一种新型的乙型肝炎病毒剪接变异体

目的 证实乙型肝炎病毒(hepatitis Bvirus，HBV)前基因组RNA在458nt～1308nt(nt，核苷酸)之间可发生剪接并产生相应的基因组剪接变异体。方法对1株分离自慢性乙型肝炎患者血清的亚基因组HBVDNA(2366bp)进行测序并以Vector NT16．0软件进行分析。将全基因组HBVDNA(3215bp)理论上可能的剪接供体及受体内的核苷酸进行定点突变并将突变株转染HepG2细胞，以位于HBVDNA458nt及1308nt两侧的特异引物检测转染后的细胞内HBV核心颗粒DNA，并以相同引物检测458nt～1308nt发生缺失的HBV DNA在不同病程的乙型肝炎患者血清中的存在情况。结果23661bp HBV DNA在458nt～1308nt之间发生缺失并符合GT-AG剪接模式。3215bp全基因组HBV DNA在459nt及1306nt分别发生G→A及A→C突变后均不能产生458nt～1308nt缺失。458nt～1308nt发生缺失的HBVDNA在慢性乙型肝炎、肝硬化及原发性肝细胞癌患者血清的检出率分别为80％、75％及70％，显著高于HBV无症状携带者10％的检出率。结论HBV前基因组RNA在458m～1308nt之间可发生剪接并产生长度为2366bp的基因组剪接变异体。该类型基因组剪接变异体基因结构特点及在HBV相关性肝病中广泛存在提示它与HBV致病性密切相关。     

注意缺陷多动障碍儿童视觉保持及运动研究

目的探讨注意缺陷多动障碍(attentiondeficithyperactivitydisorder,ADHD)儿童的视觉机能特征。方法采用本顿视觉保持测验(Bentonvisualretentiontest,BVRT)、连线测验(TrailMakingTest,TMT)及符号-数字模式测验(Symbol-DigitModalitiesTest,SDMT)3种方法,分别测试50例ADHD儿童和50例正常儿童的视知觉、视觉记忆、视空间结构、视觉运动注意和视觉扫描等。结果①ADHD组BVRT三种测验方试C、D、E正确分总分(4.9±2.0)低于对照组(7.7±1.4)(t总=8.00,P<0.01),而失误分总分(6.9±3.9)高于对照组(2.5±1.7)(t总=12.1,P<0.01)。②ADHD组完成A型TMT时间较对照组延长(94.6±51.2/56.9±23.7,t=4.61,P<0.05),所犯错误次数与对照组相比差异有显著性(0.3±0.8/0.1±0.3,Hc=4.46,P<0.05);ADHD组完成B型TMT时间较对照组延长(228.0±142.1/120.1±43.8,t=5.14,P<0.05),所犯错误次数较对照组明显增多(6.0±5.5/2.1±3.4,Hc=22.96,P<0.01)。③ADHD组SDMT得分低于对照组(65.2±29.3/97.9±18.8,t=6.65,P<0.01)。结论ADHD儿童存在视知觉、视觉记忆、视空间结构、视觉运动注意以及视觉扫描等视觉机能方面缺陷。     

脑震荡对大鼠多巴胺能神经元及神经纤维的影响

目的：探讨脑震荡(BC)鼠多巴胺能神经元和神经纤维内酪氨酸羟化酶(TH)含量的变化。方法：用金属单摆打击装置复制 BC 模型。动物随机分为对照组和伤后1～24 d 损伤组；用免疫组织化学结合图像分析方法研究 BC 后中脑黑质致密区(SNC)和腹侧被盖区(VTA)多巴胺能神经元及基底节尾壳核多巴胺能神经纤维 TH 含量的变化。结果：在 SNC 和 VTA 及基底节,BC 后1、4、8及16 d 组的 TH 免疫反应阳性明显高于对照组,其中以4d 组反应最强,24 d 组在 SNC 和基底节与对照组比较无显著性差异,但在 VTA,TH 免疫反应阳性仍高于对照组。结论：BC 后 TH 早期增高可能是脑损伤神经元过度兴奋的反应,随后持续增高一段时间可能是多巴胺能神经元上调其合成能力的一种代偿反应。     

