins/potential biomarkers in cancer, some of them also in bladder cancer.

CONCLUSION

New candidate proteins for urinary bladder cancer were identified through screening of the publicly available HPA 1.0. Although further evaluation is necessary, this strategy is promising in the search for new biomarkers, with potential to improve the management of patients with this disease.     

绿色荧光蛋白标记的GRP78蛋白表达和功能研究

目的:应用增强型绿色荧光蛋白(EGFP)来标记葡萄糖调节蛋白78(GRP78),从而研究其与免疫细胞结合的情况。方法:通过在原核表达的GRP78的C末端连接一个EGFP来显示跟踪该蛋白的表达,进而通过流式细胞仪检测该蛋白同小鼠脾脏免疫细胞结合的情况。结果:GRP-EGFP融合蛋白分子量为126kD,Western Blot证实该蛋白表达正确,并且表达GRP78-EGFP的BL21菌在紫外光激发下发射出强烈的绿色荧光;与EGFP单体相比较,GRP78-EGFP主要与中性粒细胞相结合,其平均结合率为5.15%。结论:绿色荧光蛋白标记的GRP78蛋白在原核中的表达具有天然构象并能发挥标记目的蛋白的作用。     

Gastric Bypass is not Associated with Protein Malnutrition in Morbidly Obese Patients

Background  Patients undergoing bariatric surgery with a gastric bypass lose about 66% of excess weight. Although this procedure induces weight loss, it is unknown whether it leads to protein malnutrition, which is studied here. Methods  One hundred ten obese patients (body mass index, 47.9 ± 8.6 kg/m²) undergoing gastric bypass had a measurement of plasma albumin and transthyretin (formerly prealbumin) and a calculation of nutritional risk index (NRI) before and throughout the 2 years following the surgery. Results  All but five patients lost more than 15% of initial weight; the mean loss of excess weight was 65.2 ± 26.4% at 2 years. Plasma concentrations of albumin and transthyretin decreased after surgery, but while albumin returned to initial values after 12 months, transthyretin remained low. Only one patient had an albumin below 30 g/l; another one had a transthyretin lower than 110 mg/l. All NRI scores were lower than 83.5 (62 ± 5, ranging 44–70), qualifying patients for severe malnutrition. Conclusion  Malnutrition is difficult to diagnose in obese patients undergoing surgery. The large weight loss is most often not associated with protein malnutrition. Whether gastric bypass induces protein malnutrition remains to be established.     

Pregnancy Nutritional Indices and Birth Weight After Roux-en-Y Gastric Bypass

Background  Maternal metabolic profile and nutritional course of pregnancy after bariatric interventions is incompletely known. Their impact on birth weight has also not been hitherto addressed. Aiming to document such variables, a retrospective study was undertaken. Methods  Women previously submitted to silastic ring Roux-en-Y gastric bypass, who conceived after 0–5 years (n = 14), were investigated. Intake of selected macro- and micronutrients, representative laboratory measurements, and correlation of these findings with birth weight and time to conception was documented. Results  Mean calorie intake was restricted to about 1,800 kcal/day. Protein (71 ± 17 g/day) and supplementary iron (60 mg/day) were barely adequate, and calcium and vitamin B₁₂ did not meet current recommendations, only folic acid being optimal. Biochemical monitoring reflected these inconsistencies, with occasional low values for serum albumin (4.1 ± 0.4 g/dL), hemoglobin (11.4 ± 1.5 g/dL), iron (78 ± 50 μg/dL) and vitamin B₁₂ (193 ± 102 pg/mL) but not folate. Lipids, glucose, and uric acid were much better than before the anti-obesity intervention. Reduced plasma lipids, glucose, and uric acid were associated with larger birth weight, albeit within the normal range. Conclusions  (1) Anemia as well as additional nutritional deficits during pregnancy were not totally eliminated, despite dietary guidance and micronutrient supplementation; (2) alleviation of metabolic comorbidities was demonstrated, and improved normalization predicted higher birth weight; (3) energy and folate intake was sufficient, but other nutrients probably did not reach ideal levels; (4) recent dietary guidelines for this population represent a step forward, but additional studies are needed.     

Association of bilirubin and protein thiols in relation to copper and ceruloplasmin in hyperbilirubinemic patients

Objective： Bilirubin is a double edged sword in biological system, acting as a toxic molecule and cytoprotectant. Unconjugated bilirubin is proved to show antioxidant activity in vitro and in vivo. In the current work we tried to know the relationship between both conjugated and uneonjugated bilirubin with copper and protein thiols in patients with hyperbilirubihernia. Methods： Study was conducted on 56 hyperbilirubinemie eases and 56 healthy controls. Serum copper, ceruloplasmin, protein thiols, total bilirubin, conjugated and unconjugated bilirubin, uneonjugated bilirubin/albumin ratio, total protein, albumin, AST, ALT and ALP were estimated. Results： There was significant increase in serum copper, total biliruhin, conjugated and uneonjugated bilirubin, uneonjugated bilirubin/albumin ratio, AST, ALT, and ALP, and decrease in serum ceruloplasmin, protein thiols, total protein, and albumin in hyperbilirubinemie cases when compared to healthy controls. Conjugated bilirubin correlated positively with liver enzymes AST and ALP, and negatively with protein thiols, total protein and albumin. Uneonjugated bilirubin correlated positively with ALT. Protein thiols correlated negatively with copper and positively with eeruloplasmin, and also correlated negatively with liver enzymes like AST, ALT and ALP, and positively with total protein and albumin. Conclusion： Combination of elevated levels of trace elements like copper and availability of reducing agent like bilirubin may prove deleterious by generating free radicals.     

