9.1C3单克隆抗体轻,重链可变区基因克隆及序列分析

９．１Ｃ３分子是一种参与ＮＫ、巨噬细胞及ＬＡＫ细胞杀伤过程的重要细胞表面分子，主要表达于外周血单个核细胞表面。本文运用分子生物学技术，从分泌９．１Ｃ３ＭＣＡｂ的杂交瘤细胞中提取总ＲＮＡ，经反转录、ＰＣＲ分另１１分离了９．１Ｃ３ＭｃＡｂ轻链可变区（ＶＬ）基因及重链可变区（ＶＨ）基因，并用全自动荧光ＤＮＡ序列分析仪对９．１Ｃ３ＶＨ、ＶＬ基因序列进行了分析。结果表明：９．１Ｃ３ＶＨ基因为３５１ｂＰ，编码１１７ａａ残基，９．１３ＶＬ为３２４ｂＰ，编码１０８ａａ残基；从推导出的氨基酸序列分析证实９．１Ｃ３ＶＨ、ＶＬ序列均符合小鼠ｌｇ可变区的氨基酸序列特征；根据Ｋａｂｂａｔ分类体系，９．１Ｃ３ＶＨ隶属Ｉｇ重链第Ⅱ（Ｃ）亚组，９．１Ｃ３ＶＬ隶属小鼠ＩｇＫ链第Ⅴ亚组。９．１Ｃ３ＶＨ、ＶＬ基因的克隆成功为其单链抗体的构建、表达奠定了良好的物质基础。     

Analysis of the α/β T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the α/β T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire. In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions. We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the α/β T-cell repertoire in autoimmune and infectious diseases.     

Hyperplastic foci in chronic liver disease: Their proliferative activity assessed by nucleolar organizing region

In the cirrhotic and precirrhotic liver, there may be small foci with increased cellularity and amphophilic cytoplasm. These are microscopic lesions that do not form macroscopically detectable nodules, which differ from the macroscopically apparent nodules of dysplastic nodules. In the present study, we assessed the proliferating activity of 12 hyperplastic foci in 11 patients with cirrhosis or chronic hepatitis, by staining for agyrophilic nucleolar organizing regions (AgNOR). The mean AgNOR count per nucleus in the hyperplastic foci ranged from 0.96 to 1.36 (mean, 1.13; SD 0.12), and from 0.81 to 1.06 (mean, 0.94; SD 0.08) in the controls. The AgNOR count In the hyperplastic foci was significantly higher than that In the controls (P> 30.01). Small hyperplastic foci show Increased proliferative activity. Further study on these foci is required to clarify their relation to hepatocarcinogenesis.     

Role of the 2 adenine (g.11293_11294insAA) insertion polymorphism in the 3' untranslated region of the factor VII (FVII) gene: molecular characterization of a patient with severe FVII deficiency

Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients.     

Neuronal responses to horizontal neck deflection in the group X region of the cat's medullary brainstem

Summary Neuronal responses to natural stimulation of neck proprioceptors were studied in the region of the small cell group x in the dorsolateral medullary brainstem of slightly anesthetized and paralyzed cats. Stimulation consisted of horizontal trunk rotations about C₁ with the head fixed in space. Out of 74 neurons recorded, 92% showed an increase in discharge rate with ipsilateral neck stretch and a decrease with contralateral stretch (Type N I responses); 8% showed the reverse response pattern (Type N II responses). In the primary head-to-trunk position, almost all neurons had tonic activity that probably stemmed from prestretched neck proprioceptors. Responses to sinusoidal stimulation and position trapezoids showed a static (position-sensitive) as well as a dynamic (essentially velocitysensitive) component. The relative weight of the two components varied considerably among the neurons. It was not possible to distinguish discrete neuronal populations on the basis of the dynamic characteristics. There was no evidence of a convergent input from other receptor systems, such as the horizontal canal system. Several neurons responded to muscle tapping and showed an increase of the velocity component following systemic injection of succinylcholine. We take this as evidence that they may receive input from muscle spindle receptors.Supported by the Deutsche Forschungsgemeinschaft, SFB 70/U4     

颈静脉孔区的显微解剖及定位标志研究

目的:研究颈静脉孔区的显微外科解剖,探讨寰椎横突(TPA)、头侧直肌、二腹肌沟、颈静脉突等解剖标志在颈静脉孔区病变手术中的定位意义。方法:成人头颈标本15例,男13例,女2例,红色乳胶灌注颈总动脉和椎动脉。手术显微镜下(×3～×30)逐层显露颈静脉孔区结构,明确该区显微解剖特征、相关的解剖标志及其定位意义。结果:颈静脉孔区多数重要的解剖结构均可以TPA为参照标志予以明确。二腹肌后腹位于其浅层。TPA的后方为枕下三角,三角内有椎动脉、椎静脉丛和颈1神经通过。头侧直肌起始于TPA的外表面,止于枕骨颈静脉突的下表面,可作为确定颅外颈静脉孔的解剖标志。茎突位于TPA的前方,颈内静脉、迷走神经、副神经、舌下神经穿行于茎突与TPA之间,颈内动脉位于颈内静脉的前内侧。二腹肌、颈静脉突、颈动脉嵴对定位面神经、颈静脉孔及舌咽神经等结构具有重要意义。结论:颈静脉孔区解剖结构复杂,利用寰椎横突、头侧直肌、二腹肌沟、颈静脉突等解剖标志有助于明确此区域重要的解剖结构,避免术中不必要的损伤。     

经额入路行蝶鞍区肿瘤手术的应用解剖

目的　为经额入路蝶鞍区肿瘤切除术提供解剖学资料。方法　采用解剖技术对 35个甲醛固定 ,红色明胶动脉灌注的尸头标本蝶鞍区的有关结构进行观察。结果　两侧视神经管颅口内缘间距为 11.0 9± 2 .2 2mm ,视交叉前缘至交叉前沟前缘间距为 7.0 2± 1.86mm。垂体上面与鞍隔平面有三种位置关系 :垂体上面通过鞍隔孔露出鞍隔平面以上者 3例 ( 8.75% ) ,在鞍隔平面以下者 13例 ( 37.14% ) ,与鞍隔平面平齐者 19例 ( 54.2 9% )。两侧颈内动脉在穿经海绵窦上壁处间距为 13.2 5± 2 .4 8mm ,大脑前动脉发起处 ,两侧颈内动脉之间距为 17.86± 1.55mm。结论　经额入路蝶鞍区肿瘤切除术主要是通过交叉前间隙 ,在颈内动脉之间的区域操作 ,手术中要保护颈内动脉、视神经等结构 ,以减少并发症的发生     

充满型及非充满型胆囊结石粘膜病理形态及核仁组成区的研究

目的：探讨充满型及非充满型胆囊结石粘膜组织病理学的差异及相应外科治疗措施,方法：收集1995年10月至1996年10月期间的胆囊结石标本做常规病理染色及嗜银蛋白染色分析。结果：发现充满型及非充满型结石组胆囊粘膜不典型发生率及AgNOR数目均有显著差异。结论：一经诊断为充满型胆囊结石,特别是中老年肥胖妇女,应早期手术切除病变胆囊。