站、坐位态骨盆应力分布的实验研究

目的：明确骨盆的应力分布，指导骨盆肿瘤瘤段骨切除后骨盆环的重建及人工半骨盆的设计与固定。方法：实验取完整骨盆的新鲜尸体（意外死亡）4具，采用应变计测量方法，对不同状态下骨盆的应力进行分析。结果：站立时，骶髂关节中部、耻骨支、髋关节前上方承受较大的压应变；而坐位时，坐骨支、骶髂关节中下部、耻骨支均承受较大的压应变。结论：站立时应力主要是经髋臼的前上方耻骨、骶髂关节传导，人工半骨盆置换设计与置换时，其结构应予加强，并作到确切固定，尤其是骶髂关节的中下部。     

人内质网分子伴侣BiP的克隆、表达及其在RA患者血清抗体检测中的作用

以类风湿关节炎(rheumatoid arthritis,RA)患者滑膜组织文库cDNA为模板,用PCR方法扩增得到1965 bp人内质网分子伴侣BiP的全长cDNA,将其克隆入OmicsLinkTM表达载体pReceiver-B01a,构建重组表达质粒pReceiver-B01a-BiP。重组表达质粒导入大肠杆菌BL-21(DE3)菌株中诱导表达目的蛋白,表达产物以Ni+-NTA agarose层析柱纯化。SDS-PAGE和免疫印迹法(Western blot)分析发现,表达产物以可溶性蛋白和包涵体形式共同存在,表达量约占菌体蛋白总量的20%~30%。相对分子质量约为80000,纯度达到电泳级。经免疫印迹法鉴定,获得的BiP重组蛋白可以与RA患者血清反应,检测的抗BiP抗体在RA患者血清中的阳性率为75.4%(49/65),明显高于系统性红斑狼疮(systemic lupus erythe-matosus,SLE)患者14.6%(7/48)、原发性干燥综合征(primary Sj gren’s syndrome,pSS)患者7.3%(4/55)及正常人0%(0/71)(P<0.01)。表明,经原核表达获得的BiP全长蛋白,RA患者血清抗体可以很好结合,有望应用于临床血清学诊断。     

Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array

The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilized to investigate the genetic causes of complex human diseases. Here we present a high-throughput genotyping platform that uses a one-primer assay to genotype over 10,000 SNPs per individual on a single oligonucleotide array. This approach uses restriction digestion to fractionate the genome, followed by amplification of a specific fractionated subset of the genome. The resulting reduction in genome complexity enables allele-specific hybridization to the array. The selection of SNPs was primarily determined by computer-predicted lengths of restriction fragments containing the SNPs, and was further driven by strict empirical measurements of accuracy, reproducibility, and average call rate, which we estimate to be >99.5%, >99.9%, and>95%, respectively [corrected]. With average heterozygosity of 0.38 and genome scan resolution of 0.31 cM, the SNP array is a viable alternative to panels of microsatellites (STRs). As a demonstration of the utility of the genotyping platform in whole-genome scans, we have replicated and refined a linkage region on chromosome 2p for chronic mucocutaneous candidiasis and thyroid disease, previously identified using a panel of microsatellite (STR) markers.     

高灵敏orexin A放免法的建立及其对大鼠缺血再灌注的应用

建立高灵敏orexinA放射免疫分析方法 (RIA) ,探讨肠缺血再灌注损伤对血浆及下丘脑orexinA水平的影响。采用氯胺T法制备碘标记orexinA ,抗原抗体反应采用平衡一步法 ,4℃温育 2 4h后 ,经PR试剂分离结合和游离的标记抗原 ,并用该方法测量正常人、高血脂患者血浆orexinA水平及大鼠肠缺血再灌注损伤后血浆及下丘脑组织中orexinA浓度的变化。结果表明 ,本方法测定orexinA标准曲线范围为 2 1 - 2 0 0 0 pg/mL ,最低检出量为 2 1 pg/mL ,批内和批间变异系数均小于 1 0 %。 30名正常人血浆orexinA水平为 338.4 8± 2 0 .2 4 pg/mL ,30例高血脂患者为 343.5 1± 1 5 .4 9pg/mL ;大鼠肠缺血再灌注损伤前后及不同损伤时间点orexinA的变化差异亦无显著性。本文提示orexinARIA方法灵敏度和特异性较高 ,可用于人血浆、大鼠血浆和下丘脑组织orexinA的检测     