Ag43／FGFR-1嵌合蛋白疫苗抗小鼠纤维肉瘤血管生成的实验研究

目的：利用Antigen43（Ag43）／成纤维细胞生长因子I型受体（FGFR一1）重组嵌合蛋白作为疫苗,了解其是否具有抑制小鼠肿瘤生长的作用,并初步探讨其作用机理。方法：40只BALB／c雌性小鼠接种MethA细胞后第7天,随机分为Ag43／FGFR．1蛋白免疫组（AF组）、A酗3蛋白免疫组（Ag43组）、FGFR—1蛋白免疫组（FGFR一1组）和生理盐水对照组（NS组）4组,每组10只,观察免疫治疗后的荷瘤小鼠肿瘤体积和生存曲线,分别用免疫组织化学方法检测肿瘤组织微血管密度（MVD）,免疫印迹（Western blot）方法检测抗自身FGFR—1的抗体,酶联免疫斑点试验（ELISPOT）检测分泌抗自身FGFR-1抗体的B淋巴细胞的数量。结果：Ag43／FGFR-1组与FGFR-1蛋白、A甜3蛋白、生理盐水对照组肿瘤组织MVD计数分别为11．9±2．3、59．6±3．8、60．6±1．2和61．9±3．4（P〈0．01）;与对照组相比,Ag43／FGFR-1组肿瘤体积明显变小（P〈0．01）,存活时间明显延长（P〈0．01）,血清中发现含有抗自身FGFR-1的抗体且发现小鼠脾脏中分泌抗自身FGFR-1抗体的B淋巴细胞数目明显增多（P〈0．01）。结论：Ag43／FGFR-1蛋白质疫苗能够诱导荷瘤鼠产生特异性免疫反应,从而抑制肿瘤血管生成和肿瘤的生长。     

Endothelin-1 and protein kinase C co-expression in the pathogenesis of diabetic retinopathy

Diabetic retinopathy (DR) is becoming one of the most important complications of diabetes at present. Endothelin-1 (ET-1) and protein kinase C (PKC) are the two important proteins involved in the pathogenesis of DR. A clarification on the interaction between ET-1 and PKC has not yet been made. Here, the author used a new gene ontology technology to predict the molecular function of ET-1 and PKC in an episode of co-expression. With the use of the GoFigure server, the molecular function of ET-1 and PKC is predicted. According to this study, different pathways can be derived from ET-1 and PKC; however, ET-1–PKC produces the same pathway as PKC. This could mean that the interaction between ET-1 and PKC results in increased activity of the PKC pathway but does not generate any new pathway. This finding can be a good explanation for the co-expression between ET-1 and PKC in the pathogenesis of DR.     

Effect of 8.5% and 25% caloric restriction on mitochondrial free radical production and oxidative stress in rat liver

Previous studies have consistently shown that 40% caloric restriction (CR) decreases the rate of mitochondrial ROS production and steady-state levels of markers of oxidative damage to macromolecules including mitochondrial DNA. However, few investigations have studied whether these changes also occur in lower CR regimes. This is of potential interest since moderate levels of dietary restriction are more practicable for humans. In this investigation male Wistar rats were subjected to 8.5% and 25% caloric restriction. Neither 8.5% nor 25% CR changed mitochondrial ROS production, oxygen consumption or mtDNA oxidative damage in rat liver mitochondria. However, both 8.5% and 25% CR significantly decreased the five different markers of protein oxidation, glycoxidation and lipoxidation measured, aminoadipic and glutamic semialdehyde, carboxyethyl-lysine, carboxymethyl-lysine, and malondialdehyde-lysine. The fatty acid composition of liver mitochondria was also affected and led to a moderate decrease in the degree of membrane unsaturation in both 8.5% and 25% CR. While 8.5% CR only affected complex I concentration (which was decreased), 25% CR decreased complexes I and IV and increased complexes II and III of the respiratory chain. Apoptosis-inducing factor (AIF) significantly decreased in 25% CR but not in 8.5% CR. The results show that moderate levels of caloric restriction can have beneficial effects including decreases in oxidative protein modification and a lower sensitivity of membranes to lipid peroxidation, in association with a reprogramming of the respiratory chain complexes and AIF content.