Expression,production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10⁻⁸ M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10⁻⁷ M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.     

Association of CCL5 and CCR5 Gene Polymorphisms With Periodontitis in Taiwanese

Background: It has been suggested that genetic factors may predispose individuals to periodontal diseases. The present case‐control study aims to test whether the ?403 single nucleotide polymorphism of chemokine ligand 5 (CCL5‐403) and the 32‐bp deletion of CCR5 (CCR5Δ32) polymorphisms are associated with susceptibility to chronic and aggressive periodontitis. Methods: Taiwanese participants (N = 213) were grouped into control group (CG), generalized aggressive periodontitis (GAgP), or chronic periodontitis (CP) groups. DNA samples were obtained from peripheral blood. CCL5‐403, evaluated by polymerase chain reaction‐restriction fragment length polymorphism, and CCR5Δ32, evaluated by polymerase chain reaction, were compared among the three groups. Results: There was a significant association between type of periodontitis and having allele A or G in the CCL5‐403 polymorphism. GAgP patients were 3.7 times more likely than CP patients and 2.0 times more likely than CG patients to have allele A, instead of allele G, in CCL5‐403. GAgP patients were 3.1 times more likely than CG patients to have AG versus GG genotype. GAgP patients were also 5.0 and 19.8 times more likely than CP patients to have AG and AA genotypes, respectively, compared to GG. For the CCR5Δ32 polymorphism, no association was found between the type of periodontitis and having different genotype or allele distributions among GAgP, CP, or CG patients. Conclusion: The single nucleotide polymorphism of CCL5‐403 G substitution by A may play a role in AgP; however, the CCR5Δ32 polymorphism may not.     

Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein

The deposition of amyloid-beta is a pathological hallmark of Alzheimer’s disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer’s disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer’s disease.     

桡骨远端乙状切迹背侧骨块不同治疗对腕关节功能恢复的探讨

目的探讨桡骨远端乙状切迹背侧移位骨块的不同治疗对腕关节功能恢复的影响。方法从2009年1至2012年12月通过对40例桡骨远端Frykman分型Ⅶ-Ⅷ型骨折患者手术治疗,分为可靠内固定和未可靠内固定（未固定或固定发生移位）两组进行随访,骨性愈合后患腕拍正位片并作2mm层厚的CT平扫,检查下尺桡关节恢复情况,应用握力计及采用Gartland-Werley评分系统（GW评分）对腕部进行功能评估,观察桡骨远端乙状切迹背侧移位骨块不同固定对腕关节功能恢复的影响。结果所有病例得到随访,时间为3—22个月,平均7．5个月。骨折均愈合,平均骨折愈合时间2．3个月。无感染患者、无内固定松动及腕管综合征等并发症,无腕背侧肌腱磨损疼痛或肌腱断裂者。通过Gartland—Werley评分可靠内固定组预后优良率明显提高。与未可靠内固定组比较,有统计学意义。结论桡骨远端乙状切迹背侧移位骨块给予可靠内固定术,可以增加下尺桡关节的稳定性,明显改善腕关节功能恢复。     

应用纤连蛋白片段建立椎间盘退变动物模型

目的:探讨应用N端30kDa纤连蛋白片段(Fn-f)建立模拟人类椎间盘退变规律的椎间盘退变动物模型的可行性,为椎间盘退变的防治提供实验模型及实验依据.方法:选取雄性6月龄新西兰大白兔28只,麻醉后使用30G微量注射针和微量注射器,在透视引导下经皮将25μl 1.5μmol/L Fn-f(Fn-f组)或25μl磷酸缓冲液(PBS,0.01mol/L,pH值7.2;PBS组)随机分别注射入不同节段的腰椎间盘中心区.分别于注射4、8、12、16周后获取椎间盘,对椎间盘进行组织学检测(HE、Masson三色及番红O染色),并以RT-PCR法对椎间盘聚集蛋白聚糖和Ⅱ型胶原的基因表达水平进行分析.结果:与注射PBS椎间盘相比,注射Fn-f椎间盘造模后4周时椎间盘的髓核、纤维环结构以及胞外基质蛋白聚糖等无明显差别;8周时髓核细胞数量减少、细胞簇状分布,被胞外基质分隔开来,纤维环层状结构排列部分出现紊乱,蛋白聚糖染色变浅;12和16周时髓核细胞数量明显减少,细胞变圆,呈明显的成簇聚集分布,纤维环排列明显不规整,各层间出现裂隙,甚至断裂,蛋白聚糖染色明显变浅,甚至部分未见染色.Fn-f组椎间盘聚集蛋白聚糖mRNA表达水平在8、12和16周3个时间点均明显低于PBS组(P＜0.05);Ⅱ型胶原mRNA表达水平在12和16周时明显低于PBS对照组(P＜0.05).结论:透视引导下兔椎间盘内注射N端30kDa Fn-f可诱导椎间盘产生渐进性退行性病变,该法建立的动物模型可作为研究椎间盘退变的发病机制及防治的实验模板.     