CCL25和CCL28及其相应受体CCR9和CCR10

CCL25和CCL28是CC趋化因子家族成员,主要表达于胸腺树突状细胞和黏膜上皮细胞,CCL25和CCL28相应的受体分别是CCR9和CCR10,表达于T淋巴细胞和B淋巴细胞。CCL25和CCL28及其相应受体对循环IgA浆母细胞和T淋巴细胞的归巢起重要作用,而且CCL28还具有广谱的抗微生物活性。     

Identification of antigenic epitopes in the haemagglutinin protein of H7 avian influenza virus

ABSTRACT

The H7 subtype avian influenza virus (AIV) has been reported to infect not only poultry but also humans. The haemagglutinin (HA) protein is the major surface antigen of AIV and plays an important role in viral infection. In this study, five monoclonal antibodies (mAbs, 2F8, 3F6, 5C11, 5E2 and 5C12) against the HA protein of H7 virus were produced and characterized. Epitope mapping indicated that ¹⁰³RESGSS¹⁰⁷ was the minimal linear epitope recognized by the mAbs 2F8/3F6/5C11, and mAbs 5E2/5C12 recognized the epitope 103-145aa. The protein sequence alignment of HA indicated that the two epitopes were not found in other subtypes of AIV, and none of the five mAbs cross-reacted with other subtypes, suggesting these mAbs are specific to H7 virus. The epitope ¹⁰³RESGSS¹⁰⁷ was highly conserved among Eurasian lineage strains of H7 AIV, whereas three amino acid substitutions (E104R, E104K and E104G) in the epitope occurred in 98.44% of North-American lineage strains. Any of these single mutations prevented the mutated epitope from being recognized by mAbs 2F8/3F6/5C11; thus, these mAbs can distinguish between Eurasian and North-American lineages of H7 strains. Furthermore, the mAbs 2F8, 3F6 and 5C11 could be highly blocked with H7-positive serum in blocking assays, revealing that ¹⁰³RESGSS¹⁰⁷ may be a dominant epitope stimulating the production of antibodies during viral infection. These results may facilitate future investigations into the structure and function of HA protein, as well as surveillance and detection of H7 virus.

RESEARCH HIGHLIGHTS

• Five mAbs against HA protein of H7 AIV were generated and characterized.

• Two novel epitopes ¹⁰³RESGSS¹⁰⁷ and 103-145aa were identified.

• The epitope ¹⁰³RESGSS¹⁰⁷ differs between Eurasian and North-American lineages.

• The mAbs 2F8, 3F6 and 5C11 could distinguish two lineages of H7 strains.

     

硫氧还蛋白过氧化物酶Ⅵ在小鼠卵巢、输卵管和子宫中的分布

目的探讨硫氧还蛋白过氧化物酶VI(PrxVI)卵母细胞生长发育和早期胚胎发育中的可能作用。方法用免疫组织化学方法观察6～8周雌性昆明小鼠卵巢、输卵管和子宫PrxVI的分布。结果在卵巢中,PrxVI免疫阳性见于卵巢卵母细胞和黄体;在输卵管中,输卵管上皮呈PrxVI免疫阳性反应;在子宫中,PrxVI免疫阳性反应见于子宫内膜上皮、腺上皮和子宫内膜基质细胞。结论PrxVI的定位分布提示其可能参与小鼠卵母细胞生长发育和卵母细胞成熟过程的调节,并为受精卵和早期胚胎发育提供抗氧化的良好微环境。