Thrombin Generation During Cardiopulmonary Bypass Using Heparin-Coated or Standard Circuits

For quantitative comparison of thrombin generation during cardiopulmonary bypass (CPB) with heparin-coated vs conventional CPB circuits, thrombin-antithrombin III complex (TAT) and prothrombin fragment 1+2 (F₁₊₂) were analyzed in 20 patients undergoing combined heart valve surgery and coronary artery bypass grafting (CABG), in ten cases with heparin-coated circuits (COMB-HC) and in ten with standard circuits (COMB-C). Extensive thrombin generation was found in both groups, with maximal TAT and F₁₊₂ levels at the end of CPB. Of 15 operations with only CABG, seven were performed with heparin-coated circuits and heparin dose 40% of normal (CABG-HC), and eight with standard circuits and normal heparin doses (CABG-C). TAT was maximal at the end of CPB and F₁₊₂ peaked 3 hours after protamine injection. At the end of CPB both levels were significantly higher in the CABG-HC than in the CABG-C group, though thrombin generation was less than in the COMB groups. The abundant thrombin generation during CPB thus was much more pronounced during complex operations. Use of heparin-coated circuits did not reduce thrombin generation, which was increased by 60% reduction of the systemic heparin dose. The clinical implications are still unknown, as no complications were observed.     

甲基化特异性聚合酶链反应检测粪便ppENK基因甲基化对胰腺癌诊断的可行性

目的 评价甲基化特异性聚合酶链反应(MSP)检测粪便ppENK基因高甲基化用于诊断胰腺癌的可行性.方法 收集胰腺癌患者24例和对照6例的新鲜粪便标本.采用MSP检测全部粪便标本中ppENK的甲基化状态;采用PCR检测全部粪便标本中野生型ppENK的阳性率.采用PCR-限制性片段长度多态性(RFLP)检测粪便K-ras的突变情况.将胰腺癌细胞PC3单细胞悬液加入同一健康者粪便标本,MSP法检测ppENK甲基化阳性,计算甲基化阳性时需掺入的胰腺癌细胞的最少数量.结果 30份粪便标本的甲基化检出率为0(0/30),非甲基化检出率为10％(3/30),野生型ppENK检出率为6.7％ (2/30);PCR-RFLP可检测出所选10份野生型ppENK阴性的胰腺癌标本中的8份,其中7份有K- ras第12位密码子突变.MSP方法能够检测到粪便中ppENK甲基化条带所需胰腺癌细胞的数量至少为50个/ml.结论 采用MSP方法检测胰腺癌患者粪便标本的ppENK基因甲基化状态,尚不能成为筛查和诊断胰腺癌的方法.     

福建地区口腔癌患者p53基因72密码子多态性与口腔癌易感性的研究

目的研究p53基因多态性与口腔癌的关联。 方法 对149例口腔癌病例和303例对照病例采集5 mL血液样本,提取基因组DNA,采用PCR-RFLP法检测p53基因的多态性,利用Stata12.1软件分析p53基因的多态性与口腔癌的关联,并进行基因-环境交互作用分析。 结果 p53基因Pro/Pro基因型携带者较Arg/Arg基因型携带者口腔癌的发病风险增加1.837倍(95%CI:1.016,3.325)。p53基因Pro/Pro基因型与饮酒有相乘交互作用(OR_相乘:4.375,95%CI:1.646,11.627)。 结论 p53基因Pro/Pro基因型可能是福建地区口腔癌的易感基因,Pro/Pro基因型与饮酒具有相乘交互作用。     

五种血清肿瘤标志物联合检测对肺癌的临床诊断价值

目的探讨五种血清肿瘤标志物癌胚抗原（CEA）、细胞角蛋白19片段（CYFRA21—1）、神经元特异性烯醇化酶（NSE）、糖类抗原125（CA125）和糖类抗原242（CA242）联合检测对肺癌临床诊断的价值和意义。方法选择57例初诊肺癌患者作为实验组,选择同期55例良性肺疾病患者作为对照组。采用化学发光法（CLIA）检测血清肿瘤标志物CEA、CYFRA21—1、NSE、CA125和CA242的水平,比较分析五种血清肿瘤标志物检测在肺癌临床诊断中的价值。结果实验组五种肿瘤标志物的水平均明显高于对照组（P均〈0．05）;且五种血清肿瘤标志物在不同病理类型肺癌中有不同水平,CEA、CA125、CA242在腺癌中表达较高,而CYFRA21。1在鳞癌中表达较高,NSE在小细胞癌中表达较高;联合检测时对诊断的灵敏度和准确率分别为92．8％和89．6％,明显高于各单项检查（P均〈0．05）。结论血清肿瘤标志物CEA、CYFRA21—1、NSE、CA125和CA242联合检测可以提高肺癌诊断的灵敏度和准确率,可应用于肺癌的生物学诊